(en)The present invention provides an expression cassette containing a polynucleotide coding sequence for a hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which is operably linked to a heterologous promoter. Also provided are methods of engineering plants that have reduced lignification, as well as cells, plant parts, and plant tissues from such engineered plants.

1.ApplicationNumber: US-201214232018-A
1.PublishNumber: US-2015013033-A1
2.Date Publish: 20150108
3.Inventor: LOQUÉ DOMINIQUE
EUDES AMYERICK GUILLAUME

4.Inventor Harmonized: LOQUÉ DOMINIQUE(US)
EUDES AMYERICK GUILLAUME(US)

5.Country: US
6.Claims:
(en)The present invention provides an expression cassette containing a polynucleotide coding sequence for a hydroxycinnamoyl-CoA hydratase-lyase (HCHL), which is operably linked to a heterologous promoter. Also provided are methods of engineering plants that have reduced lignification, as well as cells, plant parts, and plant tissues from such engineered plants.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit of U.S. provisional application No. 61/507,484, filed Jul. 13, 2011, which application is herein incorporated by reference.


STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with government support under Contract No. DE-AC02-05CH11231 awarded by the U.S. Department of Energy. The government has certain rights in this invention.


BACKGROUND OF THE INVENTION
Lignocellulosic plant biomass is utilized as a renewable feedstock in various agro-industrial activities. Lignin is an aromatic and hydrophobic branched polymer incrusted within biomass that negatively affects extraction and hydrolysis of polysaccharides during industrial processes. Engineering the monomer composition of lignin offers attractive potential for reducing its recalcitrance. The present invention offers a new strategy developed in Arabidopsis for the overproduction of rare lignin monomers, which incorporate as end-groups in the polymer and reduce lignin chain extension. Biosynthesis of these lignification stoppers' is achieved by expressing a bacterial hydroxycinnamoyl-CoA hydratase-lyase (HCHL) in lignifying tissues of Arabidopsis inflorescence stems. HCHL cleaves the propanoid side chain of hydroxycinnamoyl-CoA lignin precursors to produce the corresponding hydroxybenzaldehydes. Stems from plants that express HCHL accumulate higher amount of hydroxybenzaldehyde and hydroxybenzoate derivates compared to wild type plants. Part of these C 6 C 1 phenolics are alcohol-extractable from plant tissues and are released from extract-free cell walls upon mild alkaline hydrolysis. Engineered plants with intermediate HCHL activity level show no reduction of total lignin, sugar content and biomass yield compared to wild type plants. However, cell wall characterization by 2D-NMR reveals the presence of new molecules in the aromatic region and the analysis of lignin isolated from these plants revealed an increased amount of C 6 C 1 phenolic end-groups and a reduced molecular mass distribution. In addition, these engineered lines show saccharification improvement of pretreated cell wall biomass. Enhancing the incorporation of C 6 C 1 phenolic end-groups in lignin represents a promising strategy to alter lignin structure and reduce cell wall recalcitrance to enzymatic hydrolysis.
BRIEF SUMMARY OF THE INVENTION
In the first aspect, the present invention provides an isolated expression cassette comprising a polynucleotide sequence encoding a hydroxycinnamoyl-CoA hydratase-lyase (HCHL) and a heterologous promoter, and the promoter is operably linked to the polynucleotide sequence. In some embodiments, the HCHL is Pseudomonas fluorescens HCHL, which has the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the promoter used in this expression cassette is a secondary cell wall specific promoter, such as pIRX5, which is within the polynucleotide sequence set forth in SEQ ID NO:3.
In a second aspect, the present invention provides a method for engineering a plant having reduced lignification. The method includes these steps: (1) introducing the expression cassette described herein into the plant; and (2) culturing the plant under conditions under which the HCHL is expressed, thereby reducing lignification in the plant. In some embodiments, the plant is selected from the group consisting of Arabidopsis , poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.
In a third aspect, the present invention provides a plant that is engineered by the methods described herein, and a plant cell from such a plant, a seed, flower, leaf, or fruit from such a plant, a plant cell that contains the expression cassette described herein, and biomass comprising plant tissue from the plant or part of the plant described herein. Thus, the invention provides an engineered plant comprising a heterologous hydroxycinnamoyl-CoA hydratase-lyase (HCHL) operably linked to a promoter. In some embodiments, the polynucleotide encoding the heterologous HCHL is integrated into a plant genome. In some embodiments, the promoter is heterologous to the plant. In some embodiments, the promoter is an endogenous promoter. In some embodiment, the promoter is a secondary cell wall-specific promoter, such as an IRX5 promoter. In some embodiments, the HCHL is Pseudomonas fluorescens HCHL. The plant may be a monocot or a dicot. In some embodiments, the plant is selected from the group consisting of Arabidopsis , poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, and Brachypodium.
In further aspects, the invention provide methods of using an engineered plant of the invention, or parts of the plant, or plant biomass comprising material from the plant. In some embodiments, plant material is used in a saccharificatoni reaction, e.g., enzymatic saccharification, to generate soluble sugars at an increased level of efficiency as compared to wild-type plants that have not been modified to express HCHL. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase biomass yield or simplify downstream processing for wood industries (such as paper, pulping, and construction) as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase the quality of wood for construction purposes. In some embodiments the plants, parts of plants, or plant biomass material can be used in a combustion reaction, gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic or chemical). In some embodiments, the plants, plant parts, or plant biomass material are used as forage that is more readily digested compared to wild-type plants.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 . HCHL-mediated conversion of hydroxycinnamoyl-CoAs into hydroxybenzaldehydes. HCHL performs the hydration and cleavage of hydroxycinnamoyl-CoAs (R=H, coumaroyl-CoA; R=OH, caffeoyl-CoA; R=OCH 3 , feruloyl-CoA) to produce hydroxybenzaldehydes (R=H, 4-hydroxybenzaldehyde; R=OH, 3,4-dihydroxybenzaldehyde; R=OCH 3 , 4-hydroxy-3-methoxybenzaldehyde) and acetyl-CoA via the formation of the corresponding reaction intermediates 4-hydroxyphenyl-β-hydroxypropionyl-CoAs.
FIG. 2 . Analysis of HCHL expression in IRX5:HCHL lines. (A) Detection by RT-PCR of HCHL transcripts using mRNA isolated from secondary stems of five independent five-week-old transformants in the T1 generation. cDNA synthesized from mRNA purified from wild type (WT) stems were used as a negative control. Tub8-specific primers were used to assess cDNA quality for each sample. (B) Detection by western blot of HCHL tagged with the AttB2 peptide (approximate size 32 kDa) using the universal antibody and 5 μg of total protein extracted from the primary stem of five independent five-week-old IRX5:HCHL transformants in the T2 generation. A protein extract from wild type stems (WT) was used as a negative control.
FIG. 3 . Histochemical staining of stem sections from five-week-old Arabidopsis plants. (A) Mäule staining. (B) Phloroglucinol-HCl staining. (C) Toluidine blue O staining. i, interfascicular fibers; x, xylem. Bars represent 50 μm for (A) and (B), and 20 μm for (C). Note the collapsed xylem vessels (yellow arrows) observed for line IRX5:HCHL (4).
FIG. 4 . Spectral analysis of IRX5:HCHL and wild type plants. (A) Lignin and polysaccharide content in CWR of mature senesced stems from wild type (WT) and line IRX5:HCHL (2) using FT-Raman spectroscopy. Values represent integrated intensities over the range of 1555-1690 cm −1 and 1010-1178 cm −1 for lignin and polysaccharides (cellulose/hemicellulose) quantification, respectively. Values are means of three biological replicates±SE. (B) Comparison of FT-IR spectra obtained from xylem (black line) and interfascicular fibers (grey line) in basal stem sections of wild type and line IRX5:HCHL (2). A Student's t-test was performed on absorbance values of wild type versus transgenic and plotted against wave numbers. At each wavelength, the zone between −2 and +2 corresponds to non-significant differences (p-value<0.05) between the two genotypes tested. Significant positive t-values indicated a higher absorbance value in wild type than in IRX5:HCHL plants.
FIG. 5 . 2D-HSQC NMR spectra analysis of line IRX5:HCHL plants. 2D-HSQC NMR spectra of lignin from wild type (WT) stems (A) and from IRX5:HCHL (FCA1) stems (B); Difference spectrum (IRX5:HCHL (2)—wild type) showing the presence of new components in the aromatic region (C).
FIG. 6 . Polydispersity profiles of CEL lignin purified from stems of wild type and line IRX:HCHL (2) plants. SEC chromatograms were obtained using (A) UV-A 300 absorbance and (B) UV-F ex250/em450 fluorescence.
FIG. 7 . Saccharification of biomass from mature senesced stems of IRX5:HCHL and wild type plants. Amount of reducing sugars released from 10 mg of biomass after hot water, dilute alkaline, or dilute acid pretreatment followed by 72-h enzymatic hydrolysis were measured using the DNS assay. Values are means of four biological replicates±SE.
FIG. 8 . Alignment of amino acid sequences of Pseudomonas fluorescens HCHL (SEQ ID NO:1) and other homologous proteins.
FIG. 9 . Organ and tissue-specific activity of the IRX5 promoter in Arabidopsis . Line CS70758, which contains a pIRX5:GUS expression cassette, was used to localize the activity of the IRX5 promoter. Young seedlings (A and B), rosettes leaves (C and D), siliques (E and F), cauline leaves (G and H), flowers (I and J), and inflorescence stems (K and L) were incubated in the GUS assay buffer for 1 h and 8 h at 37° C. Gus activity was essentially detected in the stem xylem vessels after a 1-h incubation (K). For longer incubations (8 h), GUS staining was also observed in interfascicular fibers of the stem (L), the vascular system of young seedlings (A), siliques (F) rosette (D) and cauline leaves (H), as well as in the style and anthers (J). x: xylem vessels, if: interfascicular fibers. Scale bars: 2 mm (A-B, E-F), 4 mm (C-D, G-H), 500 μm (I-J), 100 μm (K-L).
FIG. 10 . Growth and development of IRX5:HCHL and wild type (WT) plants at different stages. (A) Three-week-old rosette (B) Five-week-old flowering stage. (C) Eight-week-old senescing stage.
FIG. 11 . Synthesis of C 6 C 1 phenolics production upon HCHL activity and probable associated enzymes. The phenylpropanoid pathway (center box) and monolignol pathway (left box) are represented. HCHL converts hydroxycinnamoyl-CoAs into their corresponding hydroxybenzaldehydes. Metabolomic data showed occurrence of hydroxycinnamic acids and alcohols, suggesting involvement of aldehyde dehydrogenases (DH) and reductases (left box). UDP-glucosyltransferases (UGT) are responsible for the formation of C6-C1 phenolic glucose conjugates. Syringaldehyde is possibly derived from vanillin and 5OH-vanillin after successive monooxyenase (Monox) and O-methyltransferase activities (OMT). Asterisks indicate compounds found in higher amount in lignin of Arabidopsis expressing HCHL. Abbreviations for enzymes are: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, 4-coumarate-CoA ligase; CCR, hydroxycinnamoyl-CoA reductase; CAD, coniferyl alcohol dehydrogenase; HCT, p-hydroxycinnamoyl-CoA:quinate shikimate p-hydroxycinnamoyl-CoA transferase; C3H, p-coumarate 3-hydroxylase; CCoAOMT, caffeoyl-CoA O-methyltransferase; FSH, ferulate 5-hydroxylase (coniferaldehyde 5-hydroxylase); COMT, caffeic acid/5-hydroxyferulic acid O-methyltransferase.
FIG. 12 . Transgenic rice lines that express pAtIRX5::HCHL.
FIG. 13 . Expression analysis of HCHL in the engineered rice lines. Results of an RT-PCR using RNA extracted from rice plants and HCHL-specific primers.
FIG. 14 . Detection of pHBA in stems from the engineered rice lines.



DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
As used herein, the term “hydroxycinnamoyl-CoA hydratase-lyase” or “HCHL” refers to an enzyme that catalyzes the hydration of the double bond of lignin precursor p-coumaroy-CoA, caffeoyl-CoA, or feruloyl-CoA thioester, which is followed by a retro aldol cleavage reaction to produce a corresponding C 6 C 1 hydroxylbenzaldehyde and acetyl-CoA. A typical HCHL within the meaning of this invention is an HCHL from bacterium Pseudomonas fluorescens (EC 4.2.1.101-trans-feruloyl-CoA hydratase), which has the amino acid sequence set forth in FIG. 11 as SEQ ID NO:1 (GenBank Accession No. CAA73502), encoded by cDNA sequence set forth in GenBank Accession No. Y13067.1 or by a codon-optimized polynucleotide sequence set forth in SEQ ID NO:2 (synthesized by GenScript, Piscatway, N.J.). In this application, the term HCHL includes polymorphic variants, alleles, mutants, and interspecies homologs to the Pseudomonas fluorescens HCHL, some examples of which are provided in FIG. 8 . A nucleic acid that encodes an HCHL refers to a gene, pre-mRNA, mRNA, and the like, including nucleic acids encoding polymorphic variants, alleles, mutants, and interspecies homologs of the particular sequences described herein. Thus, an HCHL nucleic acid (1) has a polynucleotide sequence that has greater than about 50% nucleotide sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or higher nucleotide sequence identity, preferably over a region of at least about 10, 15, 20, 25, 50, 100, 200, 500 or more nucleotides or over the length of the entire polynucleotide, to a polynucleotide sequence encoding SEQ ID NO:1 (e.g., SEQ ID NO:2 or the polynucleotide sequence set forth in Y13067.1); or (2) encodes a polypeptide having an amino acid sequence that has greater than about 50% amino acid sequence identity, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 100, 200 or more amino acids or over the length of the entire polypeptide, to a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 or to any one of the amino acid sequences shown in FIG. 8 (SEQ ID NOs:4-34). The enzymatic activity of an HCHL within the meaning of this application can be verified by functional assays known in the art or described in the example section of this application, for its ability to convert any one of lignin precursors p-coumaroy-CoA, caffeoyl-CoA, and feruloyl-CoA thioester to a corresponding C 6 C 1 hydroxylbenzaldehyde and acetyl-CoA.
The term “substantially localized,” when used in the context of describing a plant expressing an exogenous HCHL that is substantially localized to a particular tissue, refers to the enzymatic activity and modified monolignols produced therefore in substantially higher amounts in the particular cell or tissue type of interest as compared to other cell or tissue types. In some embodiments, the presence of HCHL and modified monolignols is substantially localized to the secondary cell wall of a plant cell and in the stem of a plant.
The terms “polynucleotide” and “nucleic acid” are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); positive backbones; non-ionic backbones, and non-ribose backbones. Thus, nucleic acids or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Polynucleotide sequence” or “nucleic acid sequence” includes both the sense and antisense strands of a nucleic acid as either individual single strands or in a duplex. As will be appreciated by those in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
The term “substantially identical,” used in the context of two nucleic acids or polypeptides, refers to a sequence that has at least 50% sequence identity with a reference sequence. Percent identity can be any integer from 50% to 100%. Some embodiments include at least: 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. For example, an HCHL may have an amino acid sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, the amino acid sequence of Pseudomonas fluorescens HCHL.
Two nucleic acid sequences or polypeptide sequences are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. When percentage of sequence identity is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated according to, e.g., the algorithm of Meyers & Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA).
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection.
Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=1, N=−2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10 5 , and most preferably less than about 10.
Nucleic acid or protein sequences that are substantially identical to a reference sequence include “conservatively modified variants.” With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, in a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art.
The following six groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Serine (S), Threonine (T);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
(see, e.g., Creighton, Proteins (1984)).
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C. For example, stringent conditions for hybridization, such as RNA-DNA hybridizations in a blotting technique are those which include at least one wash in 0.2×SSC at 55° C. for 20 minutes, or equivalent conditions.
The term “promoter,” refers to a polynucleotide sequence capable of driving transcription of a DNA sequence in a cell. Thus, promoters used in the polynucleotide constructs of the invention include cis- and trans-acting transcriptional control elements, translational control elements (5′UTR: untranslated region) and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. For example, a promoter can be a cis-acting transcriptional control element, including an enhancer, a promoter, a transcription terminator, an origin of replication, a chromosomal integration sequence, 5′ and 3′ untranslated regions, or an intronic or exonic sequence, which are involved in transcriptional regulation. These cis-acting sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription. Promoters are located 5′ to the transcribed gene, and as used herein, include the sequence 5′ from the translation start codon (i.e., including the 5′ untranslated region of the mRNA, typically comprising 50-200 bp). Most often the core promoter sequences lie within 1-3 kb of the translation start site, more often within 1 kbp and often within 500 bp of the translation start site. By convention, the promoter sequence is usually provided as the sequence on the coding strand of the gene it controls.
A “constitutive promoter” is one that is capable of initiating transcription in nearly all cell types, whereas a “cell type-specific promoter” initiates transcription only in one or a few particular cell types or groups of cells forming a tissue. In some embodiments, the promoter is secondary cell wall specific. Secondary cell wall is mainly composed of cellulose, hemicellulose, and lignin and is deposited in some, but not all, tissues of a plant, such as woody tissue. As used herein, a “secondary cell wall specific” promoter refers to a promoter that initiates higher levels of transcription in cell types that have secondary cell walls, e.g., lignified tissues such as vessels and fibers, which may be found in wood and bark cells of a tree, as well as other parts of plants such as the leaf stalk. In some embodiments, a promoter is secondary cell wall specific if the transcription levels initiated by the promoter in secondary cell walls are at least 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold higher or more as compared to the transcription levels initiated by the promoter in other tissues, resulting in the encoded protein substantially localized in plant cells that possess secondary cell wall, e.g., the stem of a plant. Non-limiting examples of secondary cell wall specific promoters include the promoters directing expression of genes IRX1, IRX3, IRX5, IRX7, IRX8, IRX9, IRX10, IRX14, NST1, NST2, NST3, MYB46, MYB58, MYB63, MYB83, MYB85, MYB103, PAL1, PAL2, C3H, CcOAMT, CCR1, F5H, LAC4, LAC17, CADc, and CADd. See, e.g., Turner et al 1997; Meyer et al 1998; Jones et al 2001; Franke et al 2002; Ha et al 2002; Rohde et al 2004; Chen et al 2005; Stobout et al 2005; Brown et al 2005; Mitsuda et al 2005; Zhong et al 2006; Mitsuda et al 2007; Zhong et al 2007a, 2007b; Zhou et al 2009; Brown et al 2009; McCarthy et al 2009; Ko et al 2009; Wu et al 2010; Berthet et al 2011. In some embodiments, the promoter is substantially identical to the native promoter sequence directing expression of the IRX5 gene (see, e.g., the promoter and transcriptional regulatory elements for IRX5 are contained in SEQ ID NO:3). Some of the above mentioned secondary cell wall promoter sequences can be found within the polynucleotide sequences provided herein as SEQ ID NOs:36-61. A promoter originated from one plant species may be used to direct gene expression in another plant species.
A polynucleotide is “heterologous” to an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, when a polynucleotide encoding a polypeptide sequence is said to be operably linked to a heterologous promoter, it means that the polynucleotide sequence encoding the polypeptide is derived from one species whereas the promoter sequence is derived from another, different species; or, if both are derived from the same species, the coding sequence is not naturally associated with the promoter (e.g., is a genetically engineered coding sequence, e.g., from a different gene in the same species, or an allele from a different ecotype or variety).
The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a DNA or RNA sequence if it stimulates or modulates the transcription of the DNA or RNA sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
The term “expression cassette” refers to a nucleic acid construct that, when introduced into a host cell, results in transcription and/or translation of an RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and suppression of endogenous genes (e.g., by antisense, RNAi, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical, but may be only substantially identical to a sequence of the gene from which it was derived. As explained herein, these substantially identical variants are specifically covered by reference to a specific nucleic acid sequence. One example of an expression cassette is a polynucleotide construct that comprises a polynucleotide sequence encoding a HCHL protein operably linked to a promoter that is heterologous to the plant cell into which the expression cassette may be introduced. In some embodiments, an expression cassette comprises a polynucleotide sequence encoding a HCHL protein that is targeted to a position in the genome of a plant such that expression of the HCHL polynucleotide sequence is driven by a promoter that is present in the plant.
The term “plant,” as used herein, refers to whole plants and includes plants of a variety of a ploidy levels, including aneuploid, polyploid, diploid and haploid. The term “plant part,” as used herein, refers to shoot vegetative organs and/or structures (e.g., leaves, stems and tubers), branches, roots, flowers and floral organs (e.g., bracts, sepals, petals, stamens, carpels, anthers), ovules (including egg and central cells), seed (including zygote, embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), seedlings, and plant tissue (e.g., vascular tissue, ground tissue, and the like), as well as individual plant cells, groups of plant cells (e.g., cultured plant cells), protoplasts, plant extracts, and seeds. The class of plants that can be used in the methods of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae.
The term “biomass,” as used herein, refers to plant material that is processed to provide a product, e.g., a biofuel such as ethanol, or livestock feed, or a cellulose for paper and pulp industry products. Such plant material can include whole plants, or parts of plants, e.g., stems, leaves, branches, shoots, roots, tubers, and the like.
The term “reduced lignification” encompasses both reduced size of a lignin polymer, e.g., a shorter lignin polymer chain due to a smaller number of monolignols being incorporated into the polymer, a reduced degree of branching of the lignin polymer or a reduced space filling (also called a reduced pervaded volume). Typically, a reduced lignin polymer can be shown by detecting a decrease in it molecular weight or a decrease in the number of monolignols by at least 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, or more, when compared to the average lignin molecule in a control plant. Methods for detecting reduced lignification are described in detail in the example section of this application.
As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “plant cell” includes a plurality of such plant cells.
II. Introduction
Plant cell walls are constituted by a polysaccharidic network of cellulose microfibrils and hemicellulose embedded in an aromatic polymer known as lignin. This ramified polymer is mainly composed of three phenylpropanoid-derived phenolics (i.e., monolignols) named p-coumaryl, coniferyl, and sinapyl alcohols which represent the p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignin units (Boerjan et al., 2003). Monolignols have a C 6 C 3 carbon skeleton which consists of a phenyl ring (C 6 ) and a propane (C3) side chain. Lignin is crucial for the development of terrestrial plants as it confers recalcitrance to plant cell walls. It also provides mechanical strength for upright growth, confers hydrophobicity to vessels that transport water, and acts as a physical barrier against pathogens that degrade cell walls (Boudet, 2007). Notably, lignin content and composition are finely regulated in response to environmental biotic and abiotic stresses (Moura et al., 2010).
Economically, lignocellulosic biomass from plant cell walls is widely used as raw material for the production of pulp in paper industry and as ruminant livestock feed. Plant feedstocks also represent a source of fermentable sugars for the production of synthetic molecules such as pharmaceuticals and transportation fuels using engineered microorganisms (Keasling, 2010). However, negative correlations exist between lignin content in plant biomass and pulp yield, forage digestibility, or polysaccharides enzymatic hydrolysis (de Vrije et al., 2002; Reddy et al., 2005; Dien et al., 2006; Chen and Dixon, 2007; Dien et al., 2009; Taboada et al., 2010; Elissetche et al., 2011; Studer et al., 2011). Consequently, reducing lignin recalcitrance in plant feedstocks is a major focus of interest, especially in the lignocellulosic biofuels field for which efficient enzymatic conversion of polysaccharides into monosaccharides is crucial to achieve economically and environmentally sustainable production (Carroll and Somerville, 2009).
Lignin biosynthesis is well characterized and well conserved across land plants (Weng and Chapple 2010). Genetic modifications such as silencing of genes involved in particular steps of this pathway or its regulation have been employed to reduce lignin content (Simmons et al., 2010; Umezawa, 2010) but this approach often results in undesired phenotypes such as dwarfism, sterility, reduction of plant biomass, and increased susceptibly to environmental stress and pathogens (Bonawitz and Chapple, 2010). These pleiotropic effects are generally the consequences of a loss of secondary cell wall integrity, accumulation of toxic intermediates, constitutive activation of defense responses, or depletion of other phenylpropanoid-derived metabolites which are essential for plant development and defense (Li et al., 2008; Naoumkina et al., 2010, Gallego-Giraldo et al., 2011). Alternatively, changing the recalcitrant structure and physico-chemical properties of lignin can be achieved by modifying its monomer composition. For example, incorporation of coniferyl ferulate into lignin improves enzymatic degradation of cell wall polysaccharides (Grabber et al., 2008). Recently, it has been demonstrated that enrichment in 5-hydroxy-G units and reduction in S units in lignin contribute to enhanced saccharification efficiencies without affecting drastically biomass yields and lignin content (Weng et al., 2010; Dien et al., 2011; Fu et al., 2011).
In this study, as an alternative strategy to reduce lignin recalcitrance, the inventors developed a dominant approach that uses precursors derived from the lignin biosynthetic pathways to enhance production of non-conventional monolignols, namely C 6 C 1 phenolics. These phenol units lack propane side chain and thus have different polymerization properties compared to classic C 6 C 3 monolignols. Such C 6 C 1 phenolics are usually found in trace amount in some lignins and form the so-called ‘benzyl end-groups’ (Kim et al., 2000; Ralph et al., 2001; Kim et al., 2003; Morreel et al., 2004; Ralph et al., 2008; Kim and Ralph, 2010). The inventors considered increasing C 6 C 1 end-group phenolics in lignin to reduce its polymerization degree and native branched structure. For this purpose, a hydroxycinnamoyl-CoA hydratase-lyase (HCHL, EC 4.2.2.101/EC 4.1.2.41) from Pseudomonas fluorescens was expressed in stems of Arabidopsis . HCHL is an enzyme that catalyzes the hydration of the double bond of the lignin precursor p-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA thioesters, followed by a retro aldol cleavage reaction that produces the corresponding C 6 C 1 hydroxybenzaldehydes and acetyl-CoA ( FIG. 1 ; Mitra et al., 1999). The promoter of a secondary cell wall cellulose synthase gene (Cesa4/IRX5) was used to restrict HCHL expression in lignified tissues of the stem (xylem and interfascicular fibers) and prevent depletion of hydroxycinnamoyl-CoAs in other tissues in which they are precursors of hydroxycinnamate conjugates and other derivates involved in plant defense and development (Gou et al., 2009; Luo et al., 2009; Buer et al., 2010; Milkowski and Strack, 2010). The data disclosed herein show that HCHL expression driven by the IRX5 promoter results for some lines in no significant changes in lignin content, plant development and biomass yields. It has also been demonstrated that C 6 C 1 phenolics accumulate as end-groups in the lignin of HCHL transgenics, which reduces lignin size and renders cell walls less recalcitrant to enzymatic hydrolysis.
III. Plants Having Reduced Lignification
A. Modification of Expression of an HCHL Enzyme
In one aspect, the present invention provides a method for engineering a plant having reduced lignification. This method includes these steps: first, introducing into the plant an expression cassette comprising a polynucleotide sequence encoding an HCHL enzyme and a promoter, with the coding sequence and the promoter being in an operably linked arrangement; and second, culturing the plant under conditions permissible for the expression of a functional HCHL to produce C 6 C 1 phenolics, which can be polymerized with other monolignols and thereby reducing lignification in the plant.
In particular, the present invention provides methods of engineering a plant having modified lignin polymers, which may have reduced size, molecular weight, and/or altered (especially reduced or less extensive) branching, that are substantially localized to the lignified tissue of the plant. This is achieved by first introducing into the plant an expression cassette as described above but in particular having a secondary cell wall specific promoter, and then culturing the plant under conditions in which the functional HCHL enzyme is expressed. This enzyme converts various hydroxycinnamoyl-coA into their respective hydroxybenzaldehydes that can be either directly incorporated or further modified (e.g., oxidation or reduction of the aldehyde group) by native enzymes prior to their incorporation into the lignin polymer by polymerization with native monolignols.
The expression cassette as described herein, when introduced into a plant, does not necessarily modify the lignin content. Vessel stays intact indicating that the lignin cell wall structure is still robust to prevent vessel collapse, but the lignin composition and properties are modified to a level that its recalcitrance is reduce.
One of skill in the art will understand that the HCHL that is introduced into the plant by an expression cassette described herein does not have to be identical to the Pseudomonas fluorescens HCHL, which was used in the experiments detailed in the example section of this disclosure. In some embodiments, the HCHL that is introduced into the plant by an expression cassette is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the Pseudomonas fluorescens HCHL. For example, a variant HCHL will have at least 80%, 85%, 90%, or 95% sequence identity in its amino acid residues as compared to SEQ ID NO:1, especially within one or more of the 8 highly conserved regions (shown in the 8 boxes in FIG. 8 ).
1. Hydroxycinnamoyl-CoA Hydratase-Lyase (HCHL)
In some embodiments, the expression cassette of this invention comprises a polynucleotide encoding an enzyme that produces modified monoligols that can cause reduced lignification. An example of such an enzyme is the Pseudomonas fluorescens HCHL, having the amino acid sequence set forth in SEQ ID NO:1. Additional examples of such HCHL suitable for use in the present invention include those shown in FIG. 8 . Also appropriate for use in the present invention are variants HCHL, which may be naturally occurring or recombinantly engineered, provided the variants possess (1) substantially amino acid sequence identity to an exemplary HCHL (e.g., SEQ ID NO:1) and (2) the enzymatic activity to convert at least one lignin precursor p-coumaroy-CoA, caffeoyl-CoA, or feruloyl-CoA thioester into a corresponding C 6 C 1 hydroxylbenzaldehyde, as determined by an HCHL enzymatic assay known in the art by way of various scientific publications or described herein.
Examples of naturally occurring HCHL that can be used to practice the present invention includes, p-hydroxycinnamoyl CoA hydratase/lyase (HCHL), Enoyl-CoA hydratase/isomarase (ECH), Feruloyl-CoA hydratase/lyase (FCA, FerA), as well as those named in FIG. 8 , the amino acid sequences for which are provided in SEQ ID NOs:4-34.
2. Secondary Cell Wall-Specific Promoters
In some embodiments, the polynucleotide encoding the HCHL is operably linked to a secondary cell wall-specific promoter. The secondary cell wall-specific promoter is heterologous to the polynucleotide encoding the HCHL, in other words, the promoter and the HCHL coding sequence are derived from two different species. A promoter is suitable for use as a secondary cell wall-specific promoter if the promoter is expressed strongly in the secondary cell wall, e.g., in vessel and fiber cells of the plant, but is expressed at a much lower level or not expressed in cells without the secondary cell wall.
In some embodiments, the promoter is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to the native promoter of a gene encoding a secondary cell wall cellulose synthase Cesa4/IRX5, polynucleotide sequence set forth in Genebank Accession No. AF458083 — 1 and SEQ ID NO:35, and the promoter pIRX5 is contained in SEQ ID NO:3.
In some embodiments, the secondary cell wall-specific promoter comprises SEQ ID NO:3. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 or a variant thereof. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 comprising about 50 to about 1000 or more contiguous nucleotides of SEQ ID NO:3. In some embodiments, the secondary cell wall-specific promoter comprises a subsequence of SEQ ID NO:3 comprising 50 to 1000, 50 to 900, 50 to 800, 50 to 700, 50 to 600, 50 to 500, 50 to 400, 50 to 300, 50 to 200, 50 to 100; 75 to 1000, 75 to 900, 75 to 800, 75 to 700, 75 to 600, 75 to 500, 75 to 400, 75 to 300, 75 to 200; 100 to 1000, 100 to 900, 100 to 800, 100 to 700, 100 to 600, 100 to 500, 100 to 400, 100 to 300, or 100 to 200 contiguous nucleotides of SEQ ID NO:3.
Secondary cell wall-specific promoters are also described in the art. See, for example, Mitsuda et al 2005 Plant Cell; Mitsuda et al 2007 Plant Cell; Zhou et al 2009 plant cell; Ohtani et al 2011 Plant Journal. They are contained the polynucleotide sequences provided in this application as SEQ ID NO:36-61.
It will be appreciated by one of skill in the art that a promoter region can tolerate considerable variation without diminution of activity. Thus, in some embodiments, the secondary cell wall-specific promoter is substantially identical (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical) to SEQ ID NO:3. The effectiveness of a secondary cell wall-specific promoter may be confirmed by an reporter gene (e.g., β-glucuronidase or GUS) assay known in the art or as described in the example section of this application.
B. Preparation of Recombinant Expression Vectors
Once the promoter sequence and the coding sequence for the gene of interest (e.g., a Pseudomonas fluorescens HCHL or any other HCHL as shown in FIG. 8 ) are obtained, the sequences can be used to prepare an expression cassette for expressing the gene of interest in a transgenic plant. Typically, plant transformation vectors include one or more cloned plant coding sequences (genomic or cDNA) under the transcriptional control of 5′ and 3′ regulatory sequences and a selectable marker. Such plant transformation vectors may also contain a promoter (e.g., a secondary cell wall-specific promoter as described herein), a transcription initiation start site, an RNA processing signal (such as intron splice sites), a transcription termination site, and/or a polyadenylation signal.
The plant expression vectors may include RNA processing signals that may be positioned within, upstream, or downstream of the coding sequence. In addition, the expression vectors may include regulatory sequences taken from the 3′-untranslated region of plant genes, e.g., a 3′ terminator region to increase mRNA stability of the mRNA, such as the PI-II terminator region of potato or the octopine or nopaline synthase 3′ terminator regions.
Plant expression vectors routinely also include selectable marker genes to allow for the ready selection of transformants. Such genes include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin), herbicide resistance genes (e.g., phosphinothricin acetyltransferase), and genes encoding positive selection enzymes (e.g. mannose isomerase).
Once an expression cassette comprising a polynucleotide encoding the HCHL and operably linked to a promoter (especially a secondary cell wall specific promoter) has been constructed, standard techniques may be used to introduce the polynucleotide into a plant in order to express the HCHL and effectuate reduced lignification. See, e.g., protocols described in Ammirato et al. (1984) Handbook of Plant Cell Culture—Crop Species. Macmillan Publ. Co. Shimamoto et al. (1989) Nature 338:274-276; Fromm et al. (1990) Bio/Technology 8:833-839; and Vasil et al. (1990) Bio/Technology 8:429-434.
Transformation and regeneration of plants is known in the art, and the selection of the most appropriate transformation technique will be determined by the practitioner. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG) mediated transformation; transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium tumeficiens mediated transformation. Transformation means introducing a nucleotide sequence in a plant in a manner to cause stable or transient expression of the sequence. Examples of these methods in various plants include: U.S. Pat. Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945; 5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269; 5,736,369 and 5,610,042.
Following transformation, plants can be selected using a selectable marker incorporated into the transformation vector. Typically, such a marker will confer antibiotic or herbicide resistance on the transformed plants or the ability to grow on a specific substrate, and selection of transformants can be accomplished by exposing the plants to appropriate concentrations of the antibiotic, herbicide, or substrate.
The polynucleotide sequence coding for an HCHL, as well as the polynucleotide sequence comprising a promoter (e.g., a secondary cell wall-specific promoter), can be obtained according to any method known in the art. Such methods can involve amplification reactions such as polymerase chain reaction (PCR) and other hybridization-based reactions or can be directly synthesized.
C. Plants in which Lignification can be Reduced
An expression cassette comprising a polynucleotide encoding an HCHL operably linked to a promoter, especially a secondary cell wall specific promoter, as described herein, can be expressed in various kinds of plants. The plant may be a monocotyledonous plant or a dicotyledonous plant. In some embodiments of the invention, the plant is a green field plant. In some embodiments, the plant is a gymnosperm or conifer.
In some embodiments, the plant is a plant that is suitable for generating biomass. Examples of suitable plants include, but are not limited to, Arabidopsis , poplar, eucalyptus, rice, corn, switchgrass, sorghum, millet, miscanthus, sugarcane, pine, alfalfa, wheat, soy, barley, turfgrass, tobacco, hemp, bamboo, rape, sunflower, willow, Jatropha, and Brachypodium.
In some embodiments, the plant into which the expression cassette of this invention is introduced is the same species of plant as the one from which the promoter is derived. In some embodiments, the plant into which the expression cassette is introduced is a different species of plant from the plant species the promoter is derived from.
D. Screening for Plants Having Reduced Lignification
After transformed plants are selected, the plants or parts of the plants may be evaluated to determine whether expression of the exogenous HCHL can be detected, e.g., by evaluating the level of RNA or protein, by measuring enzymatic activity of the HCHL, as well as by evaluating the size, molecular weight, content, or degree of branching in the lignin molecules found in the plants. These analyses can be performed using any number of methods known in the art.
In some embodiments, plants are screened by evaluating the level of RNA or protein. Methods of measuring RNA expression are known in the art and include, for example, PCR, northern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), and microarrays. Methods of measuring protein levels are also known in the art and include, for example, mass spectroscopy or antibody-based techniques such as ELISA, Western blotting, flow cytometry, immunofluorescence, and immunohistochemistry.
In some embodiments, plants are screened by assessing HCHL activity, and also by evaluating lignin size and composition. The enzymatic assays for HCHL are well known in the art and are described in this application. Lignin molecules can be assessed, for example, by nuclear magnetic resonance (NMR), spectrophotometry, microscopy, klason lignin assays, acetyl-bromide reagent or by histochemical staining (e.g., with phloroglucinol).
IV. Methods of Using Plants Having Reduced Lignification
Plants, parts of plants, or plant biomass material from plants having reduced lignification due to the expression of an exogenous HCHL in the secondary cell wall can be used for a variety of methods. In some embodiments, the plants, parts of plants, or plant biomass material generate less recalcitrant biomass for use in a conversion reaction as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used in a saccharification reaction, e.g., enzymatic saccharification, to generate soluble sugars at an increased level of efficiency as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase biomass yield or simplify downstream processing for wood industries (such as paper, pulping, and construction) as compared to wild-type plants. In some embodiments, the plants, parts of plants, or plant biomass material are used to increase the quality of wood for construction purposes. In some embodiments the plants, parts of plants, or plant biomass material can be used in a combustion reaction, gasification, pyrolysis, or polysaccharide hydrolysis (enzymatic or chemical). In further embodiments, the plants, parts of plants, or plant biomass is used as a forage crop and exhibit improved digestibility compared to wild-type plants.
Methods of conversion, for example biomass gasification, are known in the art. Briefly, in gasification plants or plant biomass material (e.g., leaves and stems) are ground into small particles and enter the gasifier along with a controlled amount of air or oxygen and steam. The heat and pressure of the reaction break apart the chemical bonds of the biomass, forming syngas, which is subsequently cleaned to remove impurities such as sulfur, mercury, particulates, and trace materials. Syngas can then be converted to products such as ethanol or other biofuels.
Methods of enzymatic saccharification are also known in the art. Briefly, plants or plant biomass material (e.g., leaves and stems) are optionally pre-treated with hot water, dilute alkaline, AFEX (Ammonia Fiber Explosion), ionic liquid or dilute acid, followed by enzymatic saccharification using a mixture of cell wall hydrolytic enzymes (such as hemicellulases, cellulases and beta-glucosidases) in buffer and incubation of the plants or plant biomass material with the enzymatic mixture. Following incubation, the yield of the saccharification reaction can be readily determined by measuring the amount of reducing sugar released, using a standard method for sugar detection, e.g. the dinitrosalicylic acid method well known to those skilled in the art. Plants engineered in accordance with the invention provide a higher saccharification efficiency as compared to wild-type plants, while the plants growth, development, or disease resistance is not negatively impacted.
Sugars generated from a saccharification reaction using plant biomass of the invention can be used for producing any product for which the sugars can serve as a carbon source. Examples of products include, but are not limited to, alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H 2 and CO 2 ); antibiotics (e.g., penicillin and tetracycline); vitamins (e.g., riboflavin, B12, beta-carotene), fatty acids and fatty acid derivatives (as described, e.g., in PCT/US2008/068833); isoprenyl alkanoates (as described, e.g., PCT/US2008/068756, methyl butenol (as described, e.g., PCT/US2008/068831; fatty acid esters (as described, e.g., in PCT/US2010/033299), isoprenoid-based alternative diesel fuels (as described, e.g., in PCT/US2011/059784; a polyketide synthesized by a polyketide synthase, such as a diacid (see, e.g., PCT/US2011/061900), biofuels (see, e.g., PCT/US2009/042132) and alpha-olefins (see, e.g., PCT/US2011/053787).
EXAMPLES
The following examples are provided to illustrate but not to limit the claimed invention.
Example 1
Expression of Bacterial HCHL in Arabidopsis
I. Materials and Methods
Plant Material and Growth Conditions
Arabidopsis thaliana (ecotype Columbia, Col-0) seeds were germinated directly on soil. Growing conditions were 14 h of light per day at 100 mmol m −2 s −1 , 22° C., 55% humidity. Selection of T1 and T2 homozygote transgenic plants was made on solid Murashige and Skoog vitamin medium (PhytoTechnology Laboratories) supplemented with 1% sucrose, 1.5% agar (Sigma-Aldrich) adjusted to pH 5.6-5.8, and containing 50 μg mL −1 kanamycin.
Chemicals
4-Hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, vanillic acid, 4-hydroxybenzaldehyde, vanillin, 5-hydroxyvanillin, 4-hydroxybenzyl alcohol, vanillyl alcohol, and 1-methyl-2-pyrrolidinone were purchased from Sigma-Aldrich. Vanillic acid, syringic acid, 3,4-dihydroxybenzaldehyde, syringaldehyde, and sinapyl alcohol were purchased from Alfa Aesar. 5-Hydroxyvanillic acid was obtained from Chromadex, and 3,4-dihydroxybenzyl alcohol from TCI America.
pIRX5:GUS Line and GUS Staining
Arabidopsis line CS70758 (ecotype Columbia, Col-2) was obtained from the Arabidopsis Biological Resource Center (ABRC). This line has a pMLBART plasmid containing an expression cassette consisting of the genomic fragment located upstream of the CESA4 start codon fused to the GUS gene. Histochemical GUS activity was performed as previously described (Eudes et al., 2006). Various organs of soil-grown line CS70758 were incubated for 1 h or 8 h at 37° C. in the GUS assay buffer using 5-bromo-4-chloro-3-indolyl-D-glucuronic acid (Indofine Chemical Company, Inc.) as a substrate. After staining, stem samples (1 cm) were cross-sectioned (80 μm) using a vibratome before observation under the microscope (Leica).
IRX5:HCHL Construct and Plant Transformation
For HCHL expression in Arabidopsis , the binary vector pTKan which is derived from pPZP212 was used (Hajdukiewicz et al., 2004). A Gateway cloning cassette (Invitrogen) was inserted between XhoI and PstI restriction sites to produce a pTKan-GW vector. The nucleotide sequence of the IRX5 promoter was amplified by PCR from Arabidopsis (ecotype Columbia, Col-0) genomic DNA using oligonucleotides 5′-CCCG GCGGCCGC ATGAAGCCATCCTCTACCTCGGAAA-3′ and 5′-CCCG GCTAGC GGCGAGGTACACTGAGCTCTCGGAA-3′ (NotI and NheI restriction sites underlined), and inserted between the ApaI and SpeI restriction sites of pTKan-GW to produce a pTKan-pIRX5-GW expression vector. A codon-optimized nucleotide sequence encoding the HCHL enzyme from Pseudomonas fluorescens AN103 (accession number CAA73502) for expression in Arabidopsis was synthesized without stop codon (Genescript) and amplified by PCR using oligonucleotides 5′- GGGGACAAGTTTGTACAAAAAAGCAGGCTTC ATGTCTACTTACGAGGGAAGATG G-3′ and 5′- GGGGACCACTTTGTACAAGAAAGCTGGGTC TCTCTTGTAAGCCTGGAGTCC-3′ (attb1 and attb2 sites underlined) for cloning into the Gateway pDONR221-f1 entry vector (Lalonde et al 2010). A sequence-verified HCHL entry clone was LR recombined with the pTKan-pIRX5-GW vector to generate the final IRX5:HCHL construct. The construct was introduced into wild type Arabidopsis plants (ecotype Col0) via Agrobacterium tumefaciens -mediated transformation (Bechtold and Pelletier, 1998).
RNA Extraction and RT-PCR
Total RNA (1 μg) extracted from inflorescence stems of IRX5:HCHL T1 transformants and wild type plants using the Plant RNeasy extraction kit (Qiagen) was reverse-transcribed using the Transcriptor First Strand cDNA Synthesis Kit (Roche applied Science). The obtained cDNA preparation was quality-controlled for PCR using tub8-specific oligonucleotides (5′-GGGCTAAAGGACACTACACTG-3′/5′-CCTCCTGCACTTCCACTTCGTCTTC-3′). Oligonucleotides 5′-ATGTCTACTTACGAGGGAAGATGG-3′ and 5′-TCTCTTGTAAGCCTGGAGTCC-3′ were used for the detection of HCHL expression by PCR.
Western Blot Analysis
For protein extraction, inflorescence stems of IRX5:HCHL T2 transformants and wild type plants were ground in liquid nitrogen, and 0.25 g of the resulting powder was homogenized with the extraction buffer [100 mM Tris-HCl pH 6.5, 2% (w/v) polyvinylpyrrolidone, 2% (v/v) β-mercaptoethanol, 1% (w/v) SDS] at 1400 rpm for 30 min. The mixture was centrifuged at 20,000 g for 5 min and the supernatant collected for protein quantification using the Bradford method (Bradford, 1976) and bovine serum albumin as a standard. For electrophoresis, soluble protein (5 μg) were mixed with 0.2 M Tris-HCl, pH 6.5, 8% (w/v) SDS, 8% (v/v) β-mercaptoethanol, 40% (v/v) glycerol, and 0.04% (w/v) bromophenol blue and incubated at 40° C. for 30 min. Proteins were separated by SDS-PAGE using 8-16% (w/v) polyacrylamide gradient gels (Invitrogen) and electrotransferred (100 volts, 45 min) onto PVDF membranes (Thermo Fisher Scientific). Blotted membranes were incubated 1 h in TBS-T (20 m M Tris-HCl, 150 m M NaCl, 0.1% (v/v) Tween 20, pH 7.6) containing 2% (w/v) non-fat milk powder, and incubated overnight with the universal antibody (1:20000) in TBS-T containing 2% (w/v) non-fat milk powder. Membranes were then washed in TBS-T for 30 min and incubated for 1 h with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:20000; Sigma-Aldrich) in TBS-T containing 2% (w/v) non-fat milk powder. Membranes were then washed in TBS-T for 30 min, and detection was performed by chemiluminescence using the SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).
HCHL Activity
For protein extraction, inflorescence stems of IRX5:HCHL T2 transformants and wild type plants were ground in liquid nitrogen, and 0.25 g of the resulting powder was homogenized with 25 mg of polyvinylpolypyrrolidone and 1.25 mL of extraction buffer (EB; 100 mM Tris-HCl, pH 8.5, 20 mM DTT, and 10 mM Na 2 EDTA). Extracts were shaken at 1400 rpm for 15 min at 4° C., and centrifuged for 30 min at 20,000 g at 4° C. Supernatants were collected, adjusted to 2.5 mL with EB, and applied to PD10 columns (GE healthcare) pre-equilibrated with 25 mL of EB. Proteins were eluted with 3.5 mL of EB and quantified using the Bradford method (Bradford, 1976) and bovine serum albumin as a standard.
For HCHL activity, 5 μL of protein extract was incubated for 15 min at 30° C. with 150 μM feruloyl-CoA in 100 m M Tris-HCl pH 8.5 in a total volume of 50 μL. Total amounts of protein per reaction varied from 4 to 6.5 μg. Reactions were stopped with 50 μL of cold acidified methanol (12% glacial acetic acid/88% methanol, v/v) and stored at −70° C. until LC-MS analysis.
Biomass
For biomass measurements, IRX5:HCHL and wild type plants were grown until senescence and dried stems were collected without roots, leaves and siliques before weighing. Statistical analysis was performed using ANOVA followed by Scheffe post hoc test.
Microscopy
Five-week-old plants were use for microscopy. Stem segments cut between the first and second internodes were embedded in 4% agarose. Stem semi-thin sections (100-μm thickness) were obtained using a vibratome (Leica). For toluidine blue O (TBO) staining, sections were incubated in a 0.05% (w/v) solution of TBO (Sigma-Aldrich) in water for 30 sec and rinsed with water. For Wiesner lignin staining, sections were incubated for 3 min in phloroglucinol-HCl reagent (VWR International) and rinsed with water. For Mäulne lignin staining, sections were incubated in 4% KMnO 4 for 5 min, rinsed with water, incubated in 37% HCl/H 2 O (1:1, v/v) for 2 min, and observed after addition of a drop of aqueous ammonia. Sections were immediately observed using bright field light microscopy (Leica DM4000 B).
Soluble Phenolics Extraction
For extraction of methanol soluble phenolics, approximately 200 mg of frozen stem powder was mixed with 1 mL of 80% (v/v) methanol-water and shaken for 1 h at 1400 rpm. Extracts were cleared by centrifugation (5 min, 20,000 g), mixed with 400 μL of analytical grade water and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore) prior to LC-MS analysis. Alternatively, an aliquot of the filtered extracts was dried under vacuum, resuspended with 1 N HCl and incubated at 95° C. for 3 h for acid hydrolysis. The mixture was subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and resuspended in 50% (v/v) methanol-water prior to LC-MS analysis.
Cell-Wall Bound Phenolics Extraction
For extraction of cell-wall bound phenolics, mature senesced stems were collected without the leaves and siliques, and ball-milled to a fine powder using a Mixer Mill MM 400 (Retsch) and stainless steel balls for 2 min at 30 s −1 . Extract-free cell wall residues (CWR) were obtained by sequentially washing 60 mg of ball-milled stems with 1 ml of 96% ethanol at 95° C. twice for 30 min, and vortexing with 1 mL of 70% ethanol twice for 30 sec. The resulting CWR were dried in vacuo overnight at 30° C. Approximately 6 mg of CWR was mixed with 500 μL of 2 M NaOH and shaken at 1400 rpm for 24 h at 30° C. The mixture was acidified with 100 μL of concentrated HCl, and subjected to three ethyl acetate partitioning steps. Ethyl acetate fractions were pooled, dried in vacuo, and suspended in 50% (v/v) methanol-water prior to LC-MS analysis.
LC-MS
Separation of C 6 C 1 phenolic acids and aldehydes was conducted on a Poroshell-120 column (150 mm length, 3 mm internal diameter, 2.7 μm particle size) using a 1200 Series HPLC system (Agilent Technologies Inc.). Analytes were separated using a gradient elution with mobile phase composition of 0.1% formic acid in water as solvent A, and 0.1% formic acid in acetonitrile-water (98:2, v/v) as solvent B. The elution gradient was 0-5 min 13% B, 5-7 min 50% B, 7-8 min 50% B, and 8-11 min 13% B, using a flow rate of 0.55 mL min −1 . The HPLC system was coupled to an Agilent 6210 time-of-flight (TOF) mass spectrometer (MS) via a 1:7 post-column split. Analyses were conducted using Electrospray ionization (ESI) in the positive ion mode. Detection of [M+H] + ions was carried out in full scan mode at 0.85 spectra sec and a cycle time of 1.176 sec spectrum −1 using the following parameters: capillary voltage 3500 V, fragmentor 165 V, skimmer 50 V and OCT RF 170 V, drying gas flow rate 9 L min −1 , nebulizer pressure 15 psig, drying gas temperature 325° C. Separation of C 6 C 1 phenolic alcohols was conducted on the same HPLC and MS system using the same HPLC column. Analytes were separated using gradient elution with a mobile phase composition of 0.1% formic acid in water as solvent A, and 0.1% formic acid in methanol-water (98:2, v/v) as solvent B. Elution conditions were the same as described above. Analyses were conducted using atmospheric pressure chemical ionization (APCI) in the positive ion mode. Detection of [M−H 2 O+H] + ions was carried as described above except for the following parameters: capillary voltage 3200 V, corona current 4 μA, drying gas flow rate 12 L min −1 , nebulizer pressure 30 psig, vaporizer temperature 350° C. Quantification of compounds was made by comparison with standard curves of authentic compounds prepared in 50% (v/v) methanol-water.
Lignin Analysis
Extract-free samples (CWR) of ball-milled mature senesced stems were prepared using a Soxhlet apparatus by sequentially extracting the ground material with toluene:ethanol (2:1, v/v), ethanol, and water (Sluiter et al., 2008). The determination of lignin content using the standard Klason procedure (Dente, 1992) and the thioacidolysis procedure (Lapierre et al., 1995; 1999) were carried out on CWR. The lignin-derived monomers were identified by GC-MS as their trimethyl-silylated derivatives. All the lignin analyses were performed in duplicate.
Total and Hemicellulosic Sugar Analysis
For total sugar hydrolysis, CWR of ball-milled mature senesced stems (50 mg) were swelled in 500 μL H 2 SO 4 (72%, w/v) at 30° C. for 60 min, and autoclaved at 120° C. for 1 h in dilute H 2 SO 4 (4%, w/v) after addition of deionized water (14 mL). Samples were cooled down at room temperature and filtered using pre-weighted GF/C glass microfiber filters (Whatman). Filtrates were collected and diluted 100 times with deionized water prior to HPAEC-PAD analysis. For hemicellulose hydrolysis, CWR of ball-milled mature dried stems (5 mg) were hydrolyzed in 1 ml of 2 M trifluoroacetic acid (TFA) for 1 h at 120° C. TFA was removed by drying under vacuum and the residue suspended in deionized water (1 mL) prior to HPAEC-PAD analysis.
HPAEC-PAD Analysis
Monosaccharide composition was determined by HPAEC-PAD of hydrolyzed material. Chromatography was performed on a PA20 column (Dionex) at a flow rate of 0.5 mL min −1 . Before injection of each sample (20 μL) the column was washed with 200 mM NaOH for 10 min, then equilibrated with 10 mM NaOH for 10 min. The elution program consisted of a linear gradient from 10 mM NaOH to 5 mM NaOH from 0 to 1.5 min, followed by isocratic elution with 5 mM NaOH from 1.5 to 20 min, and a linear gradient up to 800 mM NaOH from 20 to 43 min. Monosaccharides were detected using a pulsed amperometric detector (gold electrode) set on waveform A according to manufacturer's instructions. A calibration curve of monosaccharide standards that includes L -Fuc, L -Rha, L -Ara, D -Gal, D -Glc, D -Xyl, D -GalA and D -GlcA (Sigma-Aldrich) was run for verification of response factors. Statistical analysis was performed using ANOVA followed by Tukey's test.
FT-Raman and FT-IR Spectral Analyses
FT-Raman spectroscopy was conducted on CWR of ball-milled mature senesced stems (2 mg) from three independent cultures. Raman spectra were collected using a Bruker MultiRAM FT-Raman system equipped with a 1064 nm diode laser (Bruker Optics Inc.). Five spectra were acquired for each sample with spectral resolution of 4 cm −1 using a laser power of 50 mW and 256 scans to achieve good signal-to-noise ratio. White light correction of the acquired spectra was performed to correct the influence of the optics on the spectrometer. Spectra in the range of 200-3500 cm −1 were smoothed and baseline corrected using OPUS software. Lignin and polysaccharides (cellulose and hemicellulose) content were determined using integrated intensities measured over the range of 1555-1690 cm −1 and 1010-1178 cm −1 , respectively. For FT-IR spectroscopy, analyses were carried out on xylem and interfascicular fibers tissues from 50-μm thick sections of the basal region of stems of five-week-old plants. For both wild type and IRX5:HCHL (line 2), five to six sections from three different plants were analyzed. FT-IR spectra were collected from a 50 μm×50 μm window targeting xylem vessels or interfascicular fibers, and normalization of the data and statistical analysis (Student's t-test) were performed as described (Mouille et al., 2003).
Isolation of Cellulolytic Lignin (CEL) and Size Exclusion Chromatography (SEC)
CEL lignin was purified from wild type and IRX5:HCHL (line 2) plants. One gram of ball-milled mature senesced stems was mixed with 50 mM NaCl (30 ml) and incubated overnight at 4° C. After centrifugation (2,800 g, 10 min), the biomass was extracted sequentially by sonication (20 min) with 80% ethanol (three times), acetone (one time), chloroform-methanol (1:1, v/v, one time) and acetone (one time). The obtained CWR were ball-milled for 3 h per 500 mg of sample (in 10 min on/10 min off cycles) using a PM100 ball mill (Retsch) vibrating at 600 rpm with zirconium dioxide vessels (50 mL) containing ZrO 2 ball bearings (10×10 mm). Ball-milled walls (490 mg for wild type and 480 mg for IRX5:HCHL) were transferred to centrifuge tubes (50 mL) and digested four times over three days at 30° C. with crude cellulases (Cellulysin; Calbiochem; 60 mg g −1 of sample) in NaOAc pH 5.0 buffer (30 mL) under gentle rotation. The obtained CEL was washed 3 times with deionized water and lyophilized overnight. CEL recovered were 131 mg for wild type (27.3%) and 101 mg for IRX5:HCHL (20.6%). For SEC analysis, 1% (w/v) CEL lignin solutions were prepared in analytical-grade 1-methyl-2-pyrrolidinone-DMSO (1:1, v/v) and sonicated for 3 hours at 40° C.
Polydispersity of dissolved lignin was determined using analytical techniques SEC UV-F and SEC UV-A as described elsewhere (George et al., 2011, submitted). An Agilent 1200 series binary LC system (G1312B) equipped with FL (G1321A) and DA (G1315D) detectors was used. Separation was achieved with a Mixed-D column (5 mm particle size, 300 mm×7.5 mm i.d., linear molecular weight range of 200 to 400,000 u, Polymer Laboratories) at 80° C. using a mobile phase of NMP at a flow rate of 0.5 mL min −1 . Absorbance of material eluting from the column was detected at 300 nm (UV-A). Excitation 250 nm and emission 450 nm were used for UV-F detection. Intensities were area normalized and molecular mass estimates were determined after calibration of the system with polystyrene standards.
Cell Wall Pretreatments and Saccharification
Ball-milled mature senesced stems (10 mg) were mixed with 340 μL of water, 340 μL of H 2 SO 4 (1.2%, w/v), or 340 μL of NaOH (0.25%, w/v) for hot water, dilute acid, or dilute alkaline pretreatments, respectively, incubated at 30° C. for 30 min, and autoclaved at 120° C. for 1 h. After cooling down at room temperature, samples pretreated with dilute acid and dilute alkaline were neutralized with 5 N NaOH (25 μL) and 1.25 N HCl (25 μL), respectively. Saccharification was initiated by adding 635 μL of 100 m M sodium citrate buffer pH 6.2 containing 80 g ml −1 tetracycline, 5% w/w cellulase complex NS50013 and 0.5% w/w glucosidase NS50010 (Novozymes). After 72 h of incubation at 50° C. with shacking (800 rpm), samples were centrifuged (20,000 g, 3 min) and 10 μL of the supernatant was collected for reducing sugar measurement using the DNS assay and glucose solutions as standards (Miller, 1959).
Transcriptome Studies
Microarray analysis was performed on complete Arabidopsis thaliana transcriptome microarrays containing 24,576 gene-specific tags (GSTs) corresponding to 22,089 genes from Arabidopsis (Crowe et al., 2003; Hilson et al., 2004). RNA samples from three independent biological replicates were isolated and separately analyzed. For each biological replicate, RNA from the main inflorescence stem (first two internodes) of three plants were pooled. For each comparison, one technical replication with fluorochrome reversal was performed for each biological replicate (i.e. nine hybridizations per comparison). Reverse transcription of RNA was conducted in the presence of Cy3-dUTP or Cy5-dUTP (PerkinElmer-NEN Life Science Products), and hybridization and scanning of the slides were performed as described in Lurin et al. (2004).
Statistical Analysis of Microarray Data
Statistical analysis was performed with normalization based on dye swapping (i.e., four arrays, each containing 24,576 GSTs and 384 controls) as previously described (Gagnot et al., 2008). For the identification of differentially expressed genes, we performed a paired t test on log ratios, assuming that the variance of the log ratios was similar for all genes. Spots with extreme variances (too small or too large) were excluded. The raw P values were adjusted by the Bonferroni method, which controls the family-wise error rate (with a type I error equal to 5%) to minimize the number of false positives in a multiple-comparison context (Ge et al., 2003). Genes with a Bonferroni P value ≦0.05 were considered to be differentially expressed, as previously described (Gagnot et al., 2008).
Data Deposition
Microarray data from this article were deposited at GEO (http://www.ncbi.nlm.nih.gov/geo/) and at CATdb (http://urgv.evry.inra.fr/CATdb/) according to Minimum Information about a Microarray Experiment standards (MIME).
II. Results
Expression of a Bacterial HCHL Enzyme in Arabidopsis Stems
The tissue specific activity of the IRX5 promoter was verified using the beta-glucuronidase (GUS) as a reporter gene. Gus activity was essentially detected in the xylem vessels of the stem. After prolonged incubations, stem interfascicular fibers also showed strong GUS activity, and more moderate staining was observed in the vascular system of young seedlings, siliques, rosette and cauline leaves. No activity was detected in other organs or tissues except for the style and anthers ( FIG. 9 ). A codon-optimized sequence encoding HCHL from Pseudomonas fluorescens AN103 was designed and cloned downstream of the IRX5 promoter for preferential expression in lignified tissues of Arabidopsis stems. Presence of HCHL transcripts in the main stem of five independent transformants was verified by RT-PCR in the T1 generation ( FIG. 2A ). Plants homozygous for the IRX5:HCHL construct were identified in the T2 generation, and used to analyze HCHL protein expression and activity in stems. Western blotting analysis using the ‘universal antibody’ allowed detection of HCHL in stem extracts of the five selected transgenic lines ( FIG. 2B ; Eudes et al. 2010). Furthermore, HCHL activity could be detected in the stem of these lines, ranging from 0.025 to 0.16 pkat vanillin μg −1 protein using feruloyl-CoA as substrate, whereas no detectable activity was observed in protein extracts of wild type plants (Table I). Two transgenic lines showing the highest and the lowest levels of HCHL activity, and two lines exhibiting intermediate activity level were selected for detailed analysis.
Growth Characteristics and Tissue Anatomy of IRX5:HCHL Lines
IRX5:HCHL plants had growth and development characteristics visually similar to the wild type from early rosette stage and until senescence ( FIG. 10 ). However, mature senesced stems from lines IRX5:HCHL (4) and IRX5:HCHL (5) were little bit shorter (22% and 13% reduction) and had lower dry weight yield (30% and 16% reduction) compared to control plants, whereas those from lines IRX5:HCHL (1) and IRX5:HCHL (2) were not significantly different (Table II). Stem tissues of five-week-old IRX5:HCHL plants were inspected using light microscopy. Transverse stem cross-sections stained with Mäule and phloroglucinol-HCl reagents, which are indicative of S-units and hydroxycinnamaldehyde units in lignin, respectively, showed similar patterns between transgenic and wild type plants ( FIGS. 3A and 3B ). Similarly, lignin in stem sections stained with toluidine blue O did not revealed any quantitative differences between genotypes ( FIG. 3C ). A few collapsed xylem structures were, however, occasionally observed on some stem cross-sections of line IRX5:HCHL (4), but were absent in sections from other lines ( FIG. 3C ). Overall, these data suggest that lignin content is not drastically reduced in IRX5:HCHL plants.
IRX5:HCHL Lines Accumulate C 6 C 1 Soluble Phenolics
Methanol soluble fractions from stems of five-week-old wild type and IRX5:HCHL plants were extracted and analyzed by LC-MS. Analysis was performed to focus on hydroxybenzaldehydes, direct products of HCHL activity, and possible derivatives such as hydroxybenzoyl alcohols and hydroxybenzoic acids and their glucose conjugates. Trace amounts of 4-hydroxybenzaldehyde (HBAld), 3,4-dihydroxybenzaldehyde (3,4-DHBAld), and 4-hydroxybenzoic acid (HBA) were detected in IRX5:HCHL stem soluble extracts but not in wild type (Table III). Notably, much larger quantities of 4-hydroxybenzoic acid glucoside (HBAGlc) and 4-hydroxybenzoic acid glucose ester (HBAGE) were detected in IRX5:HCHL plant soluble extracts (ranging from 0.48 to 0.57 mg g −1 FW for HBAGlc, and from 0.96 to 1.65 mg g −1 FW for HBAGE), whereas trace amounts of these HBA-glucose conjugates were present in wild type extracts (Table III).
Considering that other soluble C 6 C 1 phenolics could be glycosylated, acid hydrolysis of the soluble fractions was performed to release aglycones from conjugated forms. This procedure brought down HBAGE and HBAG pools to undetectable levels, and concomitantly increased free HBA content in samples (Table IV). HBA content in the IRX5:HCHL lines ranged between 1.59 and 2.49 mg g −1 FW, which represents a 113 to 179 fold increase compared to values observed in wild type samples, and indicates that 88-94% of HBA accumulated in transgenic lines is glycosylated. In addition to HBA, other C 6 C 1 phenolics quantified in acid-treated extracts include vanillin (Van), 5-hydroxyvanillin (5OH-Van), syringaldehyde (Syrald), 5-hydroxyvanillic acid (5OH-VA), and syringic acid (SyrA), which are only detected in IRX5:HCHL extracts, as well as HBAld, 3,4-DHBAld, 3,4-dihydroxybenzoic acid (3,4-DHBA), and vanillic acid (VA), which are on average 14, 119, 1.6, and 40 times more abundant in IRX5: HCHL extracts compared to wild type, respectively (Table IV).
IRX5:HCHL Lines Show Enrichment in Cell Wall-Bound C 6 C 1 Phenolics
Extract-free cell wall residues (CWR) obtained from mature senesced stems of wild type and IRX5:HCHL plants were subjected to mild alkaline hydrolysis for the release of loosely-bound phenolics. This procedure released from the cell wall samples some HBAld, 3,4-HBAld, Van, 5OH-Van, SyrAld, HBA, VA, and SyrA, which were quantified using LC-MS analysis. 5OH-Van, undetectable in wild type cell wall, was present in that of IRX5:HCHL samples and HBAld, SyrAld, HBA, VA, and SyrA were increased on average by approx 2, 6, 68, 2 and 5 fold in cell walls of IRX5:HCHL plants compared to the wild type, respectively (Table V). These results indicate that larger amounts of C 6 C 1 phenolics are loosely-bound to cell walls in IRX5:HCHL plants. On the other hand, amount of ferulate and coumarate released from cell walls using this procedure did not differ between transgenic and wild type samples.
Spectral Analysis of IRX5:HCHL Plant Stems
Line IRX5:HCHL (2), which showed no defective xylem structures and biomass yield similar to wild type plants, was selected for further analyses. Fourier transformed Raman (FT-Raman) spectroscopy was used to determine the chemical composition of CWR obtained from senesced stems of IRX5:HCHL plants. Compared to the wild type, data showed that lignin content and amount of polysaccharides (cellulose and hemicellulose) in IRX5:HCHL plants were not significantly different ( FIG. 4A ). Moreover, Fourier transformed infrared (FT-IR) spectral analysis conducted on lignified tissues (xylem and interfascicular fibers) of transverse stem sections of five-week-old IRX5:HCHL and wild type plants revealed differences between the two genotypes ( FIG. 4B ). In particular, significant changes in spectra were observed for bands assigned to different bending or stretching of lignin (Agarwal and Atalla, 2010, Fackler et al., 2010). For example, absorptions at wavelengths 1589 cm −1 and 1506 cm −1 (aryl ring stretching), 1464 cm −1 (C—H group deformation), 1425 cm −1 (methoxyl C—H group deformation), 1379 cm −1 (aromatic skeletal vibrations combined with C—H group in plane deformation), and 1268 cm −1 (aryl ring breathing with C=O group stretch) were modified in fibers, whereas the most significant difference for xylem cell walls was observed at band 1367 cm −1 (methoxyl C—H group deformation). Overall, spectral analyses suggested compositional modifications of lignin in plants expressing HCHL.
Monosaccharide Content and Composition in IRX5:HCHL Plant Stems
Monosaccharide composition was determined after sulfuric acid hydrolysis of total cell wall polysaccharides from mature senesced stems of line IRX5:HCHL (2) and wild type plants. Although both genotypes had similar amount of total monosaccharides, IRX5:HCHL plants showed reduction in glucose (−12%) and increase in xylose (+22%) and arabinose (+16%) compared to wild type plants (Table VI). Moreover, hemicellulosic monosaccharides released from CWR using trifluoroacetic acid showed that total amount of sugar quantified in this hydrolysate was 23% higher in IRX5:HCHL stems which corresponds to higher xylose (+23%) and arabinose (+22%) contents compared to wild type (Table VI).
Incorporation of Unusual C 6 C 1 Monomers into the Lignin of IRX5:HCHL Plants
Lignin content and monomeric composition in mature senesced stems from wild type and IRX5:HCHL (2) plants was determined on CWR. In two independent cultures, klason lignin (KL) was identical and accounted for about 20% of the CWR for both wild type and IRX5:HCHL plants (Table VII). Lignin monomer composition was evaluated by thioacidolysis, a chemical degradative method that generates thioethylated monomers from lignin units involved in labile β-O-4 bonds. Data showed that total amount of conventional H, O, and S monomers released from CWR after thioacidolysis (or total yield) was reduced by 25% and 16% in the two independent cultures of IRX5:HCHL plants compared to the wild type, indicating that fewer of these three monolignols are crosslinked as β-O-4 bond in transgenics (Table VII). Considering identical KL values for both wild type and IRX5:HCHL CWR, these data indicate higher frequency of thioacidolysis-resistant bonds between lignin monomers in transgenic plants. The relative amount of G and S units recovered from this lignin fraction was unchanged, both wild type and transgenic samples showing an S/G ratio ranging between 0.34-0.36, however, molar frequency of H units was significantly higher in IRX5:HCHL plants (Table VII). Furthermore, the content of non-conventional units such as Van, Syrald, and SyrA released by thioacidolysis showed on average a 1.44-, 20.8-, and 1.65-fold increase in IRX5:HCHL plants compared to wild type plants, respectively. Interestingly, two new lignin units were released from the lignin of transgenics plants, which were identified as C 6 C 1 vanillyl alcohol (Vanalc) and syringyl alcohol (Syralc) (Table VIII). On the other hand, the content of coniferaldehyde end-groups (Cald) and VA was unchanged between the two genotypes (Table VIII). Overall, these data showed higher amount of C 6 C 1 phenolic end-groups among monomers released by thioacidolysis from IRX5:HCHL stem cell walls compared to wild type.
Lignin of IRX5:HCHL Plants has Reduced Molecular Mass
The polydispersity of cellulolytic lignin purified from wild type and IRX5:HCHL (2) stems was determined using size exclusion chromatography (SEC). Elution profiles acquired by monitoring UV-A absorbance (SEC UV-A 300 ) and UV-F fluorescence (SEC UV-F ex 250/ em 450) of the dissolved lignin revealed differences between wild type and IRX5:HCHL plants ( FIG. 6 ). First, total area corresponding to the largest mass peak detected between 7 min and 13.5 min was severely reduced in transgenics due to significant diminution of the largest lignin fragments which elute between 7 min and 9 min. Similarly, smaller molecular mass material which elutes later in a second peak between 13.5 min and 19.5 min was more abundant (increased by 27% and 16% using UV-A and UV-F detections) in IRX5:HCHL samples. Finally, the amount of the smallest lignin fragments detected between 19.5 min and 26.5 min using UV-F is increased by 55% in transgenics ( FIG. 6 ). These results demonstrate smaller chains and reduced polymerization degree in lignin purified from IRX5:HCHL plants.
IRX5:HCHL Lines Show Increased Saccharification Efficiency
To examine impact lignin size reduction on cell wall digestibility caused by the expression of the HCHL enzyme in lignifying tissues, saccharification assays were conducted biomass derived from mature senesced stems pretreated with hot water, dilute alkaline, and dilute acid. After a 72-h incubation with cellulase and glucosidase, pretreated biomass of IRX5 HCHL plants released more reducing sugars compared to wild type ( FIG. 7 ). In particular, improvement of saccharification efficiency observed for the different IRX5:HCHL lines ranged from 34% to 77% after hot water, from 43% to 71% after dilute alkaline, and from 15% to 31% after dilute acid pretreatments ( FIG. 7 ).
III. Discussion
Expression of HCHL in plants has originally been considered for in planta production of valuable and soluble compounds such as Van and HBA. Due to strong ectopic HCHL expression, however, adverse phenotypes such as chlorotic and senescing leaves, stunting, low pollen production, male sterility, collapsed xylem vessels, and reduction of biomass were observed in transgenic tobacco, and sugarcane (Mayer et al., 2001; Merali et al., 2007; McQualter et al., 2005). In this study, the inventors selected the promoter of a secondary cell wall cellulose synthase to preferentially express HCHL in the lignifying tissues of Arabidopsis stems ( FIG. 9 ). Successfully, plants transformed with the IRX5:HCHL construct were not dwarf or sterile, and young rosette leaves did not show reduced epidermal fluorescence which is symptomatic of alteration in phenylpropanoid-derived soluble phenolic pools. Although two IRX5:HCHL lines showed reduced biomass, and in one case some occasional collapsed xylem vessels caused by stronger HCHL activity and possibly modification of call wall integrity, some other IRX5:HCHL lines were comparable to wild-type plants.
As expected, the transgenic lines show increased amount of soluble C 6 C 1 aldehydes (HBAld, 3,4-DHBAld, and Van), which are produced upon HCHL activity after cleavage of hydroxybenzoyl-CoA, 3,4-dihydroxybenzoyl-CoA, and feruloyl-CoA ( FIG. 11 ). HCHL has no activity against sinapoyl-CoA, suggesting that Syrald is a conversion product of Van, which is supported by the identification of the new intermediate 5OH-Van (Mitra et al., 1999; FIG. 11 ). Similarly, the data presented herein cannot exclude that some of the 3,4-DHBald and Van accumulated in transgenics derive from HBAld after successive hydroxylation and methoxylation on the C-3 position of the phenyl ring. Interestingly, several genes encoding monooxygenases are upregulated in plants expressing HCHL, but no known or predicted O-methyltransferase showed altered expression level (Table IX). Analysis of soluble aromatics in transgenics also shows that C 6 -C 1 aldehydes are oxidized into their respective acid forms. This conversion could be a response to reduce the amount of these chemically reactive compounds since several genes from the short-chain dehydrogenase/reductase (SDR), aldo-keto reductase (AKR), and aldehyde dehydrogenase (ALDH) families are upregulated in plants expressing HCHL, ( FIG. 11 ; Kirch et al., 2004; Kavanagh et al., 2008). In particular, AKR4C9 (At3g37770) encodes an enzyme known to metabolize a range of hydroxybenzaldehydes (Simpson et al., 2009). In addition, soluble C 6 C 1 phenolics predominantly accumulate as conjugates in transgenics since we showed that glucose conjugates (phenolic glucoside and glucose ester) represented around 90% of the HBA soluble pool, presumably for vacuolar storage as previously described for other C 6 C 1 phenolics (Eudes et al., 2008). This C 6 C 1 acid glucoside accumulation is in agreement with what was observed in tobacco, sugar beet, datura and sugar cane plants expressing HCHL (Mayer et al., 2001; Mitra et al., 2002; McQualter et al., 2005; Rahman et al., 2009). Interestingly, expression analysis of HCHL plants revealed seven up-regulated genes of the UDP-glucosyltranferase (UGT) family and among them UGT75B1 and UGT73B4 were previously shown to catalyze glucose esterification and phenolic glucosylation of benzoates (Table IX; Lim et al, 2002; Eudes et al., 2008).
Furthermore, this study showed that some C 6 C 1 phenolics are released from extract-free cell wall fractions of senesced stems upon mild alkaline hydrolysis. Higher amounts of HBAld, 5OH-Van, SyrAld, HBA, VA, and SyrA were measured in the ‘loosely wall-bound’ fraction of IRX5:HCHL lines compared to wild type. Although the type of linkages involved is unclear, loosely attached C 6 C 1 phenolics were previously extracted from cell walls of Arabidopsis leaves and roots (Tan et al., 2004; Forcat et al., 2010).
The lignin from plants expressing HCHL shows increased content of C 6 C 1 phenolics. Notably, analysis of lignin monomers released after thioacidolysis identified two novel units (Vanalc and Syralc) and showed large amounts of Syrald, Van, and SyrA. This suggests part of C 6 C 1 aldehydes are converted into alcohols and acids and demonstrates that they are incorporated into the lignin as β-O-4-linked C 6 C 1 monomer end-groups in lignin ( FIG. 11 ). Due to the absence of phenyl propanoid tail, these new monolignols when incorporated in lignin end chains, should block further polymerization of the polymer and act as condensation terminator or stopper molecules. Interestingly, transgenic plants also show higher content of conventional H-units (+30%), which preferentially distribute as terminal end-groups in lignin and contribute to modifications of lignin size and structure (Lapierre, 2010; Ziebell et al., 2010). In addition, plants overproducing C 6 C 1 monolignols and with similar lignin content as wild type plants show a lower thioacidolysis release of monolignols, indicating a reduction in the availability of free propanoid tail in lignin end-chain for polymer elongation. It also indicates higher carbon-carbon linkages and increased lignin condensation degree.
It was postulated that higher incorporation of end-group units in lignin would hinder more frequently chain elongation and ultimately reduce lignin branching and polymerization degree. This hypothesis is further supported by the analysis the polydispersity of lignin in plants overproducing theses “stopper” molecules, which shows significant reduction of high molecular masses and significant increase of low molecular masses, hence supporting smaller lignin chain length. These observations are relevant for understanding the higher susceptibility of the biomass from HCHL lines to polysaccharide enzymatic hydrolysis. Although saccharification efficiency of biomass is determined by several characteristics of cell walls, the observed saccharification efficiency improvement after different pretreatments suggests that less ramified lignin would reduce cross-linkages and embedding of cell wall polysaccharides (cellulose and hemicellulose) and would favor their accessibility to hydrolytic enzymes. This hypothesis is supported by the fact that total sugar content is unchanged in cell walls of plants overproducing theses C 6 C 1 monomers.
it is concluded that in planta the over-production of lignification “stopper” molecules can be used to modify the lignin structure in order to reduce lignocellulosic biomass recalcitrance. Since this approach does not require any particular genetic background, it should be easily transferable to various energycrops. Restricting the biosynthesis of these lignification “stopper” molecules in supporting lignified tissues (i.e. schlerenchyma fibers) as well as avoiding strong production in conductive tissues (i.e. vessels) should limit the risk of adverse effects on plant development and biomass yield.
Example 2
Expression of Bacterial HCHL in Rice
This example illustrates expression of bacterial HCHL in a monocot, rice. Rice plants were transformed with the DNA constructs described in Example 1. Rice lines were engineered ( FIG. 12 ) that expressed the HCHL gene, as demonstrated by RT-PCR ( FIG. 13 ). Furthermore, evaluation of rice lines demonstrated that they accumulated pHBA (para-hydroxybenzoate) ( FIG. 14 ), which is generated from the conversion of p-coumaroyl-CoA by HCHL.
This experiment additionally demonstrated that a secondary wall promoter, pIRX5, from a dicot ( Arabidopsis in this example), can be used in a monocot (rice in this example).
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, accession numbers, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
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ILLUSTRATIVE SEQUENCES







SEQ ID NO: 1

Amino acid sequence for Pseudomonas fluorscens HCHL

(GenBank Accession No. CAA73502)

MSTYEGRWKTVKVEIEDGIAFVILNRPEKRNAMSPTLNREMIDVLETLEQDPAA



GVLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKP



TIAMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTV



GHRQSLYYIMTGKTFGGQKAAEMGLVNESVPLAQLREVTIELARNLLEKNPVVL



RAAKHGFKRCRELTWEQNEDYLYAKLDQSRLLDTEGGREQGMKQFLDDKSIKPG



LQAYKR



SEQ ID NO: 2

Polynucleotide sequence encoding SEQ ID NO: 1

(codon-optimized by GenScript)

ATGTCTACTTACGAGGGAAGATGGAAGACTGTTAAGGTTGAGATCGAGGATGGA



ATCGCTTTCGTTATCCTCAACAGACCTGAGAAGAGAAACGCTATGTCTCCTACT



CTCAACAGAGAGATGATCGATGTTCTCGAGACTCTCGAGCAGGATCCTGCTGCT



GGAGTTCTCGTTCTCACTGGAGCTGGAGAGGCTTGGACTGCTGGTATGGATCTC



AAGGAGTACTTCAGAGAGGTTGATGCTGGACCTGAGATCCTCCAGGAGAAGATC



AGAAGAGAGGCTTCTCAGTGGCAGTGGAAGCTCCTCAGAATGTACGCTAAGCCT



ACTATCGCTATGGTTAACGGATGGTGCTTCGGAGGAGGATTCTCTCCTCTCGTT



GCTTGCGATCTCGCTATCTGCGCTGATGAGGCTACTTTCGGACTCTCTGAGATC



AACTGGGGAATCCCTCCTGGAAACCTCGTTTCTAAGGCTATGGCTGATACTGTT



GGACATAGACAGTCTCTCTACTACATCATGACTGGAAAGACTTTCGGAGGACAG



AAGGCTGCTGAGATGGGACTCGTTAACGAGTCTGTTCCTCTCGCTCAGCTCAGA



GAGGTTACTATCGAGCTCGCTAGAAACCTCCTCGAGAAGAACCCTGTTGTTCTC



AGAGCTGCTAAGCATGGATTCAAGAGATGCAGAGAGCTCACTTGGGAGCAGAAC



GAGGATTACCTCTACGCTAAGCTCGATCAGTCTAGACTCCTCGATACTGAGGGA



GGAAGAGAGCAGGGTATGAAGCAGTTCCTCGATGATAAGTCTATCAAGCCTGGA



CTCCAGGCTTACAAGAGA



SEQ ID NO: 3

Polynucleotide sequence containing IRX5 promoter (pIRX5)

ATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATG



GCATGGATGCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTT



ACCACAAGAGCAACGGTTGTTTCGGGTAAATAAACTAAACTTAACCATATACAT



TAGCCTTGATTCGGTTTTTGGTTTGATTTATGGATATTAAAGATCCGAATTATA



TTTGAACAAAAAAAAATGATTATGTCACATAAAAAAAAATTGGCTTGAATTTTG



GTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTCATTTGTTTTCTGGTTAG



CTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGATTTGAAAC



AGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAG



GCACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGT



TTCAAGATCTTTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTC



GAAAATCTTATTGAAGCCGGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTC



GAATCATATTTAATAACCGCATTAAACCGAAATTTAAATTCTAATTTCACCAAA



TCAAAAAGTAAAACTAGAACACTTCAGATAAATTTTGTCGTTCTGTTGACTTCA



TTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAATAAAAACCCTAAA



AACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTTTAGGTA



AGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTC



ATTAATTTGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTT



GTGAAATCATATTTAAAACATTAATAGGTATTTATGTCTAATTTGGGGACAAAA



TAGTGGAATTCTTTATCATATCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTT



ATGATTATGTTACATGCATTGGTCCATATAAATCTATGAGCAATCAATATAATT



CCGAGCATTTTGGTATAACATAATGAGCAAGTATAACAAAAGTATCAAACCTAT



GCAGGGGAGAAGATGATGAAAAGAAGAGTGTGAGCCAATACAAAGCAGATTTGA



GGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCACAAT



GGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACT



CCATCTACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCA



GAGTTTCATTCTGATTCTTGAGTCTTTTTTTTCTCTCTCCCTCTTTTCATTTCT



GGTTTATATAACCAATTCAAATGCTTATGATCCATGCATGAACCATGATCATCT



TTGTGTTTTTTTTTCCTTCTGTATTACCATTTTGGGCCTTTGTGAAATTGATTT



TGGGCTTTTGTTATATAATCTCCTCTTTCTCTTTCTCTACCTGATTGGATTCAA



GAACATAGCCAGATTTGGTAAAGTTTATAAGATACAAAATATTAAGTAAGACTA



AAGTAGAAATACATAATAACTTGAAAGCTACTCTAAGTTATACAAATTCTAAAG



AACTCAAAAGAATAACAAACAGTAGAAGTTGGAAGCTCAAGCAATTAAATTATA



TAAAAACACTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCTG



TCTCTTGAAGCTTTCTTATGACACAAACCTTAGACCCAATTTCACTCACAGTTT



GGTACAACCTCAGTTTTCTTCACAACAAATTCAAACATCTTACCCTTATATTAC



CTCTTTATCTCTTCAATCATCAAAACACATAGTCACATACATTTCTCTACCCCA



CCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCC



SEQ ID NO: 4

Sagittula _ stellata _E-37__ZP_01746375 (amino acid sequence)

MTATEATLPANDPDLSGDNVAVAFEDGIAWVKLNRPEKRNAMSVSLAEDMNVVLD



KLEIDDRCGVLVLTGEGSAFSAGMDLKDFFRATDGVSDVERMRAYRSTRAWQWRT



LMHYSKPTIAMVNGWCFGGAFTPLICCDLAISSDDAVYGLSEINWGIIPGGVVSK



AISTLMSDRQALYYVMTGEQFGGQEAVKLGLVNESVPADKLRERTVELCKVLLEK



NPTTMRQARMAYKYIREMTWEESAEYLTAKGDQTVFVDKEKGREQGLKQFLDDKT



YRPGLGAYKR



SEQ ID NO: 5

Saccharopolyspora _ erythraea _NRRL_2338_YP_001105000

(amino acid sequence)

MSTPTTDPGTTTTPWGDTVLVDFDDGIAWVTLNRPEKRNAMNPAMNDEMVRTLDA



LEADPRCRVMVLTGAGESFSAGMDLKEYFREVDQTADPSVQIRVRRASAEWQWKR



LAHWSKPTIAMVNGWCFGGAFTPLVACDLAISDEEARYGLSEINWGIPPGGVVSR



ALAAAVSQRDALYFIMTGETFDGRRAEGMRLVNEAVPAERLRERTRELALKLAST



NPVVLRAAKVGYKIAREMPWEQAEDYLYAKLEQSQFLDAERGREKGMAQFLDDKS



YRPGLSAYSTD



SEQ ID NO: 6

Solibacter _ usitatus _Ellin6076_YP_821552

(amino acid sequence)

MDQYEEKWQTVKVEVDAEGIAWVIFNRPAKRNAMSPTLNREMAQVLETLELDAAA



KVLVLTGAGESWSAGMDLKEYFREVDGQPESHQEKIRREASLWQWKLLRMYAKPT



IAMVNGWCFGGAFSPLVACDLAIADEKAVFGLSEINWGIPPGNLVSKAVADTMGH



RKALHYIMTGETFTGAQAAEMGLVNAAVPTSELREATRTLALKLASKNPVILRAA



KHGFKRCRELTWEQNEDYLYAKLDQALHRDPEDARAEGMKQFLDEKSIKPGLQSY



KRS



SEQ ID NO: 7

Ralstonia _ solanacearum _GMI1000_NP_521786

(amino acid sequence)

MATYEGRWNTVKVDVEDGIAWVTLNRPDKRNAMSPTLNREMIDVLETLELDGDAQ



VLVLTGAGESWSAGMDLKEYFRETDGQPEIMQERIRRDCSQWQWKLLRFYSKPTI



AMVNGWCFGGAFSPLVACDLAIAADDAVFGLSEINWGIPPGNLVSKAVADTMGHR



AALHYIMTGETFTGREAAEMGLVNRSVPRERLREAVTELAGKLLAKNPVVLRYAK



HGFKRCRELSWEQNEDYLYAKVDQSNHRDPEKGRQHGLKQFLDDKTIKPGLQTYK



RA



SEQ ID NO: 8

Xanthomonas _ albilineans _YP_003377516 (amino acid sequence)

MSNYQDRWQTVQVQIDAGVAWVTLNRPEKRNAMSPTLNREMIDVLETLELDSAAE



VLVLTGAGESWSAGMDLKEYFREIDGKEEIVQERMRRDCSQWQWRLLRFYSKPTI



AAVNGWCFGGAFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAVADTMGHR



NAMLYIMTGRTFTGTEAAQMGLVNASVPRAQLRAEVTKLAQELQQKNPVVLRFAK



HGFKRCRELTWEQNEDYLYAKVDQSNHRDPEKGRQQGLKULDDKTIKPGLQTYKR



SEQ ID NO: 9

Acinetobacter _ baumannii _ATCC_17978_YP_001084143

(amino acid sequence)

MKMSYENRWETVDVKVEDGIAWVTLNRPEKKNAMSPTLNREMIDVLETLELDQNA



KVLVLTGAGDSWTAGMDLKEYFREVDTQPEIFQERIRRDSCRWQWQLLRMYSKPT



IAMVNGWCFGGGESPLVACDLAIAADEATEGLSEINWGIPPGNLVSKAMADTVGH



RASLYYIMTGKTFSGKEAETMGLVNKSVPLAQLKAEVTELANCLLEKNPVVLRTA



KNGFKRCRELTWDQNEDYLYAKLDQCIHRDTENGRQEGLKQFLDEKSIKPGLQSY



KRTG



SEQ ID NO: 10

Acinetobacter _sp._ADP1_YP_046390 (amino acid sequence)

MTYDKRWETVDVQVEHGIAWVTLNRPHKKNAMSPTLNREMIDVLETLELDSEAKV



LVLTGAGDSWTAGMDLKEYFREVDAQPEIFQERIRRDSCRWQWQLLRMYSKPTIA



MVNGWCFGGGFSPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRA



SLYYIMTGKTFTGKEAEAMGLINKSVPLAQLKAEVTELAQCLVEKNPVVLRTAKN



GEKRCRELTWDQNEDYLYAKLDQCNHRDTEGGRQEGLKQFLDEKSIKPGLQSYKR



TG



SEQ ID NO: 11

Chromohalobacter _ salexigens _DSM_3043_YP_572340

(amino acid sequence)

MSDYTNRWQTVKVDVEDGIAWVTLNRPEKRNAMSPTLNREMIDVLETIELDQDAH



VLVLTGEGESFSAGMDLKEYFREIDASPEIVQVKVRRDASTWQWKLLRHYAKPTI



AMVNGWCFGGAFSPLVACDLAIAADESVFGLSEINWGIPPGNLVSKAMADTVGHR



QALYYIMTGETFTGPQAADMGLVNQSVPRAELRETTHKLAATLRDKNPVVLRAAK



TGFKMCRELTWEQNEEYLYAKLDQAQQLDPEHGREQGLKQFLDDKSIKPGLESYR



R



SEQ ID NO: 12

Burkholderia _ cenocepacia _AU_1054_ZP_04942909

(amino acid sequence)

MSKYDNRWQTVEVKVEAGIAWVTLNRPEKRNAMSPTLNREMLEVLDAVEFDDEA



KVLVLTGAGAAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP



TIAMVNGWCFGGGFSPLVACDLAIAADDAVFGLSEINWGIPPGNLVSKAMADTV



GHRRALHYIMTGDTFTGAEAAEMGLVNSSVPLAELRDATIALAARLMDKNPVVL



RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKTIKPG



LQAYKR



SEQ ID NO: 13

Burkholderia _ ambifaria _MC40-6_YP_776799

(amino acid sequence)

MSKYDNRWQTVEVNVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA



KVLVLTGAGSAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP



TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV



GHRRALHYIMTGDTFTGVEAAEMGLVNSSVPLAGLRDATIALAARLMDKNPVVL



RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG



LQAYKR



SEQ ID NO: 14

Burkholderia _ cepacia _AMMD_YP_776799 (amino acid sequence)

MSKYDNRWQTVEVNVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA



KVLVLTGAGSAWTAGMDLKEYFREIDGGSDALQEKVRRDASEWQWRRLRMYNKP



TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV



GHRRALHYIMTGDTFTGVEAAEMGLVNSSVPLAGLRDATIALAARLMDKNPVVL



RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG



LQAYKR



SEQ ID NO: 15

Burkholderia _ thailandensis _MSMB43_ZP_02468311

(amino acid sequence)

MSKYDNRWQTVEVKVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA



KVLVLTGAGTAWTAGMDLKEYFREIDGGPDALQEKVRRDASEWQWRRLRMYGKP



TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV



GHRCALHYIMTGDTFTGVEAADMGLVNRSVPLAELRDATIALAARLIDKNPVVL



RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG



LQAYKR



SEQ ID NO: 16

Burkholderia _ ubonensis _Bu_ZP_02382374

(amino acid sequence)

MSKYENRWQTVEVKVEAGIAWVTLNRPDKRNAMSPTLNQEMLQVLDAIEFDDDA



KVLVLTGAGAAWTAGMDLKEYFREIDGGPDALQEKVRRDASEWQWRRLRMYGKP



TIAMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTV



GHRRALHYIMTGDTFTGVEAADMGLVNRSVPLAELRDATIALAARLIDKNPVVL



RAAKHGFKRSRELTWEQCEDYLYAKLDQAQLRDPERGREQGLKQFLDDKAIKPG



LQAYKR



SEQ ID NO: 17

Azotobacter _ vinelandii _AvOP_YP_002798614

(amino acid sequence)

MNKYEGRWKTVIVEIEGGIAWVTLNRPDKRNAMSPTLNREMRDVLETLEQDPAAR



VLVLTGAGSAWTAGMDLKEYFREVDAGPEILQEKIRREACEWQWKLLRMYAKPTV



AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR



QALYYIMTGKTFDGRQAAEMGLVNQSVPLAQLRETVATLCQDLLDKNPVVLRAAK



NGFKRCRELTWEQNEDYLYAKLDQSRLLDEEGGREEGMRQFLDEKSIKPGLQAYK



R



SEQ ID NO: 18

Pseudomonas _ putida _KT2440_NP_745498 (amino acid sequence)

MSKYEGRWTTVKVELEAGIAWVTLNRPEKRNAMSPTLNREMVDVLETLEQDADAG



VLVLTGAGESWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRLYAKPTI



AMVNGWCFGGGFSPLVACDLAICANEATFGLSEINWGIPPGNLVSKAMADTVGHR



QSLYYIMTGKTFDGRKAAEMGLVNDSVPLAELRETTRELALNLLEKNPVVLRAAK



NGFKRCRELTWEQNEDYLYAKLDQSRLLDTTGGREQGMKQFLDDKSIKPGLQAYK



R



SEQ ID NO: 19

Pseudomonas _ fluorescens _SBW25_YP_002872871

(amino acid sequence)

MSNYEGRWTTVKVEIEEGIAWVILNRPEKRNAMSPTLNREMIDVLETLEQDPAAG



VLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKPTI



AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR



QSLYYIMTGKTFGGQKAAEMGLVNESVPLAQLREVTIELARNLLEKNPVVLRAAK



HGFKRCRELTWEQNEDYLYAKLDQSRLLDTEGGREQGMKQFLDDKSIKPGLQAYK



R



SEQ ID NO: 20

Pseudomonas _ syringae _NP_792742 (amino acid sequence)

MSKYEGRWTTVKVEIEQGIAWVILNRPEKRNAMSPTLNREMIDVLETLEQDPEAG



VLVLTGAGEAWTAGMDLKEYFREVDAGPEILQEKIRREASQWQWKLLRMYAKPTI



AMVNGWCFGGGFSPLVACDLAICADEATFGLSEINWGIPPGNLVSKAMADTVGHR



QSLYYIMTGKTFDGKKAAEMGLVNESVPLAQLRQVTIDLALNLLEKNPVVLRAAK



HGFKRCRELTWEQNEDYLYAKLDQSRLLDKEGGREQGMKQFLDDKSIKPGLEAYK



R



SEQ ID NO: 21

Ralstonia _ eutropha _JMP134_YP_299062 (amino acid sequence)

MANYEGRWKTVKVSVEEGIAWVMFNRPEKRNAMSPTLNSEMIQVLEALELDADAR



VVVLTGAGDAWTAGMDLKEYFREVDAGPEILQEKIRRDACQWQWKLLRMYAKPTI



AMVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHR



QALHYIMTGDTFTGQQAAAMGLVNKSVPRSQLREHVLELAGKLLEKNPVVLRAAK



HGFKRSRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKSIKPGLQAYK



RA



SEQ ID NO: 22

Burkholderia _ glumae _BGR1_YP_002908688

(amino acid sequence)

MSYEGRWTTVKVTVEAGIGWVVLNRPEKRNAMSPTLNKEMIDVLETLELDDEAQV



LVLTGEGDAWTAGMDLKEYFREVDAASDVVQERIRRDASRWQWQLLRMYSKPTIA



MVNGWCFGGGESPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRQ



ALYYIMTGDTFTGKQAAQMGLVNQSVPRAALREATVALAAKLLDKNPVVLRNAKH



GFKRSRELTWEQNEDYLYAKLDQANYRDKEGGREKGLKQELDDKSIKPGLQAYKR



SEQ ID NO: 23

Burkholderia _ phytofirmans _PsJN_YP_001887778

(amino acid sequence)

MSYEGRWKTVKVDVAEGIAWVSFNRPEKRNAMSPTLNKEMIEVLEAVELDAEAQV



LVLTGEGDAWTAGMDLKEYFREVDAGPEILQEKIRRDACRWQWQLLRMYSKPTIA



MVNGWCFGGGFSPLVACDLAIAADEATFGLSEINWGIPPGNLVSKAMADTVGHRQ



ALYYIMTGETFTGQEAAQMGLVNKSVPRAELREATRALAGKLLEKNPVVLRAAKH



GFKRCRELTWDQNEDYLYAKLDQAQLRDPEGGREQGLKQFLDDKAIKPGLQTYKR



SEQ ID NO: 24

Burkholderia _ mallei _ATC_23344_YP_105383

(amino acid sequence)

MSYEGRWKTVEVIVDGAIAWVTLNRPDKRNAMSPTLNAEMIDVLEAIELDPEARVL



VLTGEGEAWTAGMDLKEYFREIDAGPEILQEKIRRDASRWQWQLLRMYAKPTIAMV



NGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQALY



YIMTGETFTGAQAAQMGLVNRSVPRAQLRDAVRALAAKLLDKNPVVLRNAKHGFKR



CRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKTIKPGLQAYRR



SEQ ID NO: 25

Burkholderia _ pseudomallei _Pasteur_ZP_01765668

(amino acid sequence)

MSYEGRWKTVEVIVDGAIAWVTLNRPDKRNAMSPTLNAEMIDVLEAVELDPEARV



LVLTGEGEAWTAGMDLKEYFREVDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA



MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQ



ALYYIMTGETFTGAQAAQMGLVNRSVPRAQLRDAVRALAAKLLDKNPVVLRNAKH



GEKRCRELTWEQNEDYLYAKLDQAQLRDPEHGREQGLKQFLDDKTIKPGLQAYRR



SEQ ID NO: 26

Burkholderia _ multivorans _ATCC_17616_YP_001583186

(amino acid sequence)

MSYEGRWKTVKVAVEGGIAWVTLNRPEKRNAMSPTLNAEMIDVLEAIELDPEAQV



LVLTGEGDAWTAGMDLKEYFREVDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA



MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRQ



ALYYIMTGDTFTGQQAAQMGLVNKSVPRAQLRDEVRALAAKLLDKNPVVIRNAKH



GFKRCRELTWEQNEDYLYAKLDQANYRDPEGGREQGLKQFLDEKSIKPGLQAYKR



SEQ ID NO: 27

Burkholderia _ vietnamiensis _G4_YP_001116289

(amino acid sequence)

MGYEGRWKTVKVEVAGGIAWVTLNRPEKRNAMSPTLNTEMIDVLEAIELDADAQV



LVLTGEGDAWTAGMDLKEYFREIDAGPEILQEKIRRDASRWQWQLLRMYAKPTIA



MVNGWCFGGGFSPLVACDLAIAADEAVFGLSEINWGIPPGNLVSKAMADTVGHRE



ALYYIMTGDTFTGQQAARMGLVNKSVPRAQLRDEVRALAAKLLDKNPVVIRNAKH



GFKRCRELTWEQNEDYLYAKLDQANYRDPEGGREQGLKQFLDDKSIKPGLQAYKR



SEQ ID NO: 28

Sphingobium _ japonicum _UT26S_YP_003543683

(amino acid sequence)

MSEYLTEGPDLSRTCVDVMFDEGIAWVTLNRPEKRNAMSPTLNSEMLAILEQLELD



PRCGVVVLTGAGDSFSAGMDLKEYFRETDGLPPAQVRRIRQTAQAWQWRTLQHFGK



PTIAMVNGWCFGGAFTPLVACDLAIAANEAVEGLSEINWGIIPGGNVTKAIQERLR



PQDAALYIMTGRNFTGEKAAQMGLVAEAVPLTDLRDHTRALALELLSKNPVVLNAA



KIALKKVADMTWDVAEDYLVAKGAQTRVADKTDGRNKGITQFLDEKSYKPGLEGYR



RDK



SEQ ID NO: 29

Xanthomonas _ axonopodis _NP_641235 (amino acid sequence)

MNEHDVVSVRIENRIAWVKFARPDKRNAMSPALNRRMMDVLDELEFDDNVGVLV



LGGEGTAWSAGMDLKEYFRETEAQGLRGVRRSQRESYGWERRLRWYQKPTIAMV



NGWCFGGGFGPLFACDLAIAADEAQFGLSEINWGILPGGGVTKVAVELLSMRDA



MWMTLTGEMVDGKKAAEWRLVNESVPLERLEARTREVAELLLRKNPVALKYAKD



AVRRVGTMTYDEAEDYLVRMQEAANSFDNNARKEGIRQFIDEKSYKPGLGEYDL



SKHSA



SEQ ID NO: 30

Xanthomonas _ campestris _ATCC_33913_NP_636201

(amino acid sequence)

MNEHDVVSVHVENRIAWVKFARPDKRNAMSPALNRRMLDVLDELEFDDNVGVLVL



GGEGTAWSAGMDLKEYFRETEAQGLRGVRRSQRESYGWFRRLRWYQKPTIAMVNG



WCFGGGEGPLFACDLAIAADEAQFGLSEINWGILPGGGVTKVAVELLSMRDAMWM



TLTGELVDGRKAAEWRLVNESVPLERLETRTREVAELLLKKNPVALKYAKDAVRR



VGTMTYDEAEDYLVRMQEAANSFDNNARKEGIRQFIDEKRYKPGLGAYEPDAGTN



SEQ ID NO: 31

Azospirillum _sp._B510_YP_003451575 (amino acid sequence)

MTQQQAAARTGTAEDVVTVELDNGVAWVTLNRPDKRNAMNPALNARMHGVLDD



LEVDDRCQVLVLTGAGESFSAGMDLKEYFRETEAKGHMATRRAQRDSYGWWRR



LRWFEKPSIAMVNGWCFGGAFSPLFACDLAVAADEAQFGLSEINWGIIPGGNV



TKVVADLMSQREAMYYILTGETFDGRKAAEMKLVNFSVPHAELRAKVRAIADN



LLEKNPQTLKAAKDAFKRVVEMPFDAAEDYLVVRQESLNYLDKSEGRKQGIKQ



FIDDKTYRPGLGAYKR



SEQ ID NO: 32

Agrobacterium _ vitis _S4_YP_002549228 (amino acid sequence)

MTVAEKSDADTVLVDIEDRIAFVTFNRPEKRNAMNPALNIRMAEVLEELEADDRC



GVLVLRGAGTSWSAGMDLQQYFRDNDDKPRHATLKSRRQSGGWWQRLTYFEKPTI



AMVNGWCFGGAFNPLVACDLAIAANEATFGLSEINWGILPGGNVTRAVAEVMNHR



DSLYYIMTGEPFGGEKARDMGLVNESVPLEELETRVRKLCASLLEKNPVTMKAAK



DTFKRVRNMPWELADDYIYAKLEQMLLLDKTRGRDEGLKQFLDDKTYRPGLGAYK



RK



SEQ ID NO: 33

Rhizobium _ etli _Brasil_5_YP_001985541 (amino acid sequence)

MTENTSPVLVEFDGGIAFVTLNRPEKRNAMNPALNARMLEVLDELEGDERCGVLV



LRGAGQSWSAGMDLKEYFRDNDDKPRDATLKARRQSGGWWGRLMYFEKPTIAMVN



GWCFGGAFTPLVSCDLAIAAEEANFGLSEINWGILPGGNVTRAVAEVMRHRDALY



YIMTGELFGGRKAAEMGLVNEAVPLVDLETRVRKICASLLEKNPVTLKAAKDTYK



RVRNLPWDLADDYIYAKLEQMLFLDKTKGRDEGLKQFLDDKTYQPGLGAYKRGR



SEQ ID NO: 34

Rhizobium _ leguminosarum _bv_trifolii_WSM1325_YP_002973001

(amino acid sequence)

MTEDKSPVLVEFDSGIAFVTLNRPEKRNAMNPALNIRMLEVLDELEGDERCGVL



VLRGAGESWSAGMDLKEYFRDNDDKPRDVTLKARRQSGNWWGRLMYFEKPTIAM



VNGWCFGGAFTPLVSCDLAIAAEEANFGLSEINWGILPGGNVTRAVAEVMRHRD



ALYYIMTGELFGGRKAAEMGLVNEAVPLAELEPRVRKICASLLEKNPVTLKAAK



DTYKRVRNLPWDLADDYIYAKLEQMLFLDKTKGRDEGLKQFLDDKTYQPGLGAY



KRGR



SEQ ID NO: 35

Amino acid sequence for IRX5

(GenBank Accession No. AF458083_1)

MEPNTMASFDDEHRHSSFSAKICKVCGDEVKDDDNGQTFVACHVCVYPVCKPCYE



YERSNGNKCCPQCNTLYKRHKGSPKIAGDEENNGPDDSDDELNIKYRQDGSSIHQ



NFAYGSENGDYNSKQQCRPNGRAFSSTGSVLGKDFEAERDGYTDAEWKERVDKWK



ARQEKRGLVTKGEQTNEDKEDDEEEELLDAEARQPLWRKVPISSSKISPYRIVIV



LRLVILVFFFRFRILTPAKDAYPLWLISVICEIWFALSWILDQFPKWFPINRETY



LDRLSMRFERDGEKNKLAPVDVFVSTVDPLKEPPIITANTILSILAVDYPVNKVS



CYVSDDGASMLLFDTLSETSEFARRWVPFCKKYNVEPRAPEFYFSEKIDYLKDKV



QTTFVKDRRAMKREYEEFKVRINALVAKAQKKPEEGWVMQDGTPWPGNNTRDHPG



MIQVYLGKEGAFDIDGNELPRLVYVSREKRPGYAHHKKAGAMNAMVRVSAVLTNA



PFMLNLDCDHYINNSKAIRESMCFLMDPQLGKKLCYVQFPQRFDGIDHNDRYANR



NIVFFDINMRGLDGIQGPVYVGTGCVFNRPALYGYEPPVSEKRKKMTCDCWPSWI



CCCCGGGNRNHHKSKSSDSSSKKKSGIKSLLSKLKKKNKKKSDDKTMSSYSRKRS



ATEAIFDLEDIEEGLEGYDELEKSSLMSQKNFEKRFGMSPVFIASTLMENGGLPE



ATNTSSLIKEAIHVISCGYEEKTEWGKEIGWIYGSVTEDILTGFRMHCRGWKSVY



CMPKRPAFKGSAPINLSDRLHQVLRWALGSVEIFFSRHCPLWYAWGGKLKILERL



AYINTIVYPFTSIPLLAYCTIPAVCLLTGKFIIPTINNFASIWFLALFLSIIATA



ILELRWSGVSINDLWRNEQFWVIGGVSAHLFAVFQGLLKVLFGVDTNFTVTSKGA



SDEADEFGDLYLFKWTTLLIPPTTLIILNMVGVVAGVSDAINNGYGSWGPLFGKL



FFAFWVIVHLYPFLKGLMGRQNRTPTIVVLWSILLASIF



SLVWVRIDPFLPKQTGPLLKQCGVDC



SEQ ID NO: 36

Polynucleotide sequence PATCESA7_PATIRX3

TGGGAACTTTCGGTACATTTTCCAATAAAATCTATATACTATAAGATATTAAAT



ATACACAAATATATCTAAGTGAATCATACAAATTATGTAGGCACACAGGAAGAG



GCTGCTGAGGCTTATGACATTGCAGCCATTAAATTCAGAGGATTAAGCGCAGTG



ACTAACTTCGACATGAACAGATACAATGTTAAAGCAATCCTCGAGAGCCCGAGT



CTACCTATTGGTAGTTCTGCGAAACGTCTCAAGGACGTTAATAATCCGGTTCCA



GCTATGATGATTAGTAATAACGTTTCAGAGAGTGCAAATAATGTTAGCGGTTGG



CAAAACACTGCGTTTCAGCATCATCAGGGAATGGATTTGAGCTTATTGCAGCAA



CAGCAGGAGAGGTACGTTGGTTATTACAATGGAGGAAACTTGTCTACCGAGAGT



ACTAGGGTTTGTTTCAAACAAGAGGAGGAACAACAACACTTCTTGAGAAACTCG



CCGAGTCACATGACTAATGTTGATCATCATAGCTCGACCTCTGATGATTCTGTT



ACCGTTTGTGGAAATGTTGTTAGTTATGGTGGTTATCAAGGATTCGCAATCCCT



GTTGGAACATCGGTTAATTACGATCCCTTTACTGCTGCTGAGATTGCTTACAAC



GCAAGAAATCATTATTACTATGCTCAGCATCAGCAACAACAGCAGATTCAGCAG



TCGCCGGGAGGAGATTTTCCGGTGGCGATTTCGAATAACCATAGCTCTAACATG



TACTTTCACGGGGAAGGTGGTGGAGAAGGGGCTCCAACGTTTTCAGTTTGGAAC



GACACTTAGAAAAATAAGTAAAAGATCTTTTAGTTGTTTGCTTTGTATGTTGCG



AACAGTTTGATTCTGTTTTTCTTTTTCCTTTTTTTGGGTAATTTTCTTATAACT



TTTTTCATAGTTTCGATTATTTGGATAAAATTTTCAGATTGAGGATCATTTTAT



TTATTTATTAGTGTAGTCDTAATTTAGTTGTATAACTATAAAATTGTTGTTTGT



TTCCGAATCATAAGTTTTTTTTTTTTTTGGTTTTGTATTGATAGGTGCAAGAGA



CTCAAAATTCTGGTTTCGATGTTAACAGAATTCAAGTAGCTGCCCACTTGATTC



GATTTGTTTTGTATTTGGAAACAACCATGGCTGGTCAAGGCCCAGCCCGTTGTG



CTTCTGAACCTGCCTAGTCCCATGGACTAGATCTTTATCCGCAGACTCCAAAAG



AAAAAGGATTGGCGCAGAGGAATTGTCATGGAAACAGAATGAACAAGAAAGGGT



GAAGAAGATCAAAGGCATATATGATCTTTACATTCTCTTTAGCTTATGTATGCA



GAAAATTCACCTAATTAAGGACAGGGAACGTAACTTGGCTTGCACTCCTCTCAC



CAAACCTTACCCCCTAACTAATTTTAATTCAAAATTACTAGTATTTTGGCCGAT



CACTTTATATAATAAGATACCAGATTTATTATATTTACGAATTATCAGCATGCA



TATACTGTATATAGTTTTTTTTTTGTTAAAGGGTAAAATAATAGGATCCTTTTG



AATAAAATGAACATATATAATTAGTATAATGAAAACAGAAGGAAATGAGATTAG



GACAGTAAGTAAAATGAGAGAGACCTGCAAAGGATAAAAAAGAGAAGCTTAAGG



AAACCGCGACGATGAAAGAAAGACATGTCATCAGCTGATGGATGTGAGTGATGA



GTTTGTTGCAGTTGTGTAGAAATTTTTACTAAAACAGTTGTTTTTACAAAAAAG



AAATAATATAAAACGAAAGCTTAGCTTGAAGGCAATGGAGACTCTACAACAAAC



TATGTACCATACAGAGAGAGAAACTAAAAGCTTTTCACACATAAAAACCAAACT



TATTCGTCTCTCATTGATCACCGTTTTGTTCTCTCAAGATCGCTGCTAATCTCC



GGCCGTCCCT



SEQ ID NO: 37

Polynucleotide sequence PATCESA8_PATIRX1

TTTAGTGCAGTCTAGGAAGACGGATCCAAAGGAGATAAACAGAGTTCAAGAAGCT



CTTAACTACTATACAATCGAATCGTCAGCCGCGCTTTTTGTTTCGTTCATGATCA



ATTTGTTTGTAACTGCGGTTTTCGCGAAAGGGTTTTATGGAACCAAACAAGCTGA



TAGTATAGGACTGGTTAACGCGGGATATTACCTACAAGAGAAATATGGCGGTGGT



GTTTTCCCGATACTATACATTTGGGGGATTGGTTTATTAGCTGCTGGACAAAGCA



GTACTATAACCGGGACTTATGCTGGACAGTTTATAATGGAAGGGTTCTTAGATCT



TCAAATGGAACAATGGCTATCAGCTTTTATAACGAGAAGCTTTGCTATTGTACCT



ACTATGTTTGTTGCTATTATGTTTAACACATCCGAGGGCTCGCTCGATGTTTTAA



ACGAATGGCTTAACATTCTTCAGTCGATGCAGATTCCTTTCGCGGTTATTCCTCT



TTTGACTATGGTTTCTAATGAACATATCATGGGTGTCTTCAAGATCGGACCTTCG



CTTGAGGTAAAGCAATTTTTTGTCATCTCTCTTTATTGTTATGTGCTTTTGATTG



TAACGAGTTAGTTGGGATCTTTGCAGAAGCTAGCTTGGACTGTGGCGGTGTTTGT



GATGATGATAAATGGGTATCTTCTTCTAGATTTCTTCATGGCTGAAGTGGAAGGG



TTTCTTGTTGGGTTTCTGGTTTTTGGTGGAGTAGTTGGATACATCAGTTTCATCA



TCTATCTTGTTTCTTATAGAAGCTCACAATCTTCTTCCTGGTCGAGTTTAGAAAT



GTCAGAGAGAGTTGTTTCCACAGAGACGTAGAAACCCATAACTTTAGTATTCTTC



AACCCTTACAACTTATCTGAGCAAAATCAGAAGGTCGAATTTGATGGATGGTTTT



GCTGTATTTGGTCAACGGTTTTATTTGAGACAGTAGACCAGAGGAAACTCAGATG



TGATGATGCAAAGACTGAATTGGTTAAGAGTGTAGATTGATTTGTTCTAACATTG



CAAATGTAGAGTAGAATTATGCAAAAAACGTTAATGAACAGAGAAGTGATTAAGC



AGAAACAAAATTAGAGAAGTGATATTATATCTCAAAATTTATTTTTGGTACAGCT



AAAGCTCAAATTGTTATAGAGATTAGAGATATTAAACCAAATGACGAGTGTTTTC



TTTAGTAGTAAACGGTGAAAATTCTCTTCTGACAAAGACAATTAAAATTTTAGGT



TTAAGACTTTAATATTTGTCACAAATTGTCATTTACCTAAATAAAAAAAAAACTA



AATATTTTTTTTAGATACATATGTGTCTTATAATTTTAACTATAAATTTTAATTT



TATGTCTTAAATAATTGTTTACACTATAAATTTAAATATTTTAATGCTAAAATTA



ATTTGATTCAAAAAAGTGATTTTAATTCTTATTTTTCTTATAGAAAGTTGGTGAT



TGAAAAGATTTACTTAAAAATTATAACAACTTCAATGGTGAATAACCCGACCCGA



ATAAACCGGATATAACAACTTCAATGTTAGCTTGATATAGAAAGTACGGTGACGC



TTAGGAGGCAAGCAAGCTAGTATCTGCCGCTGGTTAGAGACAAAGAACATGTGTC



ACTCCTCTCAACTAAAACTTTCCTTCACTTTCCCGCAAAATCATTTCAAAAAAGC



TCCAAATTTAGCTTACCCATCAGCTTTCTCAGAAAACCAGTGAAAGAAACTTCTC



AACTTCCGATTTTTCACAATCCACCAAACTTTTTTTAATAACTTTTTTTCCTCTT



ATTACAAAACCTCCACTCTCATGGCTTCTCAAACTTGTTATCCATCCAAATCTCA



ATCCCTAATTAGGGTTCATTTCTCTGTTTCTCCAAACAGGGGAATTCGAAG



SEQ ID NO: 38

Polynucleotide sequence PATNST1

GTTTGTAGAGTTGGATCAGCATCCAGATTTAAACCCTTATTTTTGTTTTTGCCAA



GCATCCAGACTTAATCCTATATTAGATACTGTATATGCATCTTGATGGAATATAG



ACTATATAGAAAGACCAAAAATGGAAGAGTACGAATAAAAATGCATAATATACCT



TGGAAATTATTCTTGGTTATTGTGAAACTTAAAACATTTCAACGAAGTCATATAC



TATTATTTAATCATTGATTTAAAATTGCTAATCAAATCACGTGTTGTTGTTATAT



ATGGATAAAGAGTTAAACTATAACACAACTGAGAAAAAAATAAAGTTATCAATTT



TGTTAAGAATCAATGAAGGTTTCACAAGACTGGGAAGAAAAAAAAATAGATATAT



GGAGTACATAAAACATTAAAATTTTGCTAAATTTTACTTTTGAACTCTATTGATT



CGGGTTGACATGATGATAATGTTACATTCGTACAATTTCACAATGAAAAAAACGA



GTACTAAATATTGTCAATCAAACATATGAATGTACAAAAATCCATAAACTCTACC



AAAATAGAATGAAGATTCTGAAATCAAACCTACTTTTTCTTTTTAATTATAAATT



CAACTATATTATAAATTTATTTATCACAAATAATAGAGGAGTGAGAATATTTTAG



ACAACGCAAATTTCTTTTATTTAGTTCTTATACTTTATTTTTTACCAAACGTTAA



TTAAAAAAATCACACATACATAATTTCTAAAAAAAATGTATTCTTCAAGTAATAT



ATCTTTCTGAGTACTAGTTTATCTATTTATCTCCGTATTTAATAATCAAAAGTTA



CGTTTAAAATAGAAACAACTTTTATCAAACAAAATATATTAGAAAACGCATGGTA



CTGGCTACTGGAAAGAATCATGACCTGTAAATTTCTACAGTTTTCCCGTTTTATA



TAGTACTTAGAAACTTTGGATTTTCATAGCGCAACCAATAAACACATGGACTTAA



GACACAAAAAAAGTTGGGTGCAATGTCATTAATCAAACTAAAAAAATAATGATTA



AAAGCATGGAATTCCGAAAACGCAACAAAATGATTCTGTGTTTAGACAAATGCAG



AAAGGCCTCTTAACTAATCTTAAATAAAGTCTTAGTTCCAACCACATAAACACTC



CTTAGCTCCATTAATTTTGGTTTTCTTAATTACGTTTCTACACAAGTACACGTAC



TTACACATACAATTCCACAGTCTAAATGATAAAACTATGTGGTTTTTGACGTCAT



CGTTACCTTTCTGTCGTCTCACCTTTATATAGTGTCTCTAACAGAACGTAACAAC



CAAATGTTTAAAAAAATAAAAACAGCACCCCTTAATTAGGCTCATTCGTTTTGCA



CTAACCATACTACAAATCATCTCGAACGATCGAGCAAAGATTTGAAAAATAAATA



AACGTATAACTCTAGAGATTTTCATTAGCTAAGAAAAGTGAAATCGATTGTTAAT



CCTATTTCAGACGGGACAGGAACACTCATTACCCAACTCTATCATCTCTCGAACA



CCAAACTATATCTACCGTTTGGGGCATTATTTCCCACTTTCTTTCGAAGACAATT



TCCCATATATAACATATACACATTATTACTAATATATTTTTATAAATTTTCGTCA



CATCCCAAAAAAAAACACTCTTTGTCACATCAACTAGTTTTTTTGTAACGATCAA



ACCTTTTCGTTTAAAAAAAAAAAACTTTTGTAGTGTAAACGTTTATTTATCGATG



AAAAAAGCCACATCTTCCGGAGGGAAACTTTTTAAGACACCCTATTTCGACTTTA



TTTTGTAAATACAGTGTGCATGTGCATATAAAGAGAGATATCATTTGTATAAATA



TCAAGAATTAGAAGAGAAAAAGAGAGAAGAAGACAATCTATTACTATTACGATGT



GTGGGTTGTTAATTTGTTTAAAGGGAGCTTTTCTATAGAGATTTTTAAGGTCAAG



GGTCATCGTTCGATGTGGGCTTGCTTCCTACAATCTAGTTGCCTTACGGGGCCTA



CTCTTTTTCTTTTGATAACTACATCACCTTTTTTTTCTCCGACAACTATATATCA



CTTTTTTTATGTTTTCCTTTTTTTCTTCACAATAATTCTTTACTCGTTGCAAATG



TAAAGATACACAAAGTTACTTATTTTGTTTACGATGGTTCTTAGTAGTTTAAAGA



ATTAATGAATAAGATAAACCTAAACTTTGAAAAGACTAAAAAAAATGTATAACAA



CATACATTATACGTATTTGAAATAGTCCAAGTGATATTATGTCATTGATATTAGC



ACAAATAATTACGATGCCTGATATTGTCACATTTGATGATTTTAAGTTCTTGTAA



AAGATAAGTGTAACTAAATCACTATAGTGAGGCCCACGTTTTAATTTCTAAACTA



ATTACAATGACAATAAAATAGCAAAACTATTTAAAACTAGACGCCAAAAAAAATT



GAAACTAATAATTGTGAAAAAAGAACAAGAGAATAATAATCATTAATAATTGACA



AGTGAAATTAATATATTGCTCTTGGAGGGTTATATTTTAATTTTCAAACTAAATA



ATGAATACAAATGGAAAAGCTAATGATAAGAGTTGAATTTTAATAATTAAGAAAA



ACAAAAAAAGGTGTACAAGGAGACACATGCGTTTTCCTCATGCATCTTGTTTTTA



TACAACAATATATATATATATATTGAGTCATTCTCTGCTAGCTCTCTCATCTCCA



ACTTTCAGTATGATATATAGTTACAATTAAATAAACCTCACATGCTCTATTCTTG



CTTGATTTTTGAGTTAATCTTGAATCTCTTTG





SEQ ID NO: 39

Polynucleotide sequence PATCESA4_PATIRX5

ATGAAGCCATCCTCTACCTCGGAAAAACTTGTTGCGAGAAGAAGACATGCGATG



GCATGGATGCTTGGATCTTTGACATTGATGACACTCTTCTCTCAACCATTCCTT



ACCACAAGAGCAACGGTTGTTTCGGGTAAATAAACTAAACTTAACCATATACAT



TAGCCTTGATTCGGTTTTTGGTTTGATTTATGGATATTAAAGATCCGAATTATA



TTTGAACAAAAAAAAATGATTATGTCACATAAAAAAAAATTGGCTTGAATTTTG



GTTTAGATGGGTTTAAATGTCTACCTCTAATCATTTCATTTGTTTTCTGGTTAG



CTTTAATTCGGTTTAGAATGAAACCGGGATTGACATGTTACATTGATTTGAAAC



AGTGGTGAGCAACTGAACACGACCAAGTTCGAGGAATGGCAAAATTCGGGCAAG



GCACCAGCGGTTCCACACATGGTGAAGTTGTACCATGAGATCAGAGAGAGAGGT



TTCAAGATCTTTTTGATCTCTTCTCGTAAAGAGTATCTCAGATCTGCCACCGTC



GAAAATCTTATTGAAGCCGGTTACCACAGCTGGTCTAACCTCCTTCTGAGGTTC



GAATCATATTTAATAACCGCATTAAACCGAAATTTAAATTCTAATTTCACCAAA



TCAAAAAGTAAAACTAGAACACTTCAGATAAATTTTGTCGTTCTGTTGACTTCA



TTTATTCTCTAAACACAAAGAACTATAGACCATAATCGAAATAAAAACCCTAAA



AACCAAATTTATCTATTTAAAACAAACATTAGCTATTTGAGTTTCTTTTAGGTA



AGTTATTTAAGGTTTTGGAGACTTTAAGATGTTTTCAGCATTTATGGTTGTGTC



ATTAATTTGTTTAGTTTAGTAAAGAAAGAAAAGATAGTAATTAAAGAGTTGGTT



GTGAAATCATATTTAAAACATTAATAGGTATTTATGTCTAATTTGGGGACAAAA



TAGTGGAATTCTTTATCATATCTAGCTAGTTCTTATCGAGTTTGAACTCGGGTT



ATGATTATGTTACATGCATTGGTCCATATAAATCTATGAGCAATCAATATAATT



CGAGCATTTTGGTATAACATAATGAGCCAAGTATAACAAAAGTATCAAACCTAT



GCAGGGGAGAAGATGATGAAAAGAAGAGTGTGAGCCAATACAAAGCAGATTTGA



GGACATGGCTTACAAGTCTTGGGTACAGAGTTTGGGGAGTGATGGGTGCACAAT



GGAACAGCTTCTCTGGTTGTCCAGTTCCCAAGAGAACCTTCAAGCTCCCTAACT



CCATCTACTATGTCGCCTGATTAAATCTTATTTACTAACAAAACAATAAGATCA



GAGTTTCATTCTGATTCTTGAGTCTTTTTTTTTCTCTCTCCCTCTTTTCATTTC



TGGTTTATATAACCAATTCAAATGCTTATGATCCATGCATGAACCATGATCATC



TTTGTGTTTTTTTTTCCTTCTGTATTACCATTTTGGGCCTTTGTGAAATTGATT



TTGGGCTTTTGTTATATAATCTCCTCTTTCTCTTTCTCTACCTGATTGGATTCA



ATATTAAGTAAGACTAAAGTAGAAATACATAATAACTTGAAAGCTACTCTAAGT



TAGAACATAGCCAGATTTGGTAAAGTTTATAAGATACAAAATACAAATTCTAAA



GAACTCAAAAGAATAACAAACAGTAGAAGTTGGAAGCTCAAGCAATTAAATTAT



ATAAAAACACTAACTACACTGAGCTGTCTCCTTCTTCCACCAAATCTTGTTGCT



GTCTCTTGAAGCTTTCTTATGACACAAACCTTAGACCCAATTTCACTCACAGTT



TGGTACAACCTCAGTTTTCTTCACAACAAATTCAAACATCTTACCCTTATATTA



CCTCTTTATCTCTTCAATCATCAAAACACATAGTCACATACATTTCTCTACCCC



ACCTTCTGCTCTGCTTCCGAGAGCTCAGTGTACCTCGCCT



SEQ ID NO: 40

Polynucleotide sequence PATGAUT8_PATIRX8

ACGAGCTGACTTGTACCGATGAGCTGGCTCTTCTGGGCGAGCTGGCTGATCTTGA



CGAGCAGACTTCTCCCGACGAGCTGACTTGTGTCGATGAGCTGGCTCTTCTGGGC



GAGTTGGCTGATCTTGACGAGCAGACTTCTCCCGACGAGCTGACTTGTGTCGATG



AGCTGGCTCTTCTGGGCGAACTGGCTGATCTTGACGAGCAGACTTCTCCCGACGA



GCTGACTTGTGCTATCCTTTCTCCAGGTCTCGAAAAAGTCCCCTTTCCCGAGACT



TTCTATTCCTTATTTATACCCGTCCGTATAGTAGGGTACGCAAGGTGAATTCTCG



AGAGTGCCCCTTTTCTACGCAGCCGAACTCACATCCTGACCAGGCCGGGCTTCGG



CCTGGTGGGCCGGCTCGAGTTCTAAAGTGATGGTCGGGGCTGGGTCGTTATTCCT



TGAAATGGGCCGGTTGATCACTGAGGCCCAATTGATGTATCAACATGTGGTTTTT



ATAAAAAGAGTCGTGAGAAGAGTTTTCTCTAAAAATCCCTTGTGTTTGGTAATCA



AACTTCATTCAACCAACGAATTCCAAAAAAACAACTAAATTGTTCGGGTATATAA



AATGATTGGTAATGATATATCCCATAGAGGCCGTAGACATAGGCCCAAAAAGTTT



CCATAACTAGCAGAAATTGAAACTTGCAAGTTGCAAATATTATTACACTGGAAAG



GCAACAAGTCTTGAAGTACAAACTACAAAGACTTCTTGTTTGGATGGGGACGACT



GACGAGTTTGAATAACTTAAGAGAAAAGGGTCGCAATCGAAATTAGACAAGAAAT



TAGTCCTCAAAAAGTAAATTCTGAAGTTGAAGCTCCAATGTCTTTGTTCAAAGAC



TTTATTTAGATGTAAAGTTATGTCTTGTAACCACCAAACAGCTCCTTTTCATCTA



CACTCCCAATTTTTTTAACATCTATGTTTTGCATTGCCTTTGACTTGTCTTTCTC



TCTCCAACTTCTCTCCTTCAACATAAAGCCAAATCCTAAATCCAAATCCCTTAAA



CCGAACCGAATTAAACCGAAGCTGTTGAACTATCGCAAAATTTCAGATCTTACTA



ATCATAAACATGTGACGTTTAATTCATTTTAAGAGTTTCATGATTTGCACTGAAT



GGTATTCCGAGTCCACCGGAAAAAAACTTTTCCTACAAGTAGAAAAAGGATAACC



CCATAAATCCAAATAACCTAACCGATCAAACATATACCAATATAAACCAAAACAA



GATTCAGATTCATCGGTTTAGTAATCGAAGTAATGTACTAATGTGTAATATTGAT



TCCACCACCAGCTTAGAGATTCGAACCAAAAACCGAATAGCGCATAACCGAGAAA



ACCCAAAGCTTCCTAACAAATACATAAAACCGTGGTGTTTCTAATTCTAACCAAC



ACACGTTTCCTTTTTATTCACAAGAAACATCAGAGTTATGATCTGCCATTAATAA



CCTAAACACAAAGCAAGGTTAGGTAAATGATATGGACCCCTAATGAATAATCATA



CAATACATAACAACGTAAGATCCAGTTTCCCTCTTCG



SEQ ID NO: 41

Polynucleotide sequence PATNST2

AACGGTGGCGTGATGGAGCTTCATCCTCCCATCTTCGCCGAATTCATCACCAACG



AATTTCCCGGCCATGTCATCCACGACTCTTTAAGCCTCCGCCACTCATCTCCACC



GCTTCTCCACGGCGAAGAACTCTTTCCCGGTAACATCTACTACCTCCTTCCTCTT



TCTTCTTCCGCAGCCGCGACCGCTCAACTGGATTCCTCCGACCAACTATCAACGC



CGTACAGAATGTCTTTCGGGAAGACGCCGATAATGGCGGCTTTGAGTGGCGGTGG



TTGTGGAGTGTGGAAGGTGAGGCTTGTGATAAGTCCGGAGCAGTTGGCGGAAATT



CTTGCGGAGGATGTGGAAACGGAAGCGTTGGTGGAAAGTGTGAGGACGGTGGCGA



AGTGTGGCGGTTACGGCTGCGGCGGAGGAGTTCATTCGAGAGCGAATTCAGACCA



GCTAAGCGTTACGAGTAGCTTTAAAGGGAAATTGTGGTAAAATTTCGAATTATGA



ATAAACTACGTTTATGTTTTAATCTGTTTCACGATTTAAGCATTTAAATTAGTAT



GTTGATTTCCGTATTCATTGAAGACTTGGAACGATTATATAAGTTTATCAACGTA



GATATATTTGAAATATCATTGTTATCTCTCATGAAACAATTAATTTATGAAGTCG



TAGACTCGTAGTTAGAGATTATTTAATCTTCCCTATTCAATGCCAAAAGTCTAGA



AGAGCAAAACAAAAGGGAGAAACTCTTTTATTTCAGGCCCAATGACACAAAGCTG



GCCAGAAACAGTTTAAGATTAGGCTAAAGTTATAAGTCCGACAAGCACGAGTGCT



AATATATATAGTTATATGACGTCTCACCATTAAGGGTTTAATAAATTTTGAAACA



CCTCAAATTAAGATTGCTTCCCATGCAAACTTCCTTCATCTTCTAGAAAAATTAC



GATTTGTAATACTTCAATTATATCATTTTAGTTTTTTGTCACTAATTATCATCAA



TTTATCATAGCTCCGTGCCGCAACAACGTTCGTTTTAATCAGATTATATATTACT



CTGCTATAAACTCAGAACCATGTTAGAAAAATGAAAAAGACATTTCAGAATATTC



ATTAACTCAAAATTTTAATCTCATGATTTAATTTTTTATTAACAATGTTATCCTA



TAGCACATGGCAAATTTGAACGGCCCTTGCGTATTAATCTATTATAATCTCAAAA



CCATGTGTAAGAAAAAGGAAATTCAGAAAATAACCTTTTGTAAATAGGCCCCCAC



AAAATCTACAACATACGTAGATACCTCCTCGCTTACAGTTGTAAACAACTGTTCA



TCTAGATTCATGCCGTCATTCAAGTTTAAATTAATACAATAATTTAAAATTTTAA



TTTGGATGAATCGAATCCACCGTCGTTTCCTGAATACCAGATAGGTTAACTTTAT



GATTAGTTCGAGTGAACCACATGCACAATATTCGAATCTTAGACATTCGTTGCAA



TGTTAACTTCACATATATTTGATAAACGCTTCTTGAATCAGATCTTAATCTCTTT



CTTTCTCTCCATCTTCTAAGGAGGTTGTGGATTATCATGTAGTATATCATTATCT



TCGCATCACCTTCAACAAGAACAAGCTACGAGCTTTAAAGTCGTATTTAACACAA



TAATGTATAAAGTCTTTCTTCATCACATCACATACATTTTTTGTTGCCATCACCC



TTCATTCACTTTTTTTGTTAACACTATTCGTTTCTATATAAAATAAAAATAAAAT



GAGGAATGTCTTGTCCATAGAGATTTTTAAGGTCGAGGGTCATCGGAGCGATGTG



GGCTTGCTTCCTACATTATAGTTGATATGTGGATCCCGCGTGGACCATATTTTTA



CCCAATAGCTACGTGCATGGTCCCACCGCTCTCTCTCACGCACTATTCCGAAATT



GCCATAAACAATTTCACCGGACAAAAAGAGCAAATAATTTCGATGTTTAATAAAG



AGACCATTAGTATATTTGACCCAAAAAAAAATAAAAAAAAAAGAGAGACATTACT



ATAACTTTTATTAGATGAAATATTGCAACATTGTATTTATAACGGATCTAATTTA



CTGAATCATATTTTTTTTCTTTGTTAAAGAGATACTGAATCATGCAGAAAAATAG



ATAGATTTTTAAATACTAGGTGAACTCATGACGAATCAACCATTACGAGAGATTT



CTGGATAAAAGCAAAAACAAAACAAAACTAACATGCTAATCTAGGCAATTAGTAG



AGCGAAAAGTCGGCAAAACCAAAGGCCGAAGAAGCTTGATCGATATACTTTTTTT



TTTTTGTTTTGGCTGGATATACTTGGTATGAACTAAGAATTAAGTAAAAACTCAT



AGGGAGTAATTTTTCGAGAAGTGCATTCACTATGAGTATAAAACAGACATTTTCA



AATTATTAAAACAAGCTCTTAGAGGCTCATATGTTTAATTGTAAGTGGCGGCTCA



TGCGAACTTATAATGAAAACATCAAATATTCGGAAAAATAATACTCCACTGTTAA



AAAGAAAACTTAACAAAGGAATTAAAAATATGAGAGCAAAAGAACACATGCATTT



TCTCATGCATGTACTATTATTTATTTTTTTGCAGAGTTGATGTAAAAAATATACA



CATATATATAGACATACTTTGGTTAGTTATAAACTCGTTCTATTTTCTTCTCCTT



TTTCTATCTTTAGCA



SEQ ID NO: 42

Polynucleotide sequence PATNST3

ATTCTACACATTCACAAAGTTTACTACACTATATATAATTTACCCAACAAACACT



TATTTTACTGCATTATTCAGTATATTATCTTACCTATAAATGTGTATCATCATCA



TCAATAACGCGATTATTTGTGCTGAAGGATTATATATTCAAAATGATCTAGTTAT



ATATGTCACATGATTGCCGTTAACAAGACACATTTGAAGAAGCTAAGCAAGAAAA



ACGGACACTITTGCGACTTGTTACATAATTTAACTTATAGGTCAAAAGAATTTGA



TTAGTCATTGCAACTACGTGTGGATGTCACTTTCTATTCAACCAAAACTCACAAT



ATTATATGATCTAGTTTTGTCGTATTACTGATTTGTATTATAAAATGTTATTTAA



TTTGAATTCTACGTAGATATTGCTCATGCATGATAGTATGTATCTAAACTATTCA



AATAACTAACTACGTGGATATTTTATAATCCAAGTAAAAAGCAGAAAGTGGGTAA



CTACGTCAGTATGACTATACTTTTATCGGAATTGCTTGACATCCAAACTTTTGCT



ATGCTTCACCAACCAATGCAGTTTCACTTAATTATTAACTATTGACTATGTCTTA



TTAAGTTAGCACTAATTCGTTAATCATTCAAAACGTTATTTGATTGAATTACATA



TTACACTCTCTTTCTGCATCACCACTCACACCATATGCAACTATAACCAACTCAT



ATTCAAATGTATTAATTGGATTTTGGTGCGAGATTAAAAATTGAAAGGAAACAAC



ACAATATGATAATGGGATAAAATCTTGAACGGAAACTCAAACTAATCCTCATAAG



GTATAACAAAATAACAATTTAAGCTAAGCACAACAACATACAAGTTCGACCTTTT



CCTTTGATGATCCAGCCCAACAGTTCTCTTATATCTCAAACCATTCGACCATTTG



AGCCAAACTAGCTAAACCTGCAGGAATCAAAACCAACAAAGATTCAGATTAGCTA



AACCGGTTTCATCCCTTTGTCACATGACTCACATCCGTCTTCTACATAACGATTT



CTAATGATGTGAGCTCTTAACTTGCTCCAGCAAGATCATCAACTTTGGAGCACCT



TCAATGATTTAGTTAACATGTTAGATAAATTAAATATTCTTGTTTCAATATATAT



CAACTTTAGTGTAAAAGCCTTAACATTCTCTTGAATATTTAATTTATTTCTCCTT



ATTTCGATTTAATGACAAATGTGAATTAATTTTTGTGATATTTTTGTTCGAAATT



AGTTTTCAGTTAATAACATACATGTGAGCATGGGACACACATGATTTAACAAAAG



GGAATGACGAAATGATATATCAAAATATTAGTATGGGAACAAATTACGAGGTGAA



ACTTCACACTCAACTCAATTAAAACTAGAATAAAGAAATGGAAAAAGTGAAAGAA



TGAGAGGTCAAATGTGGTTAATCATTATGTGGTATTAGTTAATCCATCAATTGTG



TACCCAAAAGCATGATTAAGCATAGAATTTAGAGAAACAAAACATCATTATTAAT



GTTGAAACACAAAGATCCCATCAACAGACAAATGATAAGTACAGTGCATGTAGGG



TAACAACTTTTATGTACATGTTATATACTTATATTATATAATAAGAAAACGATTA



AAGTGTCATTGCTCCAGCCTCTATTTGTAAATCATATTATATCAGTATGCTTAAT



TCCAATAATTAAGTCCATAACTAAAATATATACACATATATGTATGTTAAATGGT



TGAATATATACATATATTTTCATAAACAAATATTGCTAATTAATTCAGTTATTTG



TGTACATAATCCAACTATCACCTTTTTAGCTGGAAGTGGATATTCCAACATGTCA



GTCTGTCACTCCCACATTCATACTCTCTATTCTTTTTAGCTATTTCAATATCTAC



GGTTAAATATTAATGGCTATATAGCCTTACCCTTCATTTTAGTTTTTTTTTGGTA



TTCGCATAACCATCGAATACTCAAACTTACTATGTAAGATGGTCTGAATAACTAT



TTCCGATTTAAGATGAATAGCTAGATTGAAATATACATGCACTAATTGGACATGC



ACTAAAGGCAGAGGTGAATTAAATGATGAAATGAAGATGAAGTGTCACACTTGTG



CAAAAAGCATGTCCCCTGCTCTTCTCCGCTTGTTTCAATTTCTTTGACTTTCATC



ACGTTTTTGTCACTTAAATACACCAAAAAATATAGTACAATTAAACATCGAAAAT



CGTCCAAAAAGAAGAAAAAAAATCATGGAAAGTTCTTTCGTTAATGTTACACACA



TTATCTTGATTAGGTGACACCAGATATTAGAATAAAAATGATAGATTATGAAAAG



AAAAAAAAAATTGATGTATTTTTAGGATACATCGAAAGGAATGAACATACCAAAA



ACATGGGAAAAAATAGATAACTAATTAACATGGTAGAATGTAGATGACGTAGATC



ATGAAACGAGTGTGTGATATATTAATGAAAATTATTTTAATATACGTAGCTATAT



TAGAAAATAATTTACATTTATTTTCTTCTAAACAAATCTATACTTTATATTTACA



TACATTAGTAAAGACCAAAACACATGGAATTCAAATTCTGCAATAAGTAATTGCA



AGAAAACACAAAGATTAATCCCCCACTAAACCCGTTTATTTACGTTAGTATTTTT



CCGTTTTATACATTACACATGACATGACATTACACGTCAAAAGAAATATGTCTTA



CGTCAGAACTTACGTATGATCAAACTCGATTTAAACATAGAAACATCTGTTTACT



AAATTATACTAATTTCATAAAGACACTTTAATGCATGAACTTCTTTGTTTAAATA



ACAATTTCCCCCTTTTGGGGGCTATGTCTCGTCGAGTCCTACCACCATTATAAAT



TATCTCATCGTTTGCTTTCTTTTTTTTAAGTTGTAACCATTTCCACTCGTAATCA



TACAACTTCTCTACTCTTCTAGAGCAAAAACCCAAAAATATATTGCTATCTTCGT



TA



SEQ ID NO: 43

Polynucleotide sequence PATFRA8_PATIRX7

CTTCAAATCTCTTGTATCATTAAATAGTAACGTTTTAAATATTTTCTGGATAAGCA



TAAGTTTCTTTGAAAACTATTTTGTATATATTCCTACTTCTCCATTTTTCTAAATT



ATTTTATATTATACATAGTTTTCCAAATTATCAAACATTTTTACATGTTTTGACTA



ATAAATAAACATATTACTGCGAATTAATTAAAAATAAATATTCCACACAATAATTA



CCTTACAAGCGAATAAACTTTTACTATGTTTTCGATGTAAATTTTTCTTACATATT



TGTAACTGAAATTTCTAACTTGTTGTTTCATAAGTTTTAAAATTTATTATCTAATT



ATCTACTTTTATGTGTTCTAGAGCAAAGTGCTAAATGTATATATACTTAGATGTTG



TGTTGTAATCCAATGTCAATATAATCAATGATTTAGCTATTTGTAAACATACTAAA



TAGTATTCCACCAAAAAAAAAACATACTAAACAGTAAACAAACAGCAAAAACAAAA



TCCACATGTCCTAAAAGATAGTCTGATTTTCGTTCATAATGCTCTGGTTTTTGAAA



GATAATAATTGTGTTGTATGAGTGTATGACAAATATTCATTGGTTTGAGAAGTTAA



CAAAATTTGGTGGCTACAAATGGTTTCCTATTCGAGTTGGGTCCATTATCCCTTGG



CGTGTACGGAAATAATACCTACCCATCATAATCTGATCAAAGATGAGGTAGTCTTT



AAATAAATTTTGCGGCTTATATCAATCTTTATGTACTATAAACTGTGAACTTTTTG



TTCTTCAGGACTTCCACATCATTGCCCAATCCGGTTATACCTTCGCTAGTTAATAT



GTTAATTAACATTAAATTAAAGAGCTAACATTTCTTAGGTAGTAAAATAGAAGTTT



TGAACTACTATACTACTAACATGTGAAAATACTTTAGTCACAAATATGACAATATA



CAAATTTATTGGAATGCAAATTCTTGAATTTCAATTGTTTGAAAATTATATATTTC



TACATAACAATTCTTTATAAACTAAAAATATTAATTTTCCATGGCTATGCGTTATA



CGTATATGTCAAATATTTTTATTATTTATATAATTTTACGATAAATTAGTACTCCT



ACTTTACTATATTACTCAACACTAAAAGACCTCTTTAACTCCGCCTAACAAGATAT



GTTTTCTTTTGAATGTTTCGGTTAAACATGACAGAGATTTGTITTCTTGCTTTCGC



TCAATACATATTTGTGCTCCTTTAGAAAAGTAGTATTTCCTAACAATCCAACATTT



TCATATTTATTATATCTTTTAAATATTATCATGGTTCTTTTTCTTTCGTCATGTTT



GGCCTCTTTAAAATAATTCTTGAATTGTATGAGCATTAATCCAATAACGTCCTGAT



CCCAAAAACCTCATATTAGGTTTGAGAGTCCGAAAATATACTTTTCACATAAAGCA



CCTAAGGTGTCATACTTTAACAACTTCACAAAATATGCAAAATTTGTCATTGTCAC



TTTGAGATGTAAGTTTTTTTTTACATGCAAATAGATTGAGTCTCTTTACGTGTAAA



TTCATTTAATAAAATTGTATGGAATATCTATTTATATCATATATTTCTAACATATA



TATAAATATCTATACAAAAATACGACTTTTTGGCACATGTAATTAGAAAAATCCAC



AAGAAACAGAAAAAAGAAACACCAAATACAACGAAATGAAGAAATTATTATAAATT



TGAATGGCTTAACATCTCTTAAGAGTCAACAAGGTAAAGGATTAATTAGTAGTCTT



CATCAATCTTTCTCCACCTTCTTCTATTCCTTAATCTCCACTTTATCTCCCAAACC



CGAAAACTCCTCTICACCAACTTAAACCCTATTAACTAATCCCAACAATCAGATGT



TTCGAATTCAACAACCAGCTCAGGCCATAAGATTCATCCCGGAGAAACAAGAACG



SEQ ID NO: 44

Polynucleotide sequence PATIRX9

CGGGTTTTCGGTTCGACCCGGACTCGAAACGGGTCTAGATGAAGAAAACCTCATCT



CTTTTTGTGTCTAAGGATTTTTTGGTACTGAAACTCTCACTCTTTTTTTTGGTTCC



TCTGGTCCCTCTCTATATGATTCAGATCGAACACTGTGGTTTTATATTTTTTAATG



TTTTGTTATGTTCACACGTTGGGTTCAGAAAAATTGACGGCCGAGATCTTTTCTAT



AAGAGGAAATCGGTGGTTCTACTTAGCTAATCCTTTTTACTAGAAAAGTTTAACAT



TTTGTACTTTTTGTCTGTATGCTCTCTAGTTGTTTGTTGAGATCTCTTGCTGCTAG



ATTCACTTTTTGGGACACATTGCTTTGTATTTGAAGCTAGAAAGTTTATATCAACA



TGATCTAAAAAAGTATTTTAAGAGAACTACATTGAGGTAGTTATTTCTTTTCCTAA



ATTAGTCATTGGTAAATTACATCGTGACATTTATAGAACATTGCAGAGCATAAAAG



ATTGAAAAAAAAATGAGCTGAGATTTGTATGTATATAAAGAAAACGTATTAGCATA



GCTTTCTTTCAGATTTAACGGTGGAAATCATACAAAACTTTCTTGCAGAACAATGA



GTATATATATGAAGGACTCGTTAACGAAAATATTAGTTTAAATCTAGATATCTTCC



AGTAAAATATGAGTTTCGCCTTCGTATATGATACGGCAATAACTTTGGGACCAACT



AATTTGCATATCACATGTTGATATCTCTTTCAGTTCTACTCATTCTTTTTTTTTGA



AAACAACAAATTATTGGCTGCAAATGTTTTTTGGTTTAACTAGTGCTTCTCTAATT



GTCAAGTATCTTAGTCTAGAGTTAATTACTTAAATACTAAAAGGCTGTCGACAAAA



TCAAGCTTGAATCTCCTTGTGGTATCTTCAACTCTTCGTTGTCTGCTTACGAGTGG



TTTACTCAGTAATTATCTATAATATGTTATTTTTTTTCCCTCATCTTTTAGTTGTT



GTTTCATTACATTGAAAAGCTTGTAATGTCTTTATATGGTATATATGGATCTTATG



AGTGAGGCAAGATCCATGATGTTTTTGATCTTAGAATGTATATGATGATCTTAGAA



TGTATTTGACCGCCCACAAATTATTGTTCATTGGGATTATATCTCTAGTCCAACTC



CAAGCAATCGAAATGGGTCCTGCTTTTAAGAACAACAGTATATGTTTAAGAATAAT



AACTTTATATATTCTCGATTTTAAGATCTTTTGACAAAACCTCCTTTTCGTTAGGA



GCGTACTAATTTCCAAGTGTTTGATTAGTGGGGTCTCCGTAAATTTATTTAGAGTT



TCTATCTATTTATTAATAGCTCAATTAATTAATCTATACTGTATCTAAACATCAAT



TTATATATTTACTCTTGAGACCAAAACTGTCAATTTATAACATTGGATAGTTTCTT



AATTCTTATTATATATTTTTCAAACACTTTTCAAGACTAATCTCCACATTAGGTAC



TCTCTCTAGAGATAAAAATATTTATCAAAAACATTTTTATTTATTTATTAAGTAGT



AGATAAACTACTGTGGCAAAATCGTAAATGTCTAAATGCTGATGAATTTTTTTTGC



TGCTCCAATCTGGTTTAGTGCTCCATATACATCCACGGCCAAAATGAATCTATGGC



GGCATTAAGATTCATTAGTAAGCAACGATTATATTAATATAATTGTTTTTAGCAAT



GATTTTCCGTAATTTCCCAAATATGTTTCAGTTAATGTGTTCCAATCCCAACAACT



GGTTGTTGCAAAAGACCACCAACGCAAGCAATCATCAAACATCAAAATAATCTTAC



CTTAGCGAACAAACAATAACTACACAATTCTCATAAAGCTCTTATATATCACTAAC



TTCACACATTTTGTTTTCCACAAAAATAAAAACGGAACTCACTCAAGAAACCTTCT



TCCTTGAAGAGAGGGTT



SEQ ID NO: 45

Polynucleotide sequence PATGUT1_PATIRX10

AATAACAACCACTTAAGTTACTGCAAGTTACCACAAAGAAAAATGATCTAGCAA



ATGAGTAGCATCATATTGATCAAAGACACTGCAAGATAAAAGTCACCTTGCTAA



TGTTCGAGATAATGATAAAGTGTAGACTTGGAGCAAGAAGCCATTTAAACTAAC



AACTTCCTAATTGAGACCTTTCATGTAACTTAATGTCAAAATCACAAGCAACTA



GAGGAAGAAATAAAAATGTACCAGGTAGCTTCTTGGGCTTCCTCATGGGAACAA



ATTTGGCACCAATAGCCAACGCAATAGGAGGGCCAAAAATGAAACCTCTAGCTT



CAACACCTGCATTTACCACAACATCAATTTAGGCAGAACCAAAAATCATCCACC



AATTCATTTCAACTTTTCAGTTTAAGCTAAAGCACTCAGTATCTAAAAAGGCCA



AAAGAAACTAAATCCACAAGCTGTTAATCGATTGGAGTACCAAACAGAACCATA



CGAGTTGTTACCTGCAACAACAGATATGCCTTTATCTTTGTATCTATCAACAAA



CAAAGCAATAGTATCCTTAAAGGCCTCAGTGTCGAGAAGAAGCGTCGTTATGTC



CTGAAACATGATTCCTGCCAAGTATCCAAATTAAAACCTTAAGATCCCAACGCA



GATCAAGACTAGAGACGATATTAATCGGTATAAATGGAAAAAATGGAGACCTGG



TTTAGGGAAGTCGGGGATGACTCTAATGGAAGAGGCAATCTTAGCGATTCTGGG



ATCTTGCACATCTTCAGTCGCCATTTCACTGTCCCGACTGGCTGCTGCTTTAGC



AAAATACTCGGCGTCAGATTTGCAAACACAGAGAGACCCTAAAGACTCAATAGA



GAGACACAGTGATGAAAAAATGACCAATTTATCCCGAATGGTAACGCTTTGACG



GAATTGCCCCACGCAAGCAAAATATCTTTTTCAAAAGGAAACAAAAAGTTTAAA



AGGGAAATAGAAGGTGGTGGGGTCTACCGGCGGAGGAGAAGAGGCGGAGTGAGG



TGGTTGAACGGTGGTTTGAGAGGCGGATCGAAGGAGGAGCACGGTGGTGGTTGT



TGAGAAGACGGTTGCAAGGAACAGCACGAGCAAGACAGAGACGATGAGAAACAA



GTGGAGAAATTATTATTGTTTGCATTGTCTTTGGACTGAGAGATCTTAAAAGAG



AATGTAAATTACTTTAAACACGGAATAATGGACAAAAGCCGTGATCAATGACTT



TTCAAGTCTTAACCAAACCTATAACTCATCCATTGTTTGTTTTTTCTACATATT



TCTTCACATAAAATTGGATGATTTAGAATCTTTCAGAGTGTTCACACTCCAACA



GATTATTATCCACAATGTTATGGTTACATTTAGAGATATATAACAATGTTCATT



TCATCGTTGCTAATGACATAAAACGATCAAAAACTGAATCATAGTACTTCTTTT



ACAGTGATCTCAAATATATTAATCGCTAATCAATGAATTATGTCACCTATAATT



GTCGTATTACCAACAACTATAAAACATATATATAAAAAATTGTTGTCGTTAACT



AGTTGTTGATAGTGGCCACTCTAAAACGATCATGACCTACTACGGAAGTTATAA



CTAGTCAACGTTGGACGTTAGCAAGGCCCAATGGACATTAACTCAGCCCATAAT



AGCACGCGCCTTGTGATGTGCACCAGTTTCCGTCTTTGGTCGTTGAATTCAAGG



AAAAAAAAAGTACATCACAAGCAATTTCTTACTTATCTGTGACTTGAAGCTATT



TCTCCAATTTCGTTTTCCATCGACACTCTATTTCATTTTCACCATTCACGTCTT



CCTTCTGAATAAAATAAACCCTAAAACCTAATACCGAAGTAAACTCGTCAACCA



CTGCGCCCATGACCTCCCAACGATACTCTTCCCTTATATTCTTCCTCTTCCTTC



TTCCTTTCTGCGATCCAAACCTTCAAACACATCTCCGGTAGAT



SEQ ID NO: 46

Polynucleotide sequence PATIRX14

ACCTGCATCGAATTTATATAAATTTAAAACACATTATCATCATCTCTAACTTGAA



CTTTTAAACAAGTTTATCTTTTTGTTTCACAAAAAAAAACAAGTTTATCTTTATG



TCCCTCCTGAGACATATAAAACAAGATTATCTTTCTTCTTAGTAGGGATATAGCA



AGTCCGGACGAGATCAAAAGTAGATTGACTCTTAAGATCTTACTAAGTTTGAGCT



TGCTTTGGTTCCCACCTCTAAAAACCAGTTTTGCATAGTCTGAGACTCGTGTTAA



ATTCGATCAAATCTCTCTTTCAACGACGGTTAACTATGGACGTATTCGCAAAACA



TCACATAAAAACATCTCTAAAGTATTTGGCTATTTGCATAAATATTTCACTCTTA



CAGTCGTCAAAAGTATGAATGAACTCTACATATCGGCCCAATATGAACCAATTTG



TAAGACCATAATGGAAAGCCCATGTTTCTCTTGTGCTTGTTTTAGTTGCAGAATC



ATTAGTTCACATATTGACCGGATTATATTAGTTTTTAAAAACGCATGTATGATGT



AGTCACTGTATCATACCCAAGTTACTGTATTCATTACCCAAGTTCAAACTCGATA



AAATGCATAAACTAAACATACGTTCTTTAGCCTTTTGTTTTCACTTCAATTAACT



CATTTTGTGCGTTGTATATTTTTTTTCTTTCCAACAGCTACTTTTCTCACGTCTA



TATTTTTTACCGTTTGTGATTTTTGAGTCTCAAATATATGGAATTGTTTTTTTTA



AATGGCTACTTTCCAAAGTCTTATATTTTTTACCGTTGTAAATGTTCAGTTTCAG



ATATATATGGATTTCTTTTTCTAATGGCTACTTCTCTAACGTCTATATCTTTTAC



CGTTGTAAATTTTAAATTCTGAAATATATTACCGTTTGTGATTGAGTTCACTTGA



CACACCTTCGTTAAAAATTACACAACAAAAAGCGTTCACAATAAGCCCAATGGGC



CTAAAAGACCCTAACAATCGAACATACCCTTCTGACCAACACATTTTCTTAAGGA



GACACTGTTGGTCCATTTACTCATTTAAGTAGGATTCATAACACTTGTCATGGTC



GTCATTTCTTGTTCAAATGCCTTTTTAAGTAATAACGCAATGGAAGCATATATAT



ACTTTAAACCCACAAATTAATAATGCATATGTATCTATTTTTCTTGCATATACTA



AACATGTCTAAGTATGATATAAACTTTGACACTTTGGTGGTGCTGAGTAATCATC



ATATTTATGCTTTGTGTGCAAGTGAAAACGAACCGATAACAATCTTTAAGACTTC



CCTACCAAACCGGTTTAACCTTCACAACAAACAAACCTAGATCAATTATCTCTAA



ACCAAAACCCTTCAAACCATGTCTTTTGTCGGACCAAACTGTACTCTTATATATG



ACATGCAGATACGTCGTTTTCATGGGCCTTACTAATGGCCCATTAAAAACATTCG



TAATCAATTATTTTGGTTAGTCTTTCCCAAATTCGTCTACATTCCTCCTCGATAA



TCACTTTTAATTAAAACCATATGAATTTACGAAAAAAACAAAAACACAATTATCA



TTATGCAAAACATTTAATTCAATAAATTGAGGGATGTTTAATGTTAACACCAAAA



ATTATTACCAAAAATTGACTTCAATTAGAGACATATTAAAACGACCCTGATTTTA



CTCAAAACTTAATTGAAAGATTTAATTATCCAATAATAAAACGACACGTGTACCT



CCTTGTCGCTTTCCTCTGCTTTCTTCGATGGCGTTGCATCGAAGCATCAGAGAGA



TTGGTATGGTGGTGGTGGTGAGAGAGCAGCAACAACAGCAAGAAGAGAAAGCGAT



AATCGAACTGATTAAGATCGTGAAATCCAAGTAATCTCTGTTGCTTAATCTCAGA



TCTTTTTGATAAGGAGAAGGAAGCAGAAGAAAGAGGTCAACGAAGAAG



SEQ ID NO: 47

Polynucleotide sequence PATMYB46

GTTACACTAACGGTTTCTTGTTAGATTTAGCTGACGTGTCTTTATGAATATATAT



AGAGTTAAATTTTAATATTTTAAGAGTAGTATTACTTCATTAAAAGCTTAGTTGT



AAAATTACTAAAGATTTTCATATATTATAAACTATTTTTTCCTGGCAAACTTATA



TTATAAAATTTGTTGAGCGATTGTGTGATTCTTTCATCCACAATTAGATTAAAAA



AAATCGCAAAAAGTAATACAAGAAAAAATAATAATTTTACAAATTAATAATGATT



GTTTCTTTGGCTAAGAGTTCAGATTTGCAGAGTGTTTTTTGGTCCTTGGGCGATA



TTACGAAAAGTGAATTGTAAAGATATGTATAGATTGTGAGGAAAATGCGAGAATA



ACTGAGAGCTAGGGCTATGCATGAGATGATTGAAATATCATGAACCAAATGGTTA



GATGAGAGCTTGGAGTGAGAGGTGACACTTGTTTGAGATGGGGAATAGCGGATTA



ATGTGCTTGCATGACCTTGGTTCTGAATTTTCGATTGATGAAATCTTGCATTTCG



TTATTTTCAAACTTTGTCCACGAGTTTTACATAACTAGGTTCATTCAAGTTACAA



CTTAAATTGGTTAGCTGACGTCTTTTTTCATGCATATACAAGAGGTTGCATTTGC



AAGCTTCAAAAGAGATTACACCAAAAACAATTTCCCCTAAAGGTTAAGATATATC



TTTGGCCITCAATTCGACATTAGGAATTATGITCAAGATTCAAGATTCAGTACTA



TTCTAACTTCTTTTGTACTTTATCTATGGATGTCTTGTTTATGATTGTATAAAAA



GTTTTGTTTTTTCGGATGGGTGGGCTATTAATATTATAAATCATATAATATGAGT



GTTCTGTAAAAAAATAAAAATGATATGAGTGTAAATCGAGAACTTAAAAAATCAT



GACACACGTTTATATATTAAAGAAAAAACGAATATAAAATATATGGATAAAAGGA



GTATAACATTTTCTTCATTACAATAATTAGATTTCTTCAAGTATACGTGTTGGTG



CGCGAGAGGTGGTTGTGTGAAGCCGAAGCAAAACTTCTTGCTCGCTAAGCCTCAT



ATAACACAAAAAAAGGGTTCTGTGACACACGTCGATTTATTTTATACAATTGAAA



TATGCTTACATACGTATACAATTAATTAAATAACACAACATTTGCTTACCTTGAA



AATGAAGACATCTTTGAATAGAAATAGACATGCTCATGAATATATATATTAATGT



TATATACTATCATATATCAATGTTATATATCATATATATACACACGTAAGGTTAA



CGAATTAGATATGTCTGTAATGTATACCTTGTGAATGAAGAAACTAATAGAAATG



AGTTATATATTCAAAAAGAACAAGAAAAGAAGAAAATAAAATTAAGAACAAGTGA



AGAGCACTTCTCCTTTTTTTCTTTGATGTTTTGCATATCGGGTCTTTTTCAAAAC



CGTTTTCGTCCATGACCGATCAACTAACGTTTCTTCATTTCGTCAAATTAGTTAT



ATACAAAACATACATTTGTTGTTGGTGTATTTTATTTTATTTACCTTACACAATA



TATGCCGACAAAAAAAATGTGTTTAATTTGAAAAAGAGCCAGGGTTCGGATGTTT



TTCTTTTATGTTTTAAAACAAAGCAACACTATATTATAAATATAATATATACAAT



AAAAATATAATTAAGGAATAGAGATTAAAAAGGAAGAAGTGCAAATGGTTTTTCT



TCCCAGAATTGTAAGCAAACCATACAACCATCCCTTTCTCATCATCATCATTCTC



CCTTCATCAAGTCTTCTCTCTTTTCTCTCTCTATTATAAAACAAACTTCACTCGT



TCACATCAATGGATCCTTGAGAAAGACAAACAAATTGAAGAGAAATAATAACAAT



TAACTCAACCAAAAAT



SEQ ID NO: 48

Polynucleotide sequence PATMYB58

CAAAGACTAGAGACAGAGGCGTGCCAATAGCAACACGTTTGCTTTCGTCATGCA



AATTGGGATATTTCAACTTTCTTCCATTTTTTCAACCTAGTTTACTAAACTTTT



CTTTTTCCAGTGCGAACCTAATTGGTTCTAGTTAAAATAACATTTTCGTAAGTT



GTTCACCAAACAAAGGAACATATGATTATAACTTTACTAGAGATGCATGCACAA



TAATGCTATTGTCGAATAAATACTTATATCTTCTCCAAAAAAGTTTCTTTATTA



TGTTAGAAGATCCATCAATATACTAATTGATTTTTGGTTATATGTTTTGATTTA



AAGACAAAACTATACAGGACATGCATGTGAGAACAAAAATTGTTGTTGTTGTAG



TTGCTAGTTGAGTTTTATTTATGTTGCCAAAATAACACCATGTCAACTTTAATT



TTCGTCATATAATTTAACGTAAGCATGATGTGTTTCGTCATATCTTGTTTGGCA



TATGGAATATAAATCATACTATTGATTTGGAATCTTTAACTTAACTTCCTATTA



AGTAAGCGATTGATGCTGATATGTATGTTTCTTTAGATTGATGAACGTAATATT



AATCAGTAGTGGATATACATTGTATCTTTAGAATTTAGGTTAGTATATTATGGC



CAAAATGACTAAATTGAGTACCATAAACTAAAGTTAAAGTAGTGGTAAAAGCTT



ACGATATTGTTTTATAACAATTTTCAAAAAGTAAAAGATATATAAATGTTAGAG



GTTTTGGATAACCATATTGTTCTATAACATTTTAAACATATGTCATATATGTTT



CGTTTATAATATTTATGACTTGACCAAATAATTTGTGTATGTTATTTAAATCCA



AATATATATGAGAAATATATAGACGACATGATTAAAATTATTTAAAAGAGTCAT



GATGAGAGGGATGGAGACTAAAAAAAAGAGGAGAAAAAGATAGAACGTCGAGAA



ATGTTGTGTGTGTATAAAGTAAAGGAAAGCTAATTTGATCATTGTATTCGAAGA



AAACAAAAAAGTATACACATGTTACAGGGTTATAGGACCCATTTTCTTTAAAAT



AAATCCACTATGGACTGATGTACATATTTTTTCTTACTGTTCTTAAGCATGATT



TTATATGTATAATATGGTTATAGATTAGAATTTTATTCAGCCTTCCACGATTCT



TAACCCTAACCAGTCAATTTTTTCTTCCTTATAAATATGAGTGCCAATCGGAAG



GTGATAGCATCCTTACGTCTTGTTTGGTAGATTACTAAGTCAAGTTTTATTCAT



GAAATTTCCACTTATCAAACTTTCTCATTTTGTTAAAATTTAAAACCGTTTTTC



AAAAGTTGGTATAGCCATAGACAGAAAAAAATTATTACAATCCTATCTGATTTG



ACTCAGACACCCTAATTAGTCAAATCTCAAAATTAGCTAATATTAACTAACGAG



TTGCGCATTTTGCAGCAGTACAACAAAATTAGTCAAAATAATTTAGGATAACAG



CACTAATCACAGGAACAGGTATTTTTTTTTTTCCTTTCTTTTGACATCATAAAG



ATGGATTCAACTTATAGATTGGTCAGAGGCAATCTTTATAGGTTTCATGATTGA



ATAAAAAATATGAGACTCAGTATCTAAGTTTCAAACATGTTTCATCTGTGTTTA



GTTGATTACATTTTCATAATAGTTTATTAATGACATATAGAAATGCGAACTATA



CAATTATAAAAAAGATGTGAATTTTGCCAGATACTCATCCACAATATAGACAAG



TTTTTAACCTCAACAAATCTGATGTGACATTTGTCAATGTCTGTGGTTTATAAC



ATGTTTCTCAATGTCAGGATCACACACACCACTTCTCATGTATAAATACACATA



AAAGCAATTGGATTTGGTAAGAGGGAATCTCAAAAGTGTGTGTCTGTGAGAGAG



GAGAGAGAGAAT



SEQ ID NO: 49

Polynucleotide sequence PATMYB63

GTTGATATATATTAATATGTGTCCCTATTATGATCACACAAAACATACACATGCA



GAGCTTTATTCCAATAGCTAAAATCTGAACTTTAAAGTCAGTACACTCGAAATTG



ATATTGACGTATGTATTACTAATAGCAACATGTGTTCTTTCATCATAAGTTTACA



TAATTTTTTAATTTTATTCTACTTAATTAATGTCACAGTTTCCATCGTTTTGATA



AGGTCCATACTCCATAGGGACGTTGAAAATTTAATTTAATTTTTTCCACTCATAG



TTGTCCTTTTTTTCTTAGTAAAGTTTGGGAAAGTTTTCCCACTCATACTTGTTTG



TTCACCAACCTTCTGATTACCAAGAGTCGTATAAAAATGCAAAACTAATAGATCG



TCATTTATATATGTTGCTCCTATAGACTTTTATCGACAAAATTTTCCGAATTAAT



CATTTTGTAACTTCAATACATATACGTCCAGATATTTACCCTAGTGAAAAATATT



TCTTCTTTTTCAAACCTCTTTTCCTCTCTATTCCTCCTAAGAGCTTGTTAACGTA



ACAAAATTGTTGGGTTTATTAACTTCAATTATTGTCGATACTTAGTACTTTAAAA



TATTTGGAGTAATAGATGTAGTGATGGCTGTGTCGTAATTGCTTGAATAATTTTG



GATGGGTACAGAGGAATTAATTAAGTAATGAAGGTTTGGTGGAATTAAGTAATTA



ACGTAGCCAAGAGCCAACAACAACACCAAACCCACCAAACATTAAAAAAGTCAAA



AAGACGTAAGTCTTTGACCTCTTCCACTCTCTTTGGTCTTTAGTTTGGTGAGTTC



GTGCACTATGCTCACACACACTCCTTACGCCTTTTGGTGTTTTCGGATGTGATTA



GAAATGACTTTTTAACAGTTTTTTTTTTTTTCTGTCTCTCATTTTAATGTTATAT



TTAAGGATTATATATATTTCTGCTTTTTTGTATACAAAATATGAAAATATTCCAT



GGAGTGACGTATGGAGTGACTGCGTACTTAGTAAAACAGCATTATTAGTGAGAGT



TCATTTTTCTCGTGTTACACTGTATCTACATGATGATCACGGGACTATCTATTAT



TCAAAAGTTGGTAATTATACACTGAGCCTGATTACAGAAGACTCGCAGACAAAAA



CTAATATAATCAATTCCTCCTATGTATACCTTAAGCTAATTCTTAATTAACAAGT



TGCAGATTTACAATCTTATTTTAGTCAAAACACTACCTAATATTTTGCCACTTTA



TAACTATATATTCTTACTCCTCCAAAGTATTTTTATTAAGAAATACATAAAACTC



TTATCATTACCGCTGTAAATTCCTAAGACCATTTCAATTAACACTCGTCGACATG



TAGTAGTTTCTTACATTAGCGAAATTTATTTCAGACAATTTTATAAGATATGTCA



AATCTGATAATATTTTTAACACGAGATGCTAGTTTCCATTATTACTTGATGTCAA



AAAAGAAGAAAATATTATTTAAGGATTTTGGTTTCTAAAAACGAATGTGAAATAT



TCATGCATCGGTGTTAGAAGGAAAGATAAGTTGCATGCATCATAAGGATGCCAAA



TGAAGTAAAAATGAGAAAATGGAATCATACCAAATAATCAACCATACCACAGACA



GACAACCTTTTCCCACTCAACAAATCTGATTTGACATTTATCAATTCCTCTGTTT



ACATATTCATCTTTTCTCATGTCAAGATCACACACTCTTACCTCTCATATATATA



AAACAGAACCAAATTATCTTTGGTAAAAGTGAATCTCATCAGGAACTGAGTGATA



TAAAGTTATATATATAGAGGAGAGAGGGAGTGAGAGGGAGTGAGAGAGAGAGA



SEQ ID NO: 50

Polynucleotide sequence PATMYB83

TTTGATACAGCAACAGAAAAAAAATAAAAATACAGAAGAACATTAAGAATGATC



TTCTACCATCTGAGAATGGCAAATCCAGAAAGGATGAGAGAAGAGATGATCATG



ATAATAGACATTCCTGGGAAACAAGAGGCTCCTGTCTGTGACTCTGAATCGTAC



TGTGCGGAGGCGGTAAAGACAGAGGAGAGCGTCATTGTGGCGAAAACAGCCATC



GCTGTGTATTGATTGTAGCCAGAAGCCATGTTTACTAAATTTGACCCTCTCAAA



ACCAATTATGTCACCTTTGGCTTTGGCTTTACCAATGTTGTTGTTTTATAGGGA



AAGAAGAAGTTCGTGGGGACGTGAAGAGCATAAGGTTAATGCTCATTTCATAAA



ACCCCACTTTCTGTTTGTTGGTCAACGATTGTTATTGTAATGACTAATGACCTA



TAGAACAAAACCCATCTAACATGAATCTTCTTTTAAATGGATTTGGTGAAAAGA



CCAAGTTTTAAAATCATCATACGTGCGATGAAAGAATACCCAATTTGAAGCATG



AGCCCAATGATAGTTTATAGGCCCAAATAATTTTGATTTATAGTCACAGACAGG



ACAGGAGCCTCTTGTTTTATGAGTTGAATTGGGCCGAAGATGATACAATATAAA



GCATGAGACCAATAGAGGACTGACCAGTTTCTTACCTTCGTTCGTCGAAGAATC



GAACAGTCCCTTAATTTTTCCAGATTCAGATTAATAGCCTATGTATCATCTGTT



TGGATGTGTTAGGCTCTTTTGAATTTCTTAAAATTAGTCTAGATTTTGATTTGT



GATATCCTTGTTATACAAAATTTGAATTTTTCAGAAAATTCATACTTAATTTCA



TGGTAGACTTGTCGAACACTGTGATTTGTTTGGGAAAAAAAAGGTTTAGTTTAT



ATTCATTACGTACGTGATGCATGATGCTTAGTATGCATTAAGATAGAGTATATG



ATCCGTGCTCCATCATTACTTGCTATTATCGATCGATACTTACTATTATTGATC



CTTAAAAGCTGATTTTTGCATGCGCATTATTTTCAATATGCTATTTTGAAAATA



TTTTTTGATGATGATGATTGTTTTATTTCGGTTATAAGTTATAAACGGACTCGT



TTTTGTGATTGAATTATGGGCTTTTGATATCACATCAAATGTTATTTATGTGGA



AATGAATTGAGAAAAAATGATGATTTTATCTTGCACCTATTCTTAAGTTTGGCT



TTGATGTGTTTGGCTTTTGATGCTATATTTCTGTCAAAGAATCCTGAATTTATT



TATTTATTTAGATTCGGTTGATTGTGTCGTAAAATGGAAGTTACTTCAAAATAA



GCCTCCTTGCAAGAGTATATATACTATATTACTTTTAGATAGTGAAAATTGGTT



ATTAGTTGTCGTTTAGAAAGAAGGAAATTTTAAGAAAAAATACTGATCGTAAAC



TATAACCAATGTATGTATTAGTATACTTTGATACTTCAAACACACGTGTGTGGT



GCGGGGATGAGACAGAGAAAGAGGTTGGTCTTGTTCTTGTCTTTGACTCTAAAC



CAGTCTTTTTCGATACATTTTTCTTCACTCACAAGTCTATCATCATGTTTCTAA



CGAAGACATTTATTTTATTTATATTTTGTAACAAAAAAATGAAGACCCACCTCT



TGCTTCTTCTTCACATCCCCATTTCATCTTCTCTCTGTCTCTCTCTATTAGAGA



CTCTCTCTACTCTACCCATCAATCTCAACAAACACTACTTTCTATCTCTTTCTC



TCTTTGTCATATCCATTACGCATATTCGTATCATTCCAAAGCAATCCCCACAAA



TCATATCATCCTCTCCATCTTTCCTTGCTTCTTACAATCTCTCCAATTTCAAAT



CTGTATACTCTTCTTCAAAAAGGCTCCACCAGTCCAAA



SEQ ID NO: 51

Polynucleotide sequence PATMYB85

CTTAGCATACAATCTTTAATTTTTCATGGAAGATTTTTAAAACATTTCCGATCC



GATTAAACAAAGAAGCGAGCGAGCCACATTCTGACAATAATTAAGTAGACACTA



TGATACGACGAAGAATATTAATTTAATATTGAAAGATAGATCAATTTGTAGCAA



AACCATGAAGCCAAAATTGCAAGTCACCCACAAGTCGCAAAGATTAGAAACATA



TATTGATACAGTGATCTATACGTGTACACCATGTGTCAAATGGATATTCGTCTA



TTATTTTTCGTATCGGCGACAAAGTATTTTGTGCGGCAATTCATTATTGAAGCT



TTTACTAAGTTTCTTCTATGTTATGTAAAAAACAAATCTTACCAAAATTAGAGA



CTCGTATATAATATACTTAATAGGTTTGTTAGGGTTGCCAAAAAAAATGGTTTA



TCGCAATGGACTAAAGATCTCAATTCTCAAAACTTATCGGATTTTGCCATAGTT



GAACCGGACCAAGCCAATTATTTGAGATTCTGAAAAGAGTATTATTATGGGCAA



ATTCTGAATATTTTATGTAAAATCGGTTTTGTAAAGACTGGATCATATTTTTAT



TCGTGTTTATTTCACAGCTGATAGCGACAACAATGAAAAATTCATTTTTTTTGT



GTGTGTCATCAACTATTAGAGTCGGTGATTTATATACAGTTTTGGTGACAGAAT



AAGTGCCTACAACTTAAAACTACACTAGTTTTAGTTATCAAGATCCTTAGTACT



TAATGTTGAAATTAATACATTTTTTAATAAATAAATACAAAGTATATATTTATT



TGAAACTTGAGCAAGTATTTGAGTAAAAAAGGTATGAATCGCACGTGTGATTGC



GTACATTCGCACGCATCCTATCCTTTCACATTAGTTCCAAAGTCATTTTCACCA



ACCAAATGCGACATCTCCAATACTCCTTTCTATGATCCTACTAGCAACAGATTT



GACAAAGTAAGACAAATTATATTTCTTAACCTTAATCATTTCTGACCAAAAAAA



ACCTGAATCATTATTTATTAGAATAATCTTATTTTATCAGAATTCGTAATTCTT



TAGCTGACTAACTCCTAATTAAAATGAACCATTCAATATAAAAATATAAACGAA



CGTATTATGTATAAAGTCAGATACAGAAGATCTTCTTTGAAACTGTTGTAATTT



CCCCATCATGACACCTGTATATACATACGTACCTTAAAAAAATTCTGATCTATA



TGTACTTTTGTATGAACGAGTAATGCATAATTCTTATTTAGATTAGACATTCTT



TAATGATAAAATAGTGAAGACGGGTATTATACATATATTAAGTCACTATTAGGG



TGATTAATTGTATTTATATACCAAGAAATCTCTAAGTGACAACATTATGAGGGT



GATTAGTAGTCCGTACTGTTTTTCATTCTAACCAATCACATAAAAGAATACTAA



AAGCGACAAAAAAAACTATTATCAGCTTTTTATACCATTTTATATGTTCGTTAT



TTATACCGTTTTTAATTATTTATATGTTATCAATTACTTTTTTCATATCGACAA



AAGATTTTATAATTTTTTGTGTTACCAATCGAACCATGTATATATATATAACCG



TTACTAGTTAAAATGCTTTGCCATAATGCCACTAGAATTTTTAATAAAAGTTAC



TAAAACAATTTCGAAAATATTAAGATGTAAAGTTATTTTTTCCTGAAACCAATT



GTGGGGGAAAGGTGTGAGAAGGTTATATATAGGTGGGTGAGCTTTGGTAAGCTT



TTGACATAACTTGCAAGCTGTTGAGATTTTCCATCCTCGATAACTTTATTCTTC



CATATCTCTTCCATTTCGCTCTCTATTTCACATCCCCATATAACATAATATACA



ATCACACATATCATTTCTATATAGTATTTA



SEQ ID NO: 52

Polynucleotide sequence PATMYB103

TGGTGCCCTGGTCTACAGTTCCCTAGTTAAGATTCTATTTTGACAACAAAATTGA



GTATTCCAATCATCCATATTTGTTATAGGGAGAAATTGAGCATGCTATATACGGT



GATATATATGATATTTATTTAATATATTGAATAACAAACACAGAACTAGTGTTAT



AGGTGCAGGTATGTAAATATAATGTGATAAACATTTTTTATATAGATTGGAAAGA



ATACGAGATTGTTGTTGCTCTGTTAGAACGAACAAACAGAACTAGTGTTATAGGT



GCAGGTAAGGTAAATATAATGTGATAAACATTTTTTATATAGATTGGAACAAATA



AACATTTTTCTGTTAGAACGAACAAAGGCCTGTCAAAAAGAACAAACCTGATGTG



ACATATTACATATATGATTATAATTGATTATTGTATATATAGTATTGCATGACTA



TCTTTACAAGATTTCAATACGAAAATAAATTAAAAGGAGAAAATTTATAAACGAA



GCGATTTCATTCTCGGTAAGGTTTTCCGATATGTCTCCTAACTAAATCAAAGCCT



TGTAATTGAAACTTGTAATGATTGTTATTCTATATATCTTTGAAGAAAGCTTCGG



TATTGGACGTACTTAATATAATTGGATTTTATTTAAATTACAAAAATCACTGTAT



AATTCGGCTACATGACTTAATCAATTATTTCACGTTGAAAACAATACTATATCAA



CTTCAAATACACTCCTTGTGTATGGATTCCACAAGTTCTTTTCTATCTATAGAAA



TATAGAATCCACAAGTTCTATCTACTTTTATTAGAATTTTTTATTGTTCGTTGTT



GTTAACATAATTATAAGCAATAAATTCAAAAAAAAAAAAATCTAAAAGACACAAA



ATTTCCATCTTTGATAGGGCTTCGGAATCATTAATTACTTTTTACAAACAAAAAA



GAAGAAGATAGGGCTTCGGAAATTATTAGAAAGATGGAAGGATATTGTATTAAAT



TTGGCTTCATATTTTCCTTTGGTTTGCGGCCATCAAGTACTAGTACTACTCAGTA



CCCACAGGCCACAGGAAAAAAAGTAGTACTGCTTTATTAAGTGTGTTACGATAAA



TGGAAAGCGTTTTAGTATGTGATTACAAATTGTTGTATGTGATCATTAATTAGTT



ATTGGTCCGACTTCTAAGTTTAAATATTTTCAGAATTCAGTTAGTTTAATTATAC



ATGTTAGACGAAATGACTCTTTTTGAGCCAATATATAATGTATCTGAATTTTTCA



TTTTGAAAAATCTTTTTATAAAATAATAGGTCAACCTCGAATTATTATAATAAAA



TAAATAATTTGCGTTACTATGAAAATTAATTTACTGAATACAGTATATAGAGAGA



GATAGAAATAGAGGAAAACAGTGGATAATACATGATTAGTTGATACTCATGTGCA



GCGAGTCTATATATATTATATACTCATGATTAGTTGATACATATGTGACGATTAG



TTCTACGAATCAATCTCTAGTTTTCGTCTTAAAATCATTTGGTTTTATAGAATAT



AAGAAACAAAGAAAACAGGAACTTTCGCGGGAGACAGAAGGGTACGTGAAGAGAA



AAACACATAAAAGTGATAAGGGCTTAACGTAATAATACTACAACAAAACCTCTCT



ACGTACAACGAGTAATAACACATGAAAATAGAAAGTCGATGAGACATCGTTTTAA



GGTTAGATCGATGAAGAAATATCTCAGGCCCCACCCCTGGGACCCGACCCGACCC



GACCCGACCCGACCTTTGTCTCCTCTCCTTTTAAAAACTCTCCATTGCTTCTTTG



TCTTCTCTCTTCTATAACATAACTCAAGAAATTAAAGAAGATAGATAGAGAGAGA



GAGAGAGAGTAAAAACCTAAAGGGTGATATACTTATATAAAAATTAATTTAAGAT



TGTGATTAAGTGGTTCACTATATTTAAGTTACTTTGAGGAGCTACTAATC



SEQ ID NO: 53

Polynucleotide sequence PATCADC

AAAGCAAATCGATCTGCCAAACATATCACAGCTCTTGGAGAAAATGCAGGTCTC



TTCAGACTCTGATATTTCGGATCTCGATAGCCTTAAATTCGATGCTCCATTGCC



TAGTCATATGCAACTAAGCTTTAATTTGTTGAAATCTAGAGTCGAAACTTGTGA



CAAAAATTAGATTTTTTTTCTTACCGAGCTTTCTTCTTTGTGTTCATTGAGGCC



CAAGTATTTGTGTATTTGGACCTGAATATTCTCATACAAAGATAAATAATTATA



ATTAAATGATTTTTCGCATATAATCATTATTGTGGTATGATTAACACAGTTGGT



GTGATGACTGATTGACACAATAATCACCGTTTGGATTCGATTCCTTTAATACTT



GTCACTAGAGTTGTTTGACTAAACAGCTAACTTGTCACTAGAGTTATTGTGTTT



GTATTTTGATCTGTTATTAATCTGATTGGGTATAATTACAGATAGAGAGACATC



TATATTGTAATTAAGACAATCTTAAAGTGTAAACTAAAAAGATCTCTCTGACCT



CTGGAAAACGAAAGGTGGGTGACACATCACTCTAGCTATGAATATGATGAATAT



TCAGTACCTAACCGAACAAAGACTGGTTTGGTATTTTTATTGGAAAAAAGAGAT



AAATAATTGTGAATGTGAATTATCCTGTCTGAAAGGTAAGCTGATGACATGGCG



TTATATGATTGGACGAGCTTCAGAACAAAAGAGTAGCGTCGAATCGAATCTTTA



CCTACTACACTTTGAACTTTGAAGTACATTACCTACTTCCTCCTTGATCGAACG



TCTTTTCTCAAAACTATTTTATTTCCCCAATTAAAGTAGTGGTGATAAATTCAC



AAAAATACAAACACTTTTATTTTTGACGTCAAAAACAAATACTTCTTTGAACAG



GCTATTACAATATTTTTAAGAAAAAAGTAAGCAAAATAGTCCACAAACCAAAAT



CTGTAACATATTAAACGATTTATGTTTTTTTTTTTTTTTCTTAACTAGAGAACA



ATTCGGGCTTTTACTAAGGATGATGAGTGTAGTTACCGAATAGTGTATTCATAT



AATCTTTTAATGAGCTTAAGATATGATATTATTTCGACTAATCAGATAAGAGTA



GTTAGATAATTTCGTAATAGAGCAACTCTTTCGCAAATAAAACCATTGTAAACA



TTACCAATTAGTTTTTCTTTTTTTTTGGTCACAACCAATTAGTTTGTTTGTTCT



ATTTTATGAAGTGCGTATTAAAGCTAACGTGTTTACAGTAACGCCACACAAATA



AAAATAAAAATAATTATGTACTTTATGGATTTATAGAAAAAACAAGAATAGTCA



CCAAAAATTGATTGTGTCATATATCTTTTGTCAACTATTTTATCTTATTTTTCT



ATGGATATGTATGTCCAAAATGTTAGACAAAAAACCAAAAAATCATGTCCAAAA



TTTCGTTAGGCTGCCGATATCTCTGTTTCCCTTTCAACGACTATCTATTTAATT



ACCGTCGTCCACATTGTTTTTAATATCTTTATTCGAGGTTGGTTTAGTTTTTTT



TACCAAACTCACTTTGCTACGTTTTTGCCTTTTTGGTATGGTTGTATTTGTACC



ACCGGGAAAAAAAAGATAAGAGGTTTGGTTGGTCGAGCTTACTGATTAAAAAAT



ATACACGTCCACCAAATATTAAAACAATATATCCCATTTTTCCTCCTCTCTTTT



GGTATTACATTAATATTTTATTATTTCCCCATTTGCTCTGTATATATAAACATA



TGTCAATAGAGTGCCTCTACAGTCATGTTTCCATAGACATAATCTCTCACCATT



GTTTTTCTCTGCAAAACTAAAGAAACAAAAAAAGAAAAATCGGAGAAACCAAGA



AAAAAGAA



SEQ ID NO: 54

Polynucleotide sequence PATCADD

GCTTCGGTGATGCATTTCTCCTTCTCATCAATCATCCTAGCAATGTTTTGAAGC



TGAGAAATTCTCCACTCGTAGCTCTTCGTTCTGCCAGAGTTGAAGTTGCTTCTG



AGCTCATCTACAAGCAAAGCTGCTTCTTTTCCACTAAAGTCTGATGCTTGCTCC



TTTACCACAGCAGATAGTGTTGCATAACAAGTACTGATTCAAGACACCAAAACC



GCAATGTGAGAGACTTTAAGACTAAAAATCATGGATAAGACTAAAAAAACATGG



ATAAGTATCAACTGTTCTCACGATTATTTATTCATACCACTGTACTTAAACTTA



AAACCCACTATACTAAATAGAAAGGTAATCATCAAAAAATCAGTATGTAAAAAC



CACTTTTGTGAATAAAATATGTAAAATGGGTGAATAAAGAAATGTGCTTACAAT



TTCAACCGATAAGGGATACAAGCATTGCTGCAATATCCACCACCACCACGACGA



GATATCCGAAAAGGTGAAGTTGCAACATTTAATCTGCAACAAAAGAGGCCATTC



ATTAAAATGGTACTAATTAGATCTAATCATATCATATTGAATGACCAAATCATT



CACAGAAGCATCCATTGCTCCAATTAACATTCTAGACCAAATTCAACTTAAAGG



TAACTCTTTTATACAGGAAACCGAGAAACCGAAAACGCAATTCACATAAAAAGG



AAGGCTTGTTTGGAGAAGCAGAATCGAACAAGTCAATCTCAAACCCTGATGAGC



AGGTTTTTCAAGTTACCTGGCAGGAGAAAAACCCTTGGCAAAACAAAGGGTTTG



AATATGATTAATCTCTAGAAGCTTCGTCATGACTTGGGTTCAGTTAAAAATCTC



AAATTGGAGACATTATTGGTGTTTATATATTTGAGAGAGAGAGCCAGAGAGGAG



ACGTTGAATTGAATGAAGGGTGTGGTCGGAAGAGAAGACGTGTAGAAGAGACGA



GACAAGTAAATTTAAGCATTGGCCCCATTTACAGCCACAAGTCCGCTACAACAA



ATTATTTCCAAGAAACTCTGAGATAACGTCGTGATGAAACGGCTCATGCTGCTG



TTGTGATTCGTGAATTAGAGGTTTATCTTTTGGGTTTTTGAATGTTACTTAATT



GGACGGTCGATTTTTCAAACTGGGTGTGAAATGTGAATGGGTCATTCATAATGG



GCTTTTGTTTTAATGTGAAGCCATTCACACACTCTTTGTCCTTCTTTTCTATTA



TTCATAACTGTCACTCTTTGTTCTTCGAAATAGTAAAGAGCAAATCGATTCTTT



GTTGATCTGGGCCGTAAAATTTCCATGGTTGTGGGAAGTATTCTCGCAGCTGAT



CTGGGCCGTCAATGCTACAGTTTCATGTCAGAGAGAGGTCAAGAATCAACACGT



GGCCAACCATGATTTTAAACCAAAGCAAACACACGATTAGACCCCACATTGTTT



GTTCACCAACCCCCGTGGACCCTCCTTTAGCCGACGTGTCCACGTCAATAGTGG



TTTTTCTTCCTTTCAAAGTACACAAATTCCATTCTTTCTCATTTTACTTTTTGG



ATTACGTTGTTGTTATAAACTGGTAAAATGAATTATGAATGCAAATAAATTTCA



TTTAAGTTTTGTIGGCTICTAATATTTTTTTCACCTAAAATTCTAATAAACTAC



ACAGCCATGAGCCATCGTATGAAAAGAAGAAGAAAAAAAATGTCTTTTTCTAGA



AGGATCTTTCAACGACTAAAAAAGATTTTAAGCTTTTGACTAATTTTGTCAATA



ATATACACAAATTTACACTCAATTATAGCCATCAAATGTGTGCTATGCAGAAAC



ACCAATTATTTCATCACACATACGCATACGTTACGTTTCCAACTTTCTCTATAT



ATATATATAGTAATACACACACATAAACAGCAAAAGCGTGAAAGCAGCAGATCA



AGATAAGAAAGAAGAAAGAATCATCAAAAA



SEQ ID NO: 55

Polynucleotide sequence PATPAL1

TTTTCCCAATGATACAACTATAAATCAAAAAGAAAAAATGTACTGATAAACGAA



ACTAAACGTATAAATTAATATATTTCTTGACATAAATAGGAGGCTTTTGCCTGC



TAGTCTGCTACGATGGAAGGAAAAATGCATGCACACATGACACATGCAAAATGT



TTCAATGAAGACGCATTGCCCAATTAACCAACACACCACTTCTTCCATTCCACC



CATATTATTTATTTCTACCATTTTCTTTAATTTATTGTTTTTTCTTTGATTCAT



ACACTGTTTATGACTATTACATTTTCCCTTTCGACTAATATTAACGCGTTTAAA



CCAAAGAATGGATTTGATAATGAAATTTTATTTTATTAGCATATAGATAATGGA



TGGCTTCATGCTTGGTTTCCATGACAAGGAATGACACAAGATAATTATTTTGAA



TAAAATCATAAATATGATAATACTAGTTGTAAAAAAACTTGAGTGTTTCGTGTG



TTATTTTTCGGTTTCTTGACTTTTTATATTTCTCGTTTTTGTAATTTTAGGATG



GATTATTTAGCTTGCTTTTCTCTTTTATTACTTTCTAAAATTTTATTTATAAAC



TCATTTTTAATATATTGACAATCAATAAATGAGTTATCTTTTAATTAATAAAAA



ATTTGTAAACTCTTGTAAACAGATCATAGTCACTAAAAGCTATTATAAGTTATT



TGTAGCTATATTTTTTTATTTCATGAACTTAGGATAAGATACGAAAATGGAGGT



TATATTTACATAAATGTCACCACATTGCCTTTGTCATGCAAACGGCGTGTTGCG



TCACTCGCCTCCTATTGGGAATCTTATAATCGCGTGAATATTATTAGAGTTTGC



GATATTTCCACGTAATAGTTATCTTTCACAAATTTTATACTCAATTACAAAATC



AACGAAAATGTACATTTGTATCTTTAACTATTTACGTTTTTTTTACGTATCAAC



TTTCAGTTATATGTTTTGGATAATATATTTTTTTACTTTTGACTTTTCAGTTTT



CACCTAATGATTGGGATATACATATGCATGCATAGTTCCCATTATTTAAATGTA



AGCTAAGTGCATATGAACTGTTAGTCAAAATTACGAAGTTTATTTGTACATATA



TATAGTTATAACAAAATGGTACAGTAAATTAAACAGAACATCAAGAAAGTACAA



AAGACTGAACACAATAATTTACATGAAAACAAAACACTTAAAAAATCATCCGAT



AAAATCGAAATGATATCCCAAATGACAAAAATAACAATATAGAAAATACAAAAA



CAAAAACAAAATATGAAAGAGTGTTATGGTGGGGACGTTAATTGACTCAATTAC



GTTCATACATTATACACACCTACTCCCATCACAATGAAACGCTTTACTCCAAAA



AAAAAAAAAAAACCACTCTTCAAAAAATCTCGTAGTCTCACCAACCGCGAAATG



CAACTATCGTCAGCCACCAGCCACGACCACTTTTACCACCGTGACGTTGACGAA



AACCAAAGAAATTCACCACCGTGTTAAAATCAAATTAAAAATAACTCTCTTTTT



GCGACTTAAACCAAATCCACGAATTATAATCTCCACCACTAAAATCCATCACTC



ACTCTCCATCTAACGGTCATCATTAATTCTCAACCAACTCCTTCTTTCTCACTA



ATTTTCATTTTTTCTATAATCTTTATATGGAAGAAAAAAAGAAACTAGCTATCT



CTATACGCTTACCTACCAACAAACACTACCACCTTATTTAAACCACCCTTCATT



CATCTAATTTTCCTCAGGAACAAATACAATTCCTTAACCAACAATATTACAAAT



AAGCTCCTATCTTCTTTCTTTCTTTTAGAGATCTTGTAATCTCCTCTTAGTTAA



TCTTCTATTGTAAAACTAAGATCAAAAGTCTAA



SEQ ID NO: 56

Polynucleotide sequence PATPAL2

TTTCCCTGTTTTTTTTCCCCTCTTTCTGTTTCCCATTTGAAAGTAAAAGATCATTT



AAGCACCTAACTCAATTTTATTTTATTTTAAACACCTAATGTCATGCTCCTTGGCT



CCTTGTAATTAGTTGATCGTTTCAATTTAGACCAGCAAAACATTTTAGTATGTTCG



TAAATATTGCGTACATGCCATTTCGTTTGTCATGCAAACGGTGTGTGTTTCTTTAC



TTAGCTTCTAGTTGGTGTATATTGCGTCGCATTAATATCGGTTTACCTTCCTCCTG



TCTACGTAATGATATATTCTCCACCACAAATTTAAATTCTTATTGAAATTTCCTAA



TTTTTTAGGTAGCTCAAGGTCTCAAGTATACTACGTACCCTATTTTTTTGAATATC



TATCTATATTATAACAAGAGTTTTTCTGAGCTAGTTAATGAGATGACAATATTCTA



CATAAATAAATGACCCTCGAAAGTTTCAAGTACTTTAGGATCTGACCAAATCGGGG



TAAAACATTTTGAAACTAATTACGTTCACATCTACCATCGATGATTGACAAGCTTA



TTGTCACCTTTTATGTTAAAGTGACATGGTCTTGACGTTAATTTGCATGTTATTCT



ACATCTATAGTCCAAAGATAGCAAACCAAAGAAAAAAATTGTCACAGAGGGTTCAA



TGTTACTTAGATAGAAATGGTTCTTTACAATAATAAATTTATGTTCCATTCTTCAT



GGACCGATGGTATATATATGACTATATATATGTTACAAGAAAAACAAAAACTTATA



TTTTCTAAATATGTCTTCATCCATGTCACTAGCTCATTGTGTATACATTTACTTGC



TTCTTTTTGTTCTATTTCATTTCCTCTAACAAATTATTCCTTATATTTTGTGATGT



ACTGAATTATTATGAAAAAAAACCTTTACACTTGATAGAGAAGCATATTTGGAAAC



GTATATAATTTGTTTAATTGGAGTCACCAAAATTATACAAATCTTGTAATATCATT



AACATAATAGCAAACTAATTAAATATATGTTTTGAGGTCAAATGTTCGGTTTAGTG



TTGAAACTGAAAAAAATTATTGGTTAATAAAATTTCAAATAAAAGGACAGGTCTTT



CTCACCAAAACAAATTTCAAGTATAGATAAGAAAAATATAATAAGATAAACAATTC



ATGCTGGTTTGGTTCGACTTCAACTAGTTAGTTGTATAAGAATATATTTTTTTAAT



ACATTTTTTTAGCAACTTTTGTTTTTGATACATATAAACAAATATTCACAATAAAA



CCAAACTACAAATAGCAACTAAAATAATTTTTTGAAAACGAAATTAGTGGGGACGA



CCTTGAATTGACTGAACTACATTCCTACGTTCCACAACTACTCCCATTTCATTCCC



AAACCATAATCAATCACTCGTATAAACATTTTTGTCTCCAAAAAGTCTCACCAACC



GCAAAACGCTTATTAGTTATTACCTTCTCAATTCCTCAGCCACCAGCCACGACTAC



CTTTTCGATGCTTGAGGTTGATATTTGACGGAACACACAAATTTAACCAAACCAAA



CCAAAACCAAACGCGTTTTAAATCTAAAAACTAATTGACAAACTCTTTTTGCGACT



CAAACCAAATTCACGTTTTCCATTATCCACCATTAGATCACCAATCTTCATCCAAC



TGGTCATCATTAAACTCTCACCCACCCCTCATACTTCACTTTTTTCTCCAAAAAAT



CAAAACTTGTGTTCTCTCTTCTCTCTTCTCTTGTCCTTACCTAACAACAACACTAA



CATTGTCCTTCTTATTTAAACGTCTCTTCTCTCTTCTTCCTCCTCAGAAAACCAAA



AACCACCAACAATTCAAACTCTCTCTTTCTCCTTTCACCAAACAATACAAGAGATC



TGATCTCATTCACCTAAACACAACTTCTTGAAAACCA



SEQ ID NO: 57

Polynucleotide sequence PATC3H

ATCGTAAGTTTTTTTGTGTGTGTGTTAACAATGTACTCACTACTCACTGTTCCAT



ATTTTTGATGTACGTATATCGAAAACATTCTGCCAACAAATGCAAACATAACAAA



AGTCAAAAACAATAACATAACCGGGAATTAAACCAAAATGTAATTGCTTTTTATT



AGTGTCAGGCCTTCTGCTTAAAAATATTCTCGGCCCAGAGCCCATTAACACCTAT



CTCAATTCATATTGAAGAAAATGACTATATTACTTGACAAAAACTTTAGTCAGAA



AAATATGGAATCTCTTTCGGTACTGCTAAGTGCTAACCTTAAATAGTATAGAATT



CTTAGTTCATTCTCAAAAACATAGCTATATGTAGATTATAAAAGTTCGATATTAT



TTCCTGCAAAAGATGTTATAATGTTACAACTTACAAGAAAATGATGTATATGTAG



ATTTTATAAACTGGTACCGTAATTCATAAAAGATGGTGGTGGGTATGTATCAGTA



ACGGAACTTACATATGCGTGTGTATTACTATGTCTATATGGTGTATTCCTTTGTG



TGGAACAATGCACGTCAGAGTTGTTTATTTTCTTATAGAATTTAAGGAATCAATT



ATTGGATTTCTCAAGGTGAAAGTGGACTTCTTTGCACGCAAGGTCTAGTTGCCGA



CTTGCCGTTGCATGTAACATGATTGTTGAAATAAAGTGAATTGAGAGAAGTTTGG



CCAGACATTTTAAATTTAACCCAAAAAAAGTAGGGCCTAACACAAAATATAACCT



CTCTTTGTTCAAAGGAAATAACACCTACGTCTTATAATTGAACCAAACATTGAAT



CATTGAACTCACCTATAATAATTATAATAACACGAATTCACAAGACACCTAAAAG



AAAAAGTTCACAAAAACAAATAAAAATTTACCTCTCACCAAACACACTCACCTAC



CCGTCTGGTCCCACTGACCCCAACATACAACACCGACTCTCTCCCACACCAATTT



TTTTTTTTGGCGTTTTAAAACAAATAAACTATCTATTTTTTTTTCTTACCAACTG



ATTAATTCGTGAATAATCTATTATCTTCTTCTTTTTTTTGTGACGGATGATTAGT



GCGTGGGGAAATCAAAATTTACAAAATTTGGGATGATTCCGATTTTTGCCATTCG



ATTAATTTTGGTTAAAAGATATACTATTCATTCACCAAGTTTTCAGATGAGTCTA



AAAGATAATATCATTTCACTAGTCACTTAAAAAAAGGGTTAAAAGAACATCAATA



ATATCACTGGTTTCCTTAGGTGACCCAAAAAAAGAAGAAAAAGTCACTAGTTTCT



TTTTGGAAATTTTACTGGGCATATAGACGAAGTTGTAATGAGTGAGTTTAAATTT



ATCTATGGCACGCAGCTACGTCTGGTCGGACTATACCAAGTTACCAACTCTCTCT



ACTTCATGTGATTGCCAATAAAAGGTGACGTCTCTCTCTCTCTCACCAACCCCAA



ACCACTTTCCCCACTCGCTCTCAAAACGCTTGCCACCCAAATCTATGGCTTACGG



GGACATGTATTAACATATATCACTGAGTGAAAAGAAGGGTTTATTACCGTTGGAC



CAGTGATCAAACGTGTTTTATAAAAATTTGGAATTGAAAACATGATTTGACATTT



TTAATGATGGCAGCAGACGAAACCAACAACACTAAGTTTAACGTTCGTGGAGTAT



ACTTTTCTATTTTCGAAGAAGACATATAACTAAGCTGATTGTTATTCTTCATAGA



TTTCTTTTCACTGCGAATAAAAGTTTGTGAACATGTCACCGTTTGAACACTCAAC



AATCATAAGCGTTTTACCTTTGTGGGGTGGAGAAGATGACAATGAGAAAGTCGTC



GTACATATAATTTAAGAAAATACTATTCTGACTCTGGAACGTGTAAATAATTATC



TAAACAGATTGCGAATGTTCTCTACTTTTTTTTTGTTTACATTAAAAATGCAAAT



TTTATAACATTTTACATCGCGTAAATATTCCTGTTTTATCTATAATTAATGAAAG



CTACTGAAAAAAAACATCCAGGTCAGGTACATGTATTTCACCTCAACTTAGTAAA



TAACCAGTAAAATCCAAAGTAATTACCTTTTCTCTGGAAATTTTCCTCAGTAGTT



TATACCAGTCAAATTAAAACCTCAAATCTGAATGTTGAAAATTTGATATCCAAGA



AATTTTCTCATTGGAATAAAAGTTCAATCTGAAAATAGATATTTCTCTACCTCTG



TTTTTTTTTTTCTCCACCAACTTTCCCCTACTTATCACTATCAATAATCGACATT



ATCCATCTTTTTTATTGTCTTGAACTTTGCAATTTAATTGCATACTAGTTTCTTG



TTTTACATAAAAGAAGTTTGGTGGTAGCAAATATATATGTCTGAAATTGATTATT



TAAAAACAAAAAAAGATAAATCGGTTCACCAACCCCCTCCCTAATATAAATCAAA



GTCTCCACCACATATATCTAGAAGAATTCTACAAGTGAATTCGATTTACACTTTT



TTTTGTCCTTTTTTATTAATAAATCACTGACCCGAAAATAAAAATAGAAGCAAAA



CTTC



SEQ ID NO: 58

Polynucleotide sequence PATCCR1_PATIRX4

AAAATTGTGTCTAAGAATGTGGAACCGAGTAGTTCTCCAGAAGTCAGGTATGAA



AGTATATAAGAATTCTAGTTTTAGTTGTTTGAAAGTTTGATCCGTGAGTGAATT



AGTTCACAATTATGGATGTAGATCCTCTATGCAAACAATGAAGAAGAAAGACTC



TGTAACAGACTCCATTAAGCAAACAAAAAGAACCAAAGGTGCACTGAAGGCTGT



AAGCAATGAACCAGAAAGCACTACAGGGAAAAATCTTAAATCCTTGAAAAAGCT



GAATGGTGAACCTGATAAAACAAGAGGCAGAACTGGCAAAAAGCAGAAGGTGAC



TCAAGCTATGCACCGGAAAATCGAAAAAGATTGTGATGAGCAGGAAGACCTCGA



AACCAAAGATGAAGAAGACAGTCTGAAATTGGGGAAAGAATCAGATGCAGAGCC



TGATCGTATGGAAGATCACCAAGAATTGCCTGAAAATCACAATGTAGAAACCAA



AACTGATGGAGAAGAGCAGGAGGCAGCGAAAGAGCCAACGGCAGAGTCTAAAAC



TAATGGAGAGGAGCCAAATGCAGAACCCGAAACTGATGGAAAAGAGCATAAATC



ATTGAAGGAGCCAAATGCAGAGCCCAAATCTGATGGAGAAGAGCAGGAGGCAGC



AAAAGAGCCAAATGCTGAGCTCAAAACTGATGGAGAAAATCAGGAGGCAGCAAA



AGAGCTAACTGCAGAACGCAAAACTGATGAGGAAGAGCACAAGGTAGCTGATGA



GGTAGAGCAAAAGTCACAGAAAGAGACAAATGTAGAACCGGAAGCTGAGGGAGA



AGAGCAAAAGTCAGTGGAAGAGCCAAATGCAGAACCCAAGACCAAGGTAGAAGA



GAAAGAGTCAGCAAAAGAGCAAACTGCAGACACAAAATTGATTGAGAAGGAGGA



TATGTCTAAGACAAAGGGAGAAGAGATTGATAAAGAAACATATTCAAGCATCCC



TGAGACTGGTAAAGTAGGAAACGAAGCTGAAGAAGATGATCAGAGAGTGATTAA



GGAACTGGAAGAAGAGTCTGACAAGGCAGAAGTCAGTACTACGGTGCTTGAGGT



TGATCCATGAATGAAGGATTGTTAGGTAAATGTTAATCCAGGAAAAAAAGATTG



GTTCTTGTGGTTTAGGTAACTTATGTATTAAGTGAAGCTGCTTGTTTAGAGACT



AATGGTGTGTTTTATGAGTAGATTCTTCTGACCTATGTCTCGTTATGGAACTAG



TTTGATCTTATGTCACCTTGCTAGCAGCAGATATTGATATTTATATATTTAAGA



GACATGCGCATGAGAATGAGGGTATGGAAAAGTCCATATCAGATGACACAAACA



ATGATCGTATGTGTAGTCACTTGTGCATTTCCAGTTTTGGACATAAAATTCTGA



TATTGCATAGAAATGTTTTTAAATAACACTAATCCAAACCTAAATAAAATATCT



CTATACATCATCTAGAAATGTATGGCTTGATCAAGAATTGTAGATAATAATACC



CTGAGTTAAATGATTGTAGGTATTATTTCAGTTTTCAAAATTGTCCAAATTTAT



GAGCTATATTAAAGATAATATTTTCAATAAGGTGTGTAGTTCTAAATGTTTCTT



CTTCTTCCACCAACCCCTCTTTCTATATGTATGTTCTTTTTTCTAAAATAATTG



TTTGTTCTTTTTTAGATATATCAAATTAAATATAAAAAATATTGACAAAACTTA



TTTACCATTGTTAGGTGAACTTGGCAAGTGTGTAAATATAAAGATAACATTCCT



TTTCGTTCTTTATATATACGAAACGTACCACAAATTTCTAACTAAAGCATTCAT



AGTCTCTCGAAAGCCTCTTTTCAGAACCGAAGCTCTTTACTTTCGTCCACCGGG



AAAT



SEQ ID NO: 59

Polynucleotide sequence PATF5H

AAATTTTTGTATGAAATATTTCTTTAACGAAAATAAATTAAATAAAATTTAAAA



TTTATATTTGGAGTTCTATTTTTAATTTAGAGTTTTTATTGTTACCACATTTTT



TGAATTATTCTAATATTAATTTGTGATATTATTACAAAAAGTAAAAATATGATA



TTTTAGAATACTATTATCGATATTTGATATTATTGACCTTAGCTTTGTTTGGGT



GGAGACATGTGATTATCTTATTACCTTTTTATTCCATGAAACTACAGAGTTCGC



CAGGTACCATACATGCACACACCCTCGTGAAACGAGCGTGACTTAATATGATCT



AGAACTTAAATAGTACTACTAATTGTGTCATTTGAACTTTCTCCTATGTCGGTT



TCACTTCATGTATCGCAGAACAGGTGGAATACAGTGTCCTTGAGTTTCACCCAA



ATCGGTCCAATTTTGTGATATATATTGCGATACAGACATACAGCCTACAGAGTT



TTGTCTTAGCCCACTGGTTGGCAAACGAAATTGTCTTTATTTTTTTATGTTTTG



TTGTCAATGTGTCTTTGTTTTTAACTAGATTGAGGTTTAATTTTAATACATTTG



TTAGTTTACAGATTATGCAGTGTAATCTGATAATGTAAGTTGAACTGCGTTGGT



CAAAGTCTTGTGTAACGCACTGTATCTAAATTGTGAGTAACGACAAAATAATTA



AAATTAAAGGGACCTTCAAGTATTATTAGTATCTCTGTCTAAGATGCACAGGTA



TTCAGTAATAGTAATAAATAATTACTTGTATAATTAATATCTAATTAGTAAACC



TTGTGTCTAAACCTAAATGAGCATAAATCCAAAAGCAAAAATCTAAACCTAACT



GAAAAAGTCATTACGAAAAAAAGAAAAAAAAAAGAGAAAAAACTACCTGAAAAG



TCATGCACAACGTTCATCTTGGCTAAATTTATTTAGTTTATTAAATACAAAAAT



GGCGAGTTTCTGGAGTTTGTTGAAAATATATTTGTTTAGCCACTTTAGAATTTC



TTGTTTTAATTTGTTATTAAGATATATCGAGATAATGCGTTTATATCACCAATA



TTTTTGCCAAACTAGTCCTATACAGTCATTTTTCAACAGCTATGTTCACTAATT



TAAAACCCACTGAAAGTCAATCATGATTCGTCATATTTATATGCTCGAATTCAG



TAAAATCCGTTTGGTATACTATTTATTTCGTATAAGTATGTAATTCCACTAGAT



TTCCTTAAACTAAATTATATATTTACATAATTGTTTTCTTTAAAAGTCTACAAC



AGTTATTAAGTTATAGGAAATTATTTCTTTTATTTTTTTTTTTTTTTAGGAAAT



TATTTCTTTTGCAACACATTTGTCGTTTGCAAACTTTTAAAAGAAAATAAATGA



TTGTTATAATTGATTACATTTCAGTTTATGACAGATTTTTTTTATCTAACCTTT



AATGTTTGTTTCCTGTTTTTAGGAAAATCATACCAAAATATATTTGTGATCACA



GTAAATCACGGAATAGTTATGACCAAGATITTCAAAGTAATACTTAGAATCCTA



TTAAATAAACGAAATTTTAGGAAGAAATAATCAAGATTTTAGGAAACGATTTGA



GCAAGGATTTAGAAGATTTGAATCTTTAATTAAATATTTTCATTCCTAAATAAT



TAATGCTAGTGGCATAATATTGTAAATAAGTTCAAGTACATGATTAATTTGTTA



AAATGGTTGAAAAATATATATATGTAGATTTTTTCAAAAGGTATACTAATTATT



TTCATATTTTCAAGAAAATATAAGAAATGGTGTGTACATATATGGATGAAGAAA



TTTAAGTAGATAATACAAAAATGTCAAAAAAAGGGACCACACAATTTGATTATA



AAACCTACCTCTCTAATCACATCCCAAAATGGAGAACTTTGCCTCCTGACAACA



TTTCAGAAAATAATCGAATCCAAAAAAAACACTCAAT



SEQ ID NO: 60

Polynucleotide sequence PATLAC4

CAATTATATTTGGTTTCGATTGAAATTCAATCTAATGTGGTTAGATGAGTCCTA



TATTACCATGTCATTGTTAATACCCATTGCCAAAAATAAAAGTGAAGCAGAAGG



AGAAATTGTTTTTGTATACCCGAAGGAATTAAGATGTACGATCTTAAAATAGAC



ATTTCGGCCATCTATCAAAATAAATGTCTAAAAGTTTTGTGGTCGTCTTAAATA



CTACTTCGAGTTCAGACGTATACGTCTCACCAAAGTAATGCACATACTTGATGT



TAAGTTTATCTCTTTTTACTATTTCAAATTTCGCGTTTGACAACACTTTAAGTC



TACATTATCCATAGAGAATATAACATAAAGATCATGAACTTCTCATGAATGTAT



AAGACAAATCAAGCTTATATATGAGATCTATTTAGTAATTTGATATGTATGTAA



TATATGATAAATCTTTGATGCAATATTTTATTATGATTATTAGATATACACTAG



TCAACTTTAACTTTAGAAGATTAATCATTCCGTCGCAAACCATACCATAAATTA



GCAAGGGATCGACTTAATATCTCCGATCCGCTATATATTTAAGAAGCATTTAGA



TTGTTTATAATACATGTCATGATTTTATAATTATGTATATATAAATACTAATTG



ATGTATGAAGTACGTAGATAATGTTACGATCTATTAATCTATTTACATTAACTT



TTAATTAGTGTTGAGTAGGGAAAATTAACATATAAACCTTTAGCAGTTGGTTGT



ATTATTAAAAATAATTTGAACTTAAAATCCACCTTCGAAAAGATAAATCAAACA



AGTATAAAAAATGCTATAAATCCAGAATATTTACCTAAGGTTTTTATTCTTCTA



CTTAATAATGTAAGATAAAACCGGCACAATACTTGTTACGTATGCATGGTAGGT



ACCGCAATTGTGTAAGCAAATCGGCACAATACTAAGGTTACATATACTAACTAA



ATAAAACAATCTGATTTCAGTGACACCGTATATCTAACCTTTATTCAAATCCAA



GGGAACATGACTTGACTTCTTCTGTTGGAACTAACTCGATCCCTCAACCATCTC



CAGGGATAGAAGAGTTAGTAAAATCAAACTTGAAGTGAGGAAGTAAGCAGTTTA



ACGACTCCATATGACTACAGTTATATACAAAGTTGGGCACAAAGTACAAGTACT



AAATACTCAAAGTCAGATAATAATTTTAATAAGTACAAACTATATATATGCAGT



ACAATTATTGAGTATATATAAACGAGACTGGTGATTTGGGGCATTGTCCACCAG



GGTGTTATATCCCAATTGAAATTTGAAAATTTAAGTGTGTGAGTGTTACGACAA



AAAAAAGTGTGTGAATTGTAGGCGCGGTGAAAAGGTAAATTAAGATTGGAACTA



GAAAAATAGTTGAATATCCTTTACTAAAAGTTGTCAATTCCGGTTTTAGTAAAA



AAAAATTTTAAAATAGAAATTTTATCCAAAAGACTTCAAACACACATATTCGCA



TATATAACATAAGATATCATTTTTTGTAAACAGTTAAAAAGAAAAACACATGTT



TTTTTITTTAATTTAGAAAAAAACATGTTATTATACAAAACAGAGTTTTGCCCA



CTTTTAATATGTTATGAAAAGAAAAATGATTTTCTTGGGTTTGGTCAGAGAGAT



TGGTTGTGGTAAGAATGGGAATCTTAATTACAAAGAATTGGATTTTGGGTCGAC



CTACCACCTAAAACGACGTCGCCTCCATCTCTGGTTTCCAAATCTCTTTCTCCT



CTCCCTTTATAAGCTTGCGTTGGCCAGTCGCTCATCTCGAAAACAGAGAGAAAA



AGACTAAAAACACAGTTTAAGAAGAAGGAGAGATAGAGAGAGAAGAGAAAGATA



GAGAGGGAG



SEQ ID NO: 61

Polynucleotide sequence PATLAC17

TAAGTTTAAGTCCAATAATTTCATTTTACTAGTAAAGATCACAATGTCATTTACC



GCATTCACTTAATAATTGCTGAATTCACATAGTGCCTGTAAATTAAGACTAATTT



TAGGTTTCAAATAATTTTTCTTTTTTACATAACTTACGATCGATATTTTAAATGG



TATTGGTAAGTTTAAGGTATATAGATAGTGTGTCTAAACTAGAGTTCGTTGAAAT



TGGTCTGAGGTATAAATACCTAAAAGGTTATATATGTTTTTAGTTTAATGTAATT



CGATAAATTTTAGTCGAAACCGTTAAGAGATATCAGAATTTCGTTTTCAAATAAT



ATGGGATATAATTACCCGGGATTAACCGTACCTGATAAAATATAGCTCTCGTACG



TGTCACATGCCTAATGCCTAGTTAAACTTAAAACGAATATCTATATTTACTGTTA



TTGATTGTGAGTTACCAACTAAAATATTGTTAAAAGACATTGTAAAACTACAAAT



GGTTCGAACTGTATACTAATGATGTAAACTCGTGTTTCATCGTTATGTCCGATAT



TTTTTTCATTCAACCATTATTCAATTTCAAGATTTCTTTATTGTCTTTTTTTCTT



TCTAGAAAGCCTATATATTTAATTACCCACTTTGCATATTCAGAGGATAAGTTGA



TACGTACTTGTTAGCAACCTGTCTAGATCATCTTTTGATTGTAGATTTGACTTTA



AATTTCTCACAATTATAAATATGAAAAATAACAAGCAAAGAATTTACAAATGTAT



ATAATTATATACACGCATTGATGAATAAACATATTTAGAAAATAATGTGTTCTAA



GGAAATTTTGTGGCATTTTTTAAAAAATAATTAAACAAATAAGAATAGTGTAAAG



TTGTTTAAATATGTATGTATAAGTGGCATGCCTTTGAGGATACGAACTTAAAAGG



GAGTTAGGTAACTTGCTTGGGAAATAAAATAGCCAACCTTAATTTGAGGTTTCCT



CAATGTTCTTATCAAAAAGAATAAAAATTTCGGAAATTCCCTTCATGGATTTTGA



TATCTAACCCTAATCGTGACCTTCTTTGATAGCTACAATCTCCCTCTCTTTGCTT



ATTCCCCAAGCAATTTTAGCTTACGAATGTTTTGACTAACTCCACATCGGTTTAT



CTCTTAAGTTCCCCACCTACAAATATACAAAAAAAGAAGTAAAATAAAAATAATT



ATTAACAAACCGATGAAGTACTTATCATTTATAAACATGCTTATGAAATGTATTT



TCTAAAACATAACCGCTAACCAGAGAAGTTTCCTAGAGTTCTGCTTCAGACTCTT



TTGGTCGATCAAGAAGTCTCCAAGAGTTGTTTTTGTTGGGTCTAAACAAAACTTG



GCCAGGGAACAAATCAAACTATATTATTAATCTTCTACATCTGGTCCTAAGTTCC



TTACTATCTCATGTTAAAATTTGAAGTCTAATATACTCAAAGCTGTCAAAGAAGC



AGAACATGGAAGAGGAACTGTCATATCTGAGAAACCAAAATTGGCAATCTTGCAT



TTCATATTTAGAATCTACGCCATAGTATTGAGATGGAAACAAAGAGTTTTCGAAG



AGGGTCAAAGAGTTTGACTTATCTTTGACACCACTCATACATTAGCTGTTCATAT



AATCTAACAACTAGTCAATATCAAGTGTCTCCAAATTACGGAGAGTACTTCTCTA



CCAATTATCTTTTTGTTTTTCATAAACATTTTACTAATTGTTTTTTCTATATCTC



CTGCTCAAGCAAACACCTAACTCTCCTTTCCTATATATACACTAAAGGTTGAAAA



CAATGAATCCACAATCTACAGCAAAACATAAGCGAGGCAGAGTCTTCAGAAAACT



TACCTGCTCTAAACAACGCCTCCGTGTCCAAGCTCACTTCA







































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































TABLE I



In-vitro HCHL enzyme activities in

stems of five-week-old wild type (WT) and

IRX5:HCHL plants. Values are means of three

biological replicates.







Enzyme activity ± SE


Plant line
(pkat vanillin μg −1 protein)





WT
nd a


IRX5:HCHL (1)
0.112 ± 0.026


IRX5:HCHL (2)
0.075 ± 0.022


IRX5:HCHL (3)
0.042 ± 0.006


IRX5:HCHL (4)
0.160 ± 0.038


IRX5:HCHL (5)
0.025 ± 0.002





a nd, not detected.


































TABLE II



Height of the main inflorescence stem and total stem

dry weight of senesced wild type (WT) and IRX5:HCHL

plants. n, number of plants analyzed. Asterisks indicate

significant differences from the wild-type (*, P < 0.05, **,

P < 0.01, ***, P < 0.001).







Height (cm)
Dry weight (mg)


Plant line
Mean ± SE
Mean ± SE
n



WT
62.4 ± 4.6
477.7 ± 51.3
16

IRX5:HCHL (1)
60.3 ± 5.0
501.6 ± 62.8
14

IRX5:HCHL (2)
56.0 ± 4.6
435.3 ± 62.5
12

IRX5:HCHL (4)
48.3 ± 4.4***
335.7 ± 63.4***
15

IRX5:HCHL (5)
54.1 ± 7.6**
399.1 ± 61.1*
16



































TABLE III



Quantitative analysis of soluble phenolics in stems from five-week-old wild type (WT) and

IRX5:HCHL plants. Values are means of four biological replicates.





Mean ± SE (μg g −1 fresh weight)








Plant line
HBAld
3,4-DHBAld
HBA
HBAGlc
HBAGE



WT
nd a
nd a
nd a
2.32 ± 0.20
1.34 ± 0.41

IRX5:HCHL (1)
1.02 ± 0.07
0.33 ± 0.02
5.53 ± 0.36
544.87 ± 157.79
1653.74 ± 504.38

IRX5:HCHL (2)
0.62 ± 0.08
0.23 ± 0.02
4.77 ± 0.41
569.23 ± 138.73
1046.97 ± 439.35

IRX5:HCHL (4)
0.83 ± 0.18
0.29 ± 0.03
4.64 ± 0.57
484.06 ± 74.23
959.79 ± 189.25

IRX5:HCHL (5)
1.04 ± 0.09
0.34 ± 0.02
5.59 ± 0.27
531.29 ± 51.13
1360.03 ± 178.03



a nd, not detected


































TABLE IV



Quantitative analysis of acid-hydrolyzed soluble phenolics in stems from five-week-old

wild type (WT) and IRX5:HCHL plants. Values are means of four biological replicates.





Mean ± SE (μg g −1 fresh weight)















3,4-




3,4-




Plant line
HBAld
DHBAld
Van
5OH-Van
SyrAld
HBA
DHBA
VA
5OH-VA
SyrA



WT
0.6 ± 0.1
0.1 ± 0.0
nd a
nd a
nd a
14.0 ± 2.8
10.2 ± 2.7
5.0 ± 0.9
nd a
nd a

IRX5:HCHL
11.8 ± 2.1
14.3 ± 2.0
11.9 ± 3.8
24.3 ± 2.0
1.7 ± 0.0
2492.4 ± 534.9
17.3 ± 2.4
226.9 ± 32.6
8.1 ± 0.7
44.7 ± 7.6

(1)

IRX5:HCHL
5.7 ± 1.5
10.4 ± 2.6
3.9 ± 1.28
12.4 ± 6.1
1.6 ± 0.1
1726.1 ± 706.7
13.7 ± 3.4
175.9 ± 37.1
6.2 ± 1.5
45.9 ± 10.9

(2)

IRX5:HCHL
7.2 ± 0.8
9.9 ± 0.6
6.4 ± 1.26
10.7 ± 1.7
1.7 ± 0.1
1588.3 ± 181.1
15.4 ± 1.7
183.6 ± 19.0
5.8 ± 0.3
31.3 ± 2.4

(4)

IRX5:HCHL
9.9 ± 1.2
12.8 ± 0.7
8.0 ± 1.73
16.9 ± 2.5
1.9 ± 0.1
2061.3 ± 336.2
16.4 ± 1.2
202.3 ± 9.2
7.0 ± 0.5
39.5 ± 3.2

(5)



a nd, not detected.







































TABLE V



Quantitative analysis of cell wall-bound phenolics in stems from extract-free senesced mature

dried wild type (WT) and IRX5:HCHL plants. Values are means of four biological replicates.





Mean ± SE (μg g −1 dry weight)















3,4-




3,4-




Plant line
HBAld
DHBAld
Van
5OH-Van
SyrAld
HBA
DHBA
VA
5OH-VA
SyrA



WT
5.8 ± 0.6
1.1 ± 0.0
59.4 ± 6.5
nd a
17.8 ± 1.0
6.2 ± 0.9
nd a
24.2 ± 2.0
nd a
10.6 ± 0.3

IRX5:HCHL
11.1 ± 0.4
0.6 ± 0.0
36.9 ± 2.7
0.8 ± 0.1
107.8 ± 6.4
486.4 ± 28.2
nd a
42.2 ± 3.2
nd a
47.5 ± 2.3

(1)

IRX5:HCHL
8.9 ± 0.4
0.6 ± 0.0
25.7 ± 5.9
0.6 ± 0.1
99.9 ± 4.6
427.9 ± 49.3
nd a
39.6 ± 1.9
nd a
43.3 ± 0.9

(2)

IRX5:HCHL
9.1 ± 0.9
0.7 ± 0.0
29.9 ± 2.7
0.8 ± 0.1
122.2 ± 14.8
421.8 ± 28.2
nd a
36.8 ± 1.4
nd a
54.1 ± 6.1

(4)

IRX5:HCHL
9.1 ± 0.7
0.7 ± 0.0
45.6 ± 6.2
0.7 ± 0.0
122.4 ± 5.9
349.6 ± 27.6
nd a
47.7 ± 3.0
nd a
59.3 ± 3.1

(5)



a nd, not detected.







































TABLE VI



Chemical composition of total and hemicellulosic cell wall sugars in senesced

mature dried stems from wild type (WT) and line IRX5:HCHL (2). Values are

means ± SE of three biological replicates. Asterisks indicate significant

differences from the wild type (P < 0.001).





Mean ± SE (mg g −1 CWR)






Total Sugars
Hemicellulosic Sugars







Sugar
WT
IRX5:HCHL
WT
IRX5:HCHL



Fucose
2.23 ± 0.08
2.21 ± 0.05
1.44 ± 0.03
1.49 ± 0.05

Rhamnose
10.83 ± 0.35
11.71 ± 0.19
9.12 ± 0.23
9.76 ± 0.26

Arabinose
16.01 ± 0.56
18.58 ± 0.54*
10.15 ± 0.30
12.40 ± 0.38*

Galactose
23.06 ± 0.66
22.69 ± 0.82
15.34 ± 0.33
16.49 ± 0.50

Glucose
442.76 ± 7.09
388.66 ± 7.58*
10.09 ± 0.34
11.25 ± 0.33

Xylose
201.63 ± 1.71
245.20 ± 3.31*
114.39 ± 0.97
141.16 ± 4.20*

Galacturonic acid
93.74 ± 2.56
99.96 ± 1.52
37.13 ± 1.86
40.58 ± 1.12

Glucuronic acid
4.10 ± 0.16
4.60 ± 0.39
2.66 ± 0.17
3.12 ± 0.09

Total
794.36 ± 13.17
793.61 ± 14.85
191.32 ± 4.23
236.25 ± 6.93*













































TABLE VII



Lignin content and main H, G and S lignin-derived monomers obtained by thioacidolysis

of extract-free senesced mature dried stems from wild-type (WT) and line

IRX5:HCHL (2). Values are means ± SE from duplicate analyses.











Total yield






Klason lignin
(H + G + S)

Plant line
KL % of CWR
μmol g −1 KL
% H
% G
% S
S/G












Culture 1
WT
20.42 ± 0.14
1356 ± 40
0.98 ± 0.00
73.2 ± 0.3
25.9 ± 0.3
0.35 ± 0.01


IRX5:HCHL
20.12 ± 0.15
1014 ± 5
1.48 ± 0.04
73.7 ± 0.5
25.2 ± 0.3
0.34 ± 0.01

Culture 2
WT
20.32 ± 0.25
1238 ± 13
1.09 ± 0.00
73.8 ± 0.3
25.2 ± 0.3
0.34 ± 0.01


IRX5:HCHL
21.29 ± 0.14
1041 ± 7
1.47 ± 0.00
72.7 ± 0.1
25.9 ± 0.1
0.36 ± 0.00




































TABLE VIII



Minor monomers obtained by thioacidolysis of extract-free mature senesced dried stems from wild-type

(WT) and line IRX5:HCHL (2). Values are means ± SE of duplicate analyses. Values are expressed in μmol

g −1 KL and as a relative percentage of the total main H, G and S monomers released by thioacidolysis.











Vanalc
Syralc
Van
Syrald
Cald
VA
SyrA


μmol g −1 KL
μmol g −1 KL
μmol g −1 KL
μmol g −1 KL
μmol g −1 KL
μmol g −1 KL
μmol g −1 KL

Plant line
(% H + G + S)
(% H + G + S)
(% H + G + S)
(% H + G + S)
(% H + G + S)
(% H + G + S)
(% H + G + S)













Culture 1
WT
nd*
nd*
4.3 ± 1
0.9 ± 0.3
7.2 ± 0.6
6.7 ± 0.2
1.4 ± 0.0


IRX5:HCHL
5.0 ± 0.1
2.6 ± 0.2
(0.31)
(0.06)
(0.53)
(0.49)
(0.10)



(0.49)
(0.25)
6.5 ± 1.4
18.7 ± 3.5
7.9 ± 0.3
6.8 ± 0.2
2.2 ± 0.0





(0.64)
(1.84)
(0.77)
(0.67)
(0.21)

Culture 2
WT
nd*
nd*
4.6 ± 0.7
0.8 ± 0.3
6.9 ± 0.1
6.2 ± 0.2
1.2 ± 0.0


IRX5:HCHL
5.3 ± 0.1
2.9 ± 0.1
(0.37)
(0.06)
(0.55)
(0.50)
(0.09)



(0.50)
(0.28)
6.3 ± 0.7
16.7 ± 1.9
6.8 ± 0.1
7.0 ± 0.0
2.1 ± 0.0





(0.60)
(1.60)
(0.66)
(0.65)
(0.20)



*nd, not detected.







































TABLE IX



Comparative transcriptomics of IRX5:HCHL stems and WT. Positive and negative ratios are indicative of

upregulation and downregulation of the gene in plants expressing HCHL.






AGI Gene ID
Annotated Function
log2 ratio
P value









MONOOXYGENASES



AT1G62570
flavin-containing monooxygenase family protein

0.00E+0

AT3G28740
cytochrome P450 family protein

0.00E+0

AT4G15760
monooxygenase, putative (MO1)
0.86

AT4G37370
CYP81D8
0.72
1.20E−7

AT3G28740
cytochrome P450 family protein
0.70
5.58E−7

AT2G12190
cytochrome P450, putative
0.65
8.60E−6

AT1G69500
CYP704B1
0.58
7.38E−4

AT3G14610
CYP72A7
0.51
2.96E−2


DEHYDROGENASES/REDUCTASES

AT4G13180
short-chain dehydrogenase/reductase (SDR) family protein

0.00E+0

AT2G37770
aldo/keto reductase family protein, Transcript variant 1

0.00E+0

AT2G37770
aldo/keto reductase family protein, Transcript variant 2
0.96
0.00E+0

AT2G29350
SAG13 (Senescence-associated gene 13); short-chain dehydrogenase/reductase (SDR) family protein
0.83

AT1G14130
2-oxoglutarate and Fe(II)-dependent oxygenese superfamily protein
0.72
9.59E−8

AT1G72680
cinnamyl-alcohol dehydrogenase, putative
0.90
0.00E+0

AT1G60730
aldo/keto reductase family protein
0.65
8.30E−6

AT1G18020
FMN-linked oxidoreductases superfamily protein, Transcript variant 1
0.62
7.14E−5

AT1G18020
FMN-linked oxidoreductases superfamily protein; Transcript variant 2
0.59
4.38E−4

AT2G47130
short-chain dehydrogenase/reductase (SDR) family protein, Transcript variant 1
0.58
7.60E−4

AT2G47130
short-chain dehydrogenase/reductase (SDR) family protein, Transcript variant 2
0.58
8.36E−4

AT1G18020
FMN-linked oxidoreductases superfamily protein, Transcript variant 3
0.54
6.80E−3

AT5G14780
FDH (FORMATE DEHYDROGENASE); NAD binding/oxidoreductase, acting the CH—OH group of donors
0.53
1.24E−2

AT1G54100
ALDH7B4 (ALDEHYDE DEHYDROGENASE 7B4); 3-chloroallyl aldehyde dehydrogenase
0.51
3.00E−2


UDP-GLUCOSYLTRANSFERASES

AT1G05560
UGT75B1

0.00E+0

AT2G15490
UGT73B4

0.00E+0

AT4G34138
UGT73B1

0.00E+0

AT2G30140
UGTB7A2
0.79

AT4G34131
UGT73B3
0.58
8.09E−4

AT3G11340
UGT76B1
0.58
8.97E−4

AT4G01070
UGT72B1
0.52
2.00E−2


TRANSPORTERS

AT3G23560
ALF5 (ABERRANT LATERAL ROOT FORMATION 5), antiporter/transporter

0.00E+0

AT2G36380
PDR6 (PLEIOTROPIC DRUG RESISTANCE 6) ATPase, coupled to transmembrane movement of substances

0.00E+0

AT3G51860
CAX3 (cation exchanger 3); cation; cation antiporter

0.00E+0

AT5G65380
Multidrug and toxic compound extrusion (MATE) efflux family protein
0.92
0.00E+0

AT1G79410
ATOCT5 (organic cation/carnitine transporter 5)
0.89
0.00E+0

AT5G13750
ZIFL1 (ZINC INDUCED FACILITOR-LIKE 1); tetracycline:hydrogen antiporter/transporter
0.78

AT1G76520
auxin efflux carrier family protein
0.70
4.27E−7

AT1G76530
auxin efflux carrier family protein
0.69
8.91E−7

AT4G18197
AT4G18200/PUP7 (purine permease 7); purine transporter
0.64
1.62E−5

AT4G28390
AAC3 (ADP/ATP CARRIER 3); ATP:ADP antiporter/binding
0.62
6.57E−5

AT5G45380
DUR3 (DEGRADATION OF UREA 3); sodium:solute:symporter family protein
0.61
1.05E−4

AT3G18830
PLT5 (POLYOL TRANSPORTER 5)
0.57
1.56E−3

AT2G17500
auxin efflux carrier family protein
0.55
3.26E−3


DETOXIFICATION

AT1G17170
ATGSTU24 (Glutathione S-transferase (class tau) 24)

0.00E+0

AT2G29420
ATGSTU7 (GLUTATHIONE S-TRANSFERASE 25)

0.00E+0

AT2G47730
ATGSTF8 (GLUTATHIONE S-TRANSFERASE 8)

0.00E+0

AT4G02520
ATGSTF2 (Glutathione S-transferase (class phi) 2)
0.78

AT3G09270
ATGSTU8 (Glutathione S-transferase (class tau) 8)
0.65
1.43E−5

AT2G29490
ATGSTU1 (GLUTATHIONE S-TRANSFERASE 19)
0.54
7.21E−3

AT4G19880
unknown protein, Glutathione S-transferase family protein
0.76

AT5G39050
ATPMaT1 (phenolic glucoside malonyltransferase 1); transferase family protein
0.77

AT5G39090
ATPMaT1-like; transferase family protein
0.52
2.13E−2


JASMONIC ACID METABOLISM

AT1G76680
OPR1 (12-oxophytodienoate reductase 1)

0.00E+0

AT5G54206
12-oxophytodienoate reductase-related
0.99
0.00E+0


STRESS INDUCIBLE/DEFENSE/SENESCENCE

AT5G49480
ATCP1 (CA2+-BINDING PROTEIN 1); calcium ion binding, NaCl stress inducible

0.00E+0

AT1G35260
Bet v I allergen family protein, defense response
0.88
0.00E+0

AT3G62550
universal stress protein (USP) family protein, Adenine nucleotide alpha-like protein
0.87
0.00E+0

AT1G73500
ATMKK9 (Arabidopsis thaliana MAP kinase kinase 9)
0.80

AT4G02380
SAG21 (SENESCENCE-ASSOCIATED GENE 21)
0.77

AT3G04720
PR4 (PATHOGENESIS-RELATED 4), similar to the antifungal chitin-binding protein hevein
0.64
2.04E−5

AT1G75270
DHAR2; glutathione dehydrogenase (ascorbate)
0.61
1.17E−4

AT1G70530
CRK3 (CYSTEINE-RICH RLK (RECEPTOR-LIKE PROTEIN KINASE) 3), protein kinase family protein
0.60
2.36E−4

AT3G50970
LTI3O/XERO2 (LOW TEMPERATURE-INDUCED 30); dehydrin stress-related
0.58
8.28E−4

AT5G27760
hypoxia-responsive family protein
0.54
6.87E−3

AT3G56710
SIB1 (SIGMA FACTOR BINDING PROTEIN 1); binding
0.51
2.30E−2


MISCELLANEOUS


Transcription factor

AT5G63790
ANAC102 (Arabidopsis NAC domain containing protein 102); transcription factor. Transcript variant 1

0.00E+0

AT1G77450
ANAC032 (Arabidopsis NAC domain containing protein 32); transcription factor

0.00E−0

AT5G63790
ANAC102 (Arabidopsis NAC domain containing protein 102); transcription factor, Transcript variant 2
0.65
1.26E−5

AT1G01720
ATAF1 (Arabidopsis NAC domain containing protein 2); transcription factor
0.54
7.23E−3


Glycine-rich protein

AT2G05380
GRP3S (GLYCINE-RICH PROTEIN 3 SHORT ISOFORM) Transcript variant 1

0.00E+0

AT2G05380
GRP3S (GLYCINE-RICH PROTEIN 3 SHORT ISOFORM) Transcript variant 2

0.00E+0

AT2G05530
glycine-rich protein
0.96
0.00E+0

AT2G05540
glycine-rich protein
0.90
0.00E+0


Auxin metabolism

AT3G44300
NIT2 (NITRILASE 2)

0.00E+0

AT3G44310
NIT1 (NITRILASE 1)
0.51
3.32E−2


Other

AT5G30870
transposable element gene; pseudogene, hypothetical protein

0.00E+0

AT3G14990
4-methyl-5(b-hydroxyethyl)-thiazole monophosphate biosynthesis protein, putative

0.00E+0

AT1G65280
heat shock protein binding/unfolded protein binding

0.00E+0

AT4G16190
cysteine proteinase, putative
0.89
0.00E+0

AT1G02850
glycosyl hydrolase family 1 protein BGLU11
0.86

AT1G17860
trypsin and protease inhibitor family protein/Kunitz family protein
0.86

AT3G49780
ATPSK4 (PHYTOSULFOKINE 4 PRECURSOR); growth factor
0.82

AT2G41380
embryo-abundant protein-related, methyltransferase activity
0.82

AT3G24420
hydrolase, alpha/beta fold family protein
0.79

AT5G52810
ornithine cyclodeaminase/mu-crystallin family protein
0.67
2.65E−6

AT5G17380
pyruvate decarboxylase family protein
0.64
1.84E−5

AT1G23890
NHL repeat-containing protein
0.59
3.35E−4

AT4G28380
leucine-rich repeat family protein, zinc ion binding
0.59
3.52E−4

AT4G01870
tolB protein-related
0.59
4.41E−4

AT1G37130
NIA2 (NITRATE REDUCTASE 2)
0.62
6.75E−5

AT1G24610
SET domain-containing protein, unknown protein
0.58
9.14E−4

AT4G11600
ATGPX6 (GLUTATHIONE PEROXIDASE 6); glutathione peroxidase
0.52
1.83E−2


UNKNOWN

AT5G61820
unknown protein

0.00E+0

AT1G76600
unknown protein

0.00E+0

AT1G76960
unknown protein
0.71
2.01E−7

AT4G17840
unknown protein
0.67
2.77E−6

AT1G21680
unknown protein
0.61
1.38E−4

AT5G40960
unknown protein, DUF3339
0.59
5.08E−4

AT4G08555
unknown protein
0.58
8.16E−4

AT2G30690
unknown protein, DUF593
0.53
8.86E−3

AT5G66052
unknown protein
0.50
4.05E−2


CARBOHYDRATE METABOLISM

AT2G06850
EXGT-A1 (ENDO-XYLOGLUCAN TRANSFERASE); hydrolase, acting on glycosyl bonds
−0.51
2.51E−2

AT3G52840
BGAL2 (beta-galactosidase 2), Glycoside hydrolase family 35, putative lactase
−0.52
1.83E−2

AT3G01345
Glycoside hydrolase family 35, beta-galactosidase putative
−0.53
1.13E−2

AT3G53190
pectate lyase family protein
−0.56
1.69E−3

AT5G03350
legume lectin family protein, carbohydrate binding
−0.57
1.28E−3

AT1G26810
GALT1 (galactosyltransferase 1), Glycoside transferase family 31
−0.61
1.02E−4

AT1G19600
pfkB-type carbohydrate kinase family protein
−0.63
3.41E−5

AT4G28250
ATEXPB3 (ARABIDOPSIS THALIANA EXPANSIN B3)
−0.79

AT3G30720
unknown protein, QUA-QUINE STARCH (QQS)
−1.08
0.00E+0


MISCELLANEOUS

AT4G27440
PORB (PROTOCHLOROPHYLLIDE OXIDOREDUCTASE B); protochlorophyllide reductase
−0.50
4.45E−2

AT5G02890
HXXXD-type acyl-transferase family protein
−0.50
3.86E−2

AT1G18950
aminoacyl-tRNA synthetase family
−0.51
3.29E−2

AT5G47330
palmitoyl protein thioesterase family protein
−0.53
1.07E−2

AT1G03870
FLA9 (FLA9)
−0.54
4.82E−3

AT1G20530
unknown protein, DUF630 and DUF632
−0.55
4.67E−3

ATCG00470
ATP SYNTHASE EPSILON CHAIN, rotational mechanism
−0.55
3.12E−3

AT5G51720
unknown protein, 2 iron, 2 sulfur cluster binding
−0.56
2.02E−3

ATCG00330
RPS14, CHLOROPLAST RIBOSOMAL PROTEIN S14
−0.58
8.70E−4

ATCG00340
D1 subunit of photosystem I and II reaction centers, Transcript variant 1
−0.62
5.42E−5

AT2G38870
serine-type endopeptidase inhibitor activity, pathogenesis-related peptide of the PR-6 proteinase inhibitor family
−0.64
1.54E−5

ATCG00340
D1 subunit of photosystem I and II reaction centers, Transcript variant 2
−0.71
2.75E−7