(en)Described herein are compositions comprising at least one auxin transport inhibitor for pre-treating a plant or seed to increase saccharification, or saccharide release by hydrolysis, the at least one auxin transport inhibitor being in an amount effective to increase sugar release from a plant tissue by hydrolysis. Also described are plant mutations, and methods to screen for such plant mutations, having an improved sugar release phenotype. The described compositions, methods and plant mutations are particularly useful for producing biofuel crops, such as maize, to improve sugar extractability from lignocellulosic biomass and hence, the efficiency of bioethanol production overall.

1.ApplicationNumber: US-201715824013-A
1.PublishNumber: US-2018171381-A1
2.Date Publish: 20180621
3.Inventor: BONETTA, Dario Torquato
McCOURT, Peter John
VIDAURRE, Danielle
STAMATIOU, George

4.Inventor Harmonized: BONETTA DARIO TORQUATO(CA)
MCCOURT PETER JOHN(CA)
VIDAURRE DANIELLE(CA)
STAMATIOU GEORGE(CA)

5.Country: US
6.Claims:
(en)Described herein are compositions comprising at least one auxin transport inhibitor for pre-treating a plant or seed to increase saccharification, or saccharide release by hydrolysis, the at least one auxin transport inhibitor being in an amount effective to increase sugar release from a plant tissue by hydrolysis. Also described are plant mutations, and methods to screen for such plant mutations, having an improved sugar release phenotype. The described compositions, methods and plant mutations are particularly useful for producing biofuel crops, such as maize, to improve sugar extractability from lignocellulosic biomass and hence, the efficiency of bioethanol production overall.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation that claims benefit under 35 U.S.C. § 120 of co-pending U.S. application Ser. No. 14/388,089 filed Sep. 25, 2014, which is a 35 U.S.C. § 371 National Phase Entry Application of International Application No. PCT/CA13/00289 filed Mar. 26, 2013, and which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 61/615,530 filed Mar. 26, 2012 the contents of which are incorporated herein by reference in their entireties.


SEQUENCE LISTING
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 22, 2014, is named 924270WO_ST25.txt and is 257,246 bytes in size.
FIELD OF THE INVENTION
The present invention is directed to compositions and methods for improving saccharide extraction from biomass, as well as to methods for identifying mutations that affect saccharide extraction. More particularly, the invention relates to compositions comprising auxin transport inhibitors, methods relating thereto, mutant plant varieties, and methods of genetic screening for such mutations that affect saccharification in plant tissue.
BACKGROUND OF THE INVENTION
Plant biomass and in particular cellulosic ethanol has gained considerable interest as a stable, environmentally benign source of energy that could partially offset fossil fuels. However, the encapsulation of cellulose and branched polysaccharides collectively known as hemicellulose lignin, together with the crystalline nature of cellulose, make the biochemical conversion of lignocellulosic biomass to biofuels a costly and energy inefficient process. The recalcitrance of lignocellulose has led to the development of a variety of technologies that usually involve the deconstruction of plant cell walls through acid, thermochemical, or enzymatic hydrolysis. For example, hemicellulose can be hydrolyzed by dilute acid treatments, but these conditions are not severe enough for cellulose hydrolysis. Increasing acid concentrations or carrying out acid treatments at high temperature and pressure improves sugar yields from cellulose, but both processes are corrosive and increase costs. Unfortunately, enzymatic approaches of digesting lignocellulose are still in their infancy. Moreover, the protective nature of the cell wall to cellulases means digestion is slow and inefficient. As a consequence, acid hydrolysis pretreatments are often used to depolymerize and solubilize hemicelluloses.
The lack of energy efficient and environmentally friendly conversion of lignocellulosic polymers into fermentable sugars, or saccharification, has spurred interest in using genetic and genomic approaches that modify the cell wall for industrial processing. Often these approaches have involved manipulating known cell wall synthesis or degradation enzymes. Although these rational approaches are promising they depend on a prior molecular knowledge of the genes of interest, usually followed by reverse genetics to test functionality.
Most approaches to genetically improving conversion of lignocellulosic biomass into a fermentable sugar source take advantage of our understanding of cell wall polymer synthesis. This usually involves manipulating glycosyltransferases and glycan synthases that are involved in polymerizing polysaccharides or modulating levels of lignin. However, the rudimentary knowledge about the regulation of this complex matrix limits this approach. For example, estimates of over 1000 cell wall proteins in Arabidopsis alone make it difficult to know which ones will functionally influence saccharification. Furthermore, over 700 genes are annotated as encoding putative glycosyltransferases or glycosyl hydrolases.
By contrast, forward genetic screens, which inherently have no mechanistic bias have the potential to uncover novel processes that could improve saccharification. The limitation of forward screens, however, is designing specific high throughput assays, followed by efficient molecular identification of the genes involved. In this latter case, however, the recent development of next generation sequencing technologies to identify mutant alleles has greatly reduced this bottleneck.
SUMMARY OF THE INVENTION
The invention is directed to a use of an auxin transport inhibitor in the pretreatment of a plant tissue to increase the sugar released from the plant tissue through hydrolysis.
The invention is further directed to the use of a genetically modified plant that has disrupted auxin transport to increase the sugar released from the plant through hydrolysis.
The invention is further directed to the use of a genetically modified plant that contains cell wall defects to increase the sugar released from the plant through hydrolysis.
The invention is further directed to the use of genetically modified plant tissue with increased starch accumulation to increase the sugar released from the plant through hydrolysis.
The invention is further directed to the use of any of the forgoing in production of bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textile, chemical, biocosmetic, and feed stock production.
The invention is further directed to a method of identifying plant genotypes that show an improved sugar release under mild acid treatment comprising the following steps:
a) providing a plurality of mutated plant seeds;
b) germinating the mutated plant seeds;
c) retrieving samples from each mutated plant seed;
d) submerging the samples in a weak acid;
e) incubating the samples with a colorimetric reagent in a concentrated acid; and
f) measuring the colour absorbance to determine the relative concentration of the sugar release.
The invention is further directed to a screening method to identify new plant cellulose synthase (CESA) alleles wherein mutagenized plants are screened with a cellulose biosynthetic inhibitor (CBI).
The invention is further directed to the use of an X-ray diffractometer to measure the proportion of crystalline cellulose relative to the proportion of amorphous cellulose in plant stem tissue.
The invention is further directed to the use of forward genetic screens for identifying mutants with improved saccharification from plant tissues.
The invention is further directed to the use of a forward genetic screen for identifying mutations that show increased sugar release from plant biomass as compared with wild types, under mild acid hydrolysis conditions.
The invention is further directed to a method of identifying genes involved with saccharification by means of a genetic screen.
According to an aspect of the invention, there is provided a composition for pre-treating a plant tissue to increase saccharide, or sugar, release from said plant tissue by hydrolysis, the composition comprising at least one auxin transport inhibitor in an amount effective to increase sugar release from said plant tissue by hydrolysis.
In a further aspect of the invention, there is also provided a method of pre-treating a plant tissue to increase saccharide release the said plant tissue by hydrolysis, the method comprising administering a composition as defined herein in an amount effective to increase sugar release from the plant, or tissues thereof, by hydrolysis.
Also provided is a method of screening for plants having an increased saccharide release phenotype, a reduced cellulose crystallinity phenotype, or both. The method comprises:
treating at least one plant or plant seed with at least one cellulose biosynthetic inhibitor (CBI) in an amount effective to select for CBI-resistance in the plant or plant seed; germinating the plant seeds and/or incubating the plant and selecting for CBI-resistant mutant plants, or seeds thereof; and measuring saccharide release, cellulose crystallinity, or both, in the CBI-resistant mutant plants to identify an increased saccharide release phenotype, a reduced cellulose crystallinity phenotype, or both.

Other details and aspects of the invention will be apparent from the following description of these compositions, uses and methods, as well as the mutant plants and genes described in detail throughout this application.



BRIEF DESCRIPTION OF THE FIGURES
This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
These and other features of the invention will become more apparent from the description, in which reference is made to the following drawings wherein:
FIGS. 1A-1B illustrate methodology and results of screening for wall hydrolysis sensitive (whs) mutants. FIG. 1A (PRIOR ART) is a schematic of the production of ethanol from cellulosic biomass. For biomass pretreatment, dilute sulphuric acid is used to solubilize the hemicellulosic fraction and to disrupt the crystalline structure of cellulose so that hydrolyzing enzymes can easily access and convert cellulose to fermentable sugars. FIG. 1B illustrates the results of measuring hexose content in known cell wall mutants subjected to acid hydrolysis using 1M H 2 SO 4 at 21 days after germination (DAG). Of the 30 cell wall mutants tested, only mur11-1 showed a significant difference in cell wall accessibility relative to wild type. All experiments were repeated at least three times with similar results. Dotted line denotes wild type levels (Results are averages ±s.d. (n=4). *, P<0.05 using Student's t-test.) FIG. 1C shows the results of measuring hexose content in mur11-1 and sac9-3 (SALK_058870) relative to wild type. Leaf discs were assayed for increased saccharification using 1M H 2 SO 4 at 21 days. (Results are averages ±s.d. (n=8-10).)
FIGS. 2A-2C illustrate the results of characterizing whs mutants. FIG. 2A shows three-week old Arabidopsis plants grown in 96-well flats at 22° C. under a 16 h/8 h light/dark cycle (top panel). Leaf 3 or 4 was excised from 21 day-old plants using a hole punch and subjected to acid hydrolysis using 1 M H 2 SO 4 . c; cotyledon, leaf numbers indicated (middle panel). Results of colorimetric anthrone assay illustrating that whs mutants release more sugars and turn a blue/green colour. Yellow indicates baseline levels of sugar release (bottom panel). FIG. 2B shows the hierarchical cluster analysis of monosaccharide composition analysis by gas chromatography of whs mutants in 21 day-old seedlings. Values are shown as a percentage relative to wild type. Yellow indicates high expression and blue indicates low expression. FIG. 2C shows a clustered heatmap of hexose content from 63 whs mutants subjected to acid hydrolysis of fresh leaf tissue using 1M H 2 SO 4 , acid hydrolysis of senesced whole plant tissues using 0.2 M H 2 SO 4 , enzymatic assays using cellulase, cellulase+xylanase and cellulase+peroxidase and starch staining of 14 day-old seedlings. Values are shown as a percentage relative to wild type. Yellow indicates high expression and black indicates low expression.
FIGS. 3A-3C illustrate the starch analysis of whs mutants mur11, dpe2 and sex4. FIG. 3A shows the acid hydrolysis of fresh leaf disc tissue from known starch mutants using 1 M H 2 SO 4 . (Results are averages ±s.d. (n=4); all experiments were repeated at least three times with similar results.) FIG. 3B shows the treatment of senesced material from starch mutants with α-amylase and the quantification of the amount of starch released using the anthrone method. (Results are averages ±s.d. (n=4); all experiments were repeated two times with similar results.) FIG. 3C shows the assay of the tissue by acid hydrolysis for residual hexose release using 1 M H 2 SO 4 , post-amylase treatment. (Results are averages ±s.d. (n=3).)
FIGS. 4A-4D illustrate the analysis of pin-shaped inflorescence mutants and NPA treatment, resulting in increased saccharification in Arabidopsis and maize. FIG. 4A shows senesced tissue from Arabidopsis pin-shaped inflorescence mutants subjected to 0.2 M acid hydrolysis. (Results are averages ±s.d. (n=3); all experiments were repeated three times with similar results.) Inset shows representative pin-shaped inflorescence in Arabidopsis . FIG. 4B shows maize inflorescence mutants bif2 and bat subjected to 0.2 M H 2 SO 4 acid hydrolysis. (Results are averages ±s.d. (n=3-4). N, phenotypically normal siblings.) Inset shows representative maize inflorescence mutant. FIG. 4C shows wild type (Col-0) Arabidopsis 28 day-old seedlings grown on MS media supplemented with 0, 1 or 5 μM NPA and subjected to 0.2 M H 2 SO 4 acid hydrolysis. (Results are averages ±s.d. (n=4). *, P<0.001 and **, P<0.005 using Student's t-test; all experiments were repeated two times with similar results.)
FIG. 4D shows two maize cultivars treated with 120 μM NPA for 2 weeks and subjected to 0.2 M H 2 SO 4 acid hydrolysis. (Results are averages ±s.d. (n=6-9).)
FIG. 5 shows absorbance readings from anthrone acid hydrolysis as quantified against a glucose curve. Candidate whs mutants are considered as releasing a significant amount of sugars when readings measure 2 or more standard deviations above wild type (Abs 660 nm 0.12±0.002).
FIG. 6 shows the map based cloning of cell wall accessible genes.
FIG. 7 shows the wall hydrolysis sensitivity of the SAC domain family in Arabidopsis using the following T-DNA insertions: sac1-1 (SALK_070875), sac1-2 (SALK_020109), sac2-1 (SALK_099031), sac2-2 (SALK_091926), sac3-1 (SALK_023548), sac3-2 (SALK_049623), sac4-1 (SALK_119184), sac4-2 (SALK_005871), sac4-3 (SALK_056500), sac5-1 (SALK_012372), sac5-2 (SALK_125856), sac6-1 (SALK_021488), sac6-2 (SALK_136049), sac7-1 (SALK_000558), sac7-2 (SALK_092575), sac8-1 (SALK_062145) and sac8-2 (SALK_115643). Leaf disc tissue from 21 day-old plants was assayed using 1 M H 2 SO 4 . (Results are averages ±s.d. (n=3-4).)
FIG. 8 shows the wall hydrolysis sensitivity of auxin response factor mutants. Leaf disc tissue from 21 day-old plants was assayed using 1 M H 2 SO 4 . (Results are averages ±s.d. (n=4-8).)
FIG. 9 shows the relative cellulose crystallinity of wt (Col, Ler) and mutant lines. “C” refers to Col-0; “L” refers to Ler; each instance of “f” denotes a fxr mutant line; and each instance of “ix” denotes an ixr mutant line.
FIG. 10 shows the percent total sugar releases following hydrolysis of wt (Col, Ler) and mutant stem tissue using different treatments.



DETAILED DESCRIPTION
Described herein are compositions, methods, mutant genes, cells, plants and other materials which are useful to increase carbohydrate availability for saccharification, in particular, through pre-treatment of a plant with an auxin transport inhibitor.
Saccharification is generally known as the process of breaking a complex carbohydrate (such as starch or cellulose) into its monosaccharide components. By increasing carbohydrate availability for saccharification, the compositions, methods, mutant genes, cells, plants and other materials described in this application can be used for a variety of industrial processes. For instance, they may be used to pretreat feedstock typically used in the biofuels industry for production of bioethanol. They may be employed in the production of biomass which is, for example, useful in producing biofuels, bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textile, chemical, biocosmetics, and in other feed stock production.
The compositions and methods described herein are applicable in a variety of plant species. Of interest are the monocotyledonous plants, e.g. corn ( Zea mays ), sugar cane ( Saccharum sp.), switchgrass ( Panicum virgatum ) and other grass species ( Miscanthus ), and other species used in bioethanol production. However, the present invention is also applicable in dicotyledonous plants, e.g. Arabidopsis, . . .
In certain embodiments of the invention, the auxin transport inhibitor may include at least one of the following: 1-N-Naphthylphthalmaic acid (NPA), 2-{(E)-1-[4-(3,5-difluorophenyl) semicarbazono] ethyl}nicotinic acid (diflufenzopyr), 2,3,5-triiodobenzoic acid (TIBA), 9-hydroxyfluorene-9-carboxylic acid (HFCA), p-chlorophenoxyisobutyric acid (PCIB), 2-carboxyphenyl-3-phenylpropane-1,2-dione (CPD), chlorflurenol, quimerac, tricyclopyr, CPIB, quercetin, genistein, including agriculturally acceptable salts, esters, or derivatives thereof.
Chemical structures for some of the above-listed compounds, and certain additional examples of auxin transport inhibitors, include the following:
















In certain preferred embodiments of the invention, the auxin transport inhibitor may be of a phthalamate (e.g. 1-N-naphthylphthalmaic acid (NPA)) or semicarbazone (2-{(E)-1-[4-(3,5-difluorophenyl)semicarbazono] ethyl}nicotinic acid (diflufenzopyr)) class of auxin transport inhibitor.
In certain other embodiments of the invention, which are non-limiting, the auxin transport inhibitor may be of the following molecular class of auxin transport inhibitors:









including agriculturally acceptable salts, esters, or derivatives thereof. The term “Ar” represents “aryl”, and refers to a monovalent unsaturated aromatic carbocyclic group having a single ring (e.g. phenyl) or multiple condensed rings (e.g. naphthyl or anthryl), which can optionally be unsubstituted or substituted with, e.g., halogen (for instance F, Cl, Br, or I), alkyl (for instance, a lower alkyl group), alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term “alkyl” refers to a cyclic, branched, or straight chain alkyl group containing only carbon and hydrogen, and unless otherwise mentioned contains one to twelve carbon atoms. This term is further exemplified by groups such as methyl, ethyl, n-propyl, isobutyl, t-butyl, pentyl, pivalyl, heptyl, adamantyl, and cyclopentyl. Alkyl groups can either be unsubstituted or substituted with one or more substituents, e.g. halogen, alkyl, alkoxy, alkylthio, trifluoromethyl, acyloxy, hydroxy, mercapto, carboxy, aryloxy, aryloxy, aryl, arylalkyl, heteroaryl, amino, alkylamino, dialkylamino, morpholino, piperidino, pyrrolidin-1-yl, piperazin-1-yl, or other functionality.
The term “lower alkyl” refers to a cyclic, branched or straight chain monovalent alkyl radical of one to seven carbon atoms. This term is further exemplified by such radicals as methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), cyclopropylmethyl, i-amyl, n-amyl, hexyl and heptyl. Lower alkyl groups can also be unsubstituted or substituted, where a specific example of a substituted alkyl is 1,1-dimethyl heptyl.
The auxin transport inhibitor may, in certain embodiments of the invention, be Naptalam, which is also known as N-1-naphthylphthalamic acid of the chemical formula:









including agriculturally acceptable salts, esters, or derivatives thereof.
Certain auxin transport inhibitors, including NPA and diflufenzopyr, may have functional groups which can be ionized, and accordingly can also be used in the form of an agriculturally acceptable salt. In general, an “agriculturally acceptable” salt will be a salt form whose cation has no adverse effect on the action of the active compound. For example, agriculturally acceptable cations may include ions of the alkali metals, such as lithium, sodium and potassium; of the alkaline earth metals, such as calcium and magnesium; of the transition metals, such as manganese, copper, zinc and iron; ammonium; substituted ammonium (organoammonium) ions in which one to four hydrogen atoms are replaced by C 1 -C 8 -alkyl, hydroxy-C 1 -C 4 -alkyl, in particular hydroxy-C 2 -C 4 -alkyl, C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, in particular C 1 -C 4 -alkoxy-C 2 -C 4 -alkyl, hydroxy-C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl, in particular hydroxy-C 2 -C 4 -alkoxy-C 2 -C 4 -alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, pentylammonium, hexylammonium, heptylammonium, 2-hydroxyethylammonium (olamine salt), 2-(2-hydroxyethoxy)eth-1-ylammonium (diglycolamine salt), di(2-hydroxyeth-1-yl)ammonium (=diethanolammonium salt or diolamine salt), tri(2-hydroxyethyl)ammonium (=triethanolammonium salt or trolamine salt), mono-, di- and tri(hydroxypropyl)ammonium (=mono-, di- and tripropanolammonium), benzyltrimethylammonium, benzyltriethylammonium; phosphonium ions; or sulfonium ions, preferably tri(C 1 -C 4 -alkyl)sulfonium such as trimethylsulfonium, and sulfoxonium ions, preferably tri (C 1 -C 4 -alkyl)sulfoxonium.
Auxin transport inhibitors, including N-1-naphthylphthalamic acid, may also carry a carboxyl group that can also be employed in the form of agriculturally acceptable derivatives, for example as amides such as mono- or di-C 1 -C 6 -alkylamides or arylamides, as esters, for example as allyl esters, propargyl esters, C 1 -C 10 -alkyl esters or alkoxyalkyl esters, and also as thioesters, for example as C 1 -C 10 -alkyl thioesters. Preferred mono- and di-C 1 -C 6 -alkylamides are the methyl- and the dimethylamides. Preferred arylamides are, for example, the anilidines and the 2-chloroanilides. Preferred alkyl esters are, for example, the methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, mexyl (1-methylhexyl) or isooctyl (2-ethylhexyl) esters. Preferred C 1 -C 4 -alkoxy-C 1 -C 4 -alkyl esters are the straight-chain or branched C 1 -C 4 -alkoxyethyl esters, for example the methoxyethyl, ethoxyethyl or butoxyethyl (butoyl) esters. An example of the straight-chain or branched C 1 -C 10 -alkyl thioesters is the ethyl thioester. Preferred derivatives are the esters.
The compositions of the invention preferably comprise N-1-naphthylphthalamic acid, or a salt or ester thereof. Suitable salts of N-1-naphthylphthalamic acid include those salts where the counterion is an agriculturally acceptable cation. In certain non-limiting embodiments, suitable salts of N-1-naphthylphthalamic acid may include the alkali metal salts, in particular the sodium and the potassium salts, and the ammonium or substituted ammonium salts, in particular the ammonium salt, the diethanolammonium salt, the diglycolammonion salt, the isopropylammonium salt, the dimethylammonium salt or the triethanolammonium salt.
The above-described compositions may be applied using any number of techniques as would be customary to one of skill in the art. Without wishing to be limiting in any way, the compositions may be applied e.g. by spraying or foliar application. A variety of spray application techniques are known and would be apparent to those of skill in the art. For example, the composition may be applied with water as a carrier, and applied to the soil and/or the plants at desired spray rates. In other embodiments of the invention, the composition may be applied by foliar application using an appropriate spray mixture.
It is also envisioned that the auxin transport inhibitor described herein may be used in combination with other compounds or agents, for instance, herbicidal agents, compound synergistic, fertilizers and the like. Such combinations may be formulated into a single composition, or applied separately.
Also provided herein is a method of pre-treating a plant to increase saccharide release from a plant tissue by hydrolysis, the method comprising administering an auxin transport inhibitor, or a composition as described herein, in an amount effective to increase sugar release from the plant tissue by hydrolysis.
In an embodiment of the above method, the auxin transport inhibitor or composition is administered in an amount effective to increase saccharide release from cellulose, starch, or both, in said plant tissue.
In addition, the method may further comprise a step of hydrolyzing cellulose, starch, or both, from the plant tissue, to produce monosaccharides, disaccharides, polysaccharides, or a combination thereof.
In a further non-limiting embodiment, the auxin transport inhibitor or composition may be applied by spraying, foliar application, or a combination thereof.
Also provided herein is a method of screening for plants having an increased saccharide release phenotype, a reduced cellulose crystallinity phenotype, or both, the method comprising:
treating at least one plant or plant seed with at least one cellulose biosynthetic inhibitor (CBI) in an amount effective to select for CBI-resistance in said plant or plant seed; germinating the plant seeds and/or incubating the plant and selecting for CBI-resistant mutant plants, or seeds thereof; and measuring saccharide release, cellulose crystallinity, or both, in the CBI-resistant mutant plants to identify an increased saccharide release phenotype, a reduced cellulose crystallinity phenotype, or both.

In a non-limiting embodiment of the method, the cellulose crystallinity may be measured using an X-ray diffractometer, for example, to determine a proportion of crystalline cellulose relative to a proportion of amorphous cellulose in a tissue of said CBI-mutagenized plant.
In a further non-limiting embodiment of the method, the tissue may be a stem and/or leaf tissue.
Without wishing to be limiting, the cellulose biosynthetic inhibitor may be of a nitrile, benzamide, triazolocarboxamide, or quinoline carboxylic acid class of cellulose biosynthetic inhibitor. For example, the cellulose biosynthetic inhibitor may be one or more of dichlobenil, chlorthiamid, isoxaben, flupoxam, quinclorac, or a salt, ester, or derivative thereof. In particular embodiments, the cellulose biosynthetic inhibitor may preferably comprise isoxaben or flupoxam.
Also described are uses of the compositions described herein for pre-treating a plant or plant tissue to increase saccharide release from the plant tissue by hydrolysis. For example, the plant or plant tissue may comprise biomass, e.g. for production of biofuel (such as bioethanol), bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textiles, monosaccharides, disaccharides, polysaccharides, other chemicals, as well as biocosmetics.
Also described herein are plant mutations which result in improved saccharide release upon hydrolysis treatment. Without limitation, the mutations may include one or more of the following mutations in maize or Arabidopsis genes, or equivalent genes having corresponding gene products in other plant species:
barren inflorescence2 (bif2), comprising a mutation in the bif2 sequence corresponding to SEQ ID NO: 1 reducing or substantially inhibiting bif2 function; barren stalk1 (BA1), comprising a mutation in the BA1 sequence corresponding to SEQ ID NO: 3, reducing or substantially inhibiting BA1 function; mur11-1 comprising a mutation corresponding to R278H in SEQ ID NO: 5, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant mur11-1 polypeptide or fragment thereof; pid-100 comprising a mutation corresponding to D223N in SEQ ID NO: 7, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant pid-100 polypeptide or fragment thereof; dpe2-100, comprising a mutation in the dpe2-100 sequence which reduces or substantially inhibits dpe2-100 function, such as but not limited to the W323Stop mutation in SEQ ID NO: 9, including nucleotides encoding the mutant dpe2-100 sequence; dpe2-101 comprising a mutation corresponding to R561K in SEQ ID NO: 11, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant dpe2-101 polypeptide or fragment thereof; sex4-100, comprising a mutation in the sex4-100 sequence which reduces or substantially inhibits sex4-100 function, such as but not limited to the sex4-100 splice junction mutant corresponding to SEQ ID NO: 13, or a fragment thereof containing a mutation corresponding to G2194A in SEQ ID NO: 13, including nucleic acid sequences that are 80% identical (or 85%, more particularly 90%, even more particularly 99% identical) thereto; fpx 2-1 comprising a mutation corresponding to G1013R in SEQ ID NO: 15, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 2-1 polypeptide or fragment thereof; fpx 2-2 comprising a mutation corresponding to P1010L in SEQ ID NO: 17, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 2-2 polypeptide or fragment thereof; fpx 2-3 comprising a mutation corresponding to G1009D in SEQ ID NO: 19, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 2-3 polypeptide or fragment thereof; fpx 1-1 comprising a mutation corresponding to 51040L in SEQ ID NO: 21, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 1-1 polypeptide or fragment thereof; fpx 1-2 comprising a mutation corresponding to 51037F in SEQ ID NO: 23, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 1-2 polypeptide or fragment thereof; fpx 1-3 comprising a mutation corresponding to S983F in SEQ ID NO: 25, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant fpx 1-3 polypeptide or fragment thereof; ixr1-3 comprising a mutation corresponding to G998S in SEQ ID NO: 27, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr1-3 polypeptide or fragment thereof; ixr1-4 comprising a mutation corresponding to R806K in SEQ ID NO: 29, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr1-4 polypeptide or fragment thereof; ixr1-5 comprising a mutation corresponding to L797F in SEQ ID NO: 31, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr1-5 polypeptide or fragment thereof; ixr1-6 comprising a mutation corresponding to S377F in SEQ ID NO: 33, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr1-6 polypeptide or fragment thereof; ixr1-7 comprising a mutation corresponding to R276H in SEQ ID NO: 35, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr1-7 polypeptide or fragment thereof; and ixr2-2 polypeptide comprising a mutation corresponding to S1002F in SEQ ID NO: 37, including polypeptides, polypeptide fragments, and nucleotides encoding the mutant ixr2-2 polypeptide or fragment thereof.

The above listed mutant nucleotide and polypeptide sequences may, in certain embodiments, be provided in isolated form, and may have 80% identity to their respective sequences listed, whereas in other embodiments the sequence identity may be higher, including 85%, 90%, or even 99% identical, including identity ranges intervening these integers. In addition, these same mutations may be made in corresponding sequences from other species, including both monocot and dicot species such as but not limited to corn ( Zea mays ), sugar cane ( Saccharum sp.), switchgrass ( Panicum virgatum ) and other grass species ( Miscanthus ), other species used in bioethanol production, as well as Arabidopsis and other dicotyledonous plant species.
Each of the above-listed mutants may also be provided in the form, for example, of a plant or seed thereof having a phenotype characterized by increased saccharide release from plant tissue by hydrolysis. In one non-limiting example, which can be applied throughout the above list of mutations, the plant or seed thereof may comprise a mutant barren inflorescence2 (bif2) gene comprising a mutation in the bif2 sequence corresponding to SEQ ID NO: 1 which reduces or substantially inhibits bif2 function. The plant or seed thereof may accordingly be used to produce biomass for production of bioethanol, bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textiles, monosaccharides, disaccharides, polysaccharides, or biocosmetics, preferably for production of bioethanol. The plant or seed thereof may also be provided, in non-limiting embodiments, in a commercial package comprising the plant or seed thereof, wherein the commercial package is for producing biomass for production of bioethanol, bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textiles, monosaccharides, disaccharides, polysaccharides, or biocosmetics.
Also provided herein are vectors, such as but not limited to plasmids, which include a nucleic acid or encoding a polypeptide sequence of one or more of the mutants described herein. Host cells comprising such vectors, or a nucleic acid encoding a polypeptide sequence of one or more of the mutants described herein are also provided. Similarly, seeds and plants may be provided which comprise such vectors and/or nucleic acids.
The seeds or plants containing these mutant sequences, or which express the mutant polypeptides described herein, have a phenotype which is characterized by an increased saccharide release from the plant tissue by hydrolysis.
Thus, the nucleic acids or polypeptides, the vectors, the host cells, the seeds and plants described herein can be used to produce plant tissues with a phenotype characterized by increased saccharide release by hydrolysis. These nucleic acids, polypeptides, vectors, host cells, seeds and plants are especially useful in producing biomass for production of biofuels (such as bioethanol), as well as bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textiles, monosaccharides, disaccharides, polysaccharides, and biocosmetics.
Experiments
A high-throughput strategy, using the model plant Arabidopsis , was used to identify mutants with improved sugar release from plant biomass. Molecular analysis showed a variety of processes, including starch degradation, cell wall composition and polar transport of the plant hormone auxin, can contribute to this improved saccharification. Genetic or chemical inhibition of polar auxin transport in maize is also shown to result in increased sugar release from plant tissues. This information not only uncovers new functions that contribute to cell wall integrity but also demonstrates that information gleaned from genetic approaches involving Arabidopsis can be directly translated to monocotyledonous biofuel crops, such as but not limited to maize, to improve sugar extractability from lignocellulosic biomass.
The high throughput strategy involved a forward genetic screen to identify genotypes that showed an improved sugar release under mild acid treatment, and identified a large collection of lines. The frequency of mutant identification (0.3%) and lack of many alleles within the collection suggested the screen was not saturated, and that more genetic variation remains to be discovered.
The identification of mutants that over-accumulate starch in vegetative tissues presents an unforeseen approach with respect to the improvement of fermentable sugars for biofuel production. Because starch is a simple easily accessible glycopolymer compared to lignocellulose, it is efficiently converted to sugar for ethanol production. However, unlike reproductive tissues such as corn kernels, starch levels in stems and leaves are limited, and therefore these vegetative tissues have not previously been considered a useful starch based feedstock.
The inventors have shown that genetically increasing vegetative starch levels can contribute to the overall fermentable sugar yields during acid pretreatment. Because this sugar source is not lignocellulosic, in principle its genetic manipulation should be a stackable trait with other lignocellulosic feedstock technologies. The observation that only some starch excess mutants were identified in the screens, however, suggests that the relationship between starch and acid-dependent sugar release is complex. Without wishing to be bound by theory, it is possible that certain mutants accumulate starch as a secondary consequence of a mutation. For example, not all sugar release from mur11 mutants is explained through starch accumulation, which is consistent with this mutant also having a defective cell wall. It is also possible that various starch accumulating mutants accumulate slightly different forms of starch, and that these forms may not be equally accessible to mild acid hydrolysis.
An association between cell walls and auxin has existed for some time with respect to the role of this hormone in cell expansion. More recently, the demonstration that mutating the cellulose synthase gene CESA results in mislocalization of PIN1 efflux carriers further suggests a close linkage between auxin transport and cell wall synthesis. As shown in the experiments below, pinoid and additional pin-shaped inflorescence mutants have increased cell wall accessibility, which reveals an important role for auxin in maintaining the integrity of the cell wall. Interestingly, this association is limited to auxin mutants that display a pin-shaped inflorescence phenotype, which may mean that altering cell wall integrity contributes to aberrant inflorescence development.
The acid hydrolysis screen only identified pinoid loss-of-function mutants. Presumably, additional Arabidopsis mutants that form pin-shaped inflorescences such as pint or mp were not found because, unlike pinoid, these mutants are completely penetrant and therefore infertile. Although this makes propagation of these lines problematic, the pin-shaped phenotype may have advantages with respect to preventing gene flow among commercially grown transgenic crops.
The inventors also show that treatment of wild type Arabidopsis and maize plants with the polar auxin transport inhibitor, 1-N-Naphthylphthalamic acid (NPA), also results in increased saccharification. In contrast to making transgenic plants, which can be costly, time-consuming and often involve constitutive phenotypes, chemically-induced phenotypes using compounds such as NPA allows for more tailored temporal and spatial control of the cell wall composition. Moreover, NPA, which is already an approved pre-emergence herbicide, can be applied broadly, for example, to bio-energy crops that have rudimentary genetics, or that are difficult to transform.
Finally, the ability to increase saccharification using NPA suggests chemical genetic screening using Arabidopsis can be applied to develop further chemical leads that may be useful in pretreatment lignocellulosic processing. The experiments presented here show that the results obtained in Arabidopsis can be successfully translated to maize, and thus other monocot species, such as but not limited to sugarcane ( Saccharum sp.), Miscanthus or switchgrass, are expected to show similar results.
Example 1: Screening for Wall Hydrolysis Sensitive Mutants
A colorimetric assay was developed that allowed for the visualization of saccharification from plant tissue incubated in dilute acid at room temperature for one hour.
Using an anthrone reagent, which turns blue or green in the presence of sugars, (in this example, hexoses,) an average sugar release (4.1±0.1 μg sugar/leaf disc) from 100 wild type leaf samples was determined ( FIG. 5 ). With this baseline, the assay was applied against a collection of 30 known cell wall mutants as indexed by the Plant Cell Wall Biosynthesis Research Network (WallBioNet) ( FIG. 1( b ) ).
Table 1 shows known cell wall mutants and their gene products. MUR11 was molecularly identified in this study and is shown in the table in bold.







TABLE 1



Mutant
AGI
GENE



csld3-1
At3g03050
CELLULOSE SYNTHASE-LIKE 3

eld1-1
At3g08550
ELONGATION DEFECTIVE 1

fk
At3g52940
FACKEL

irx1
At4g18780
IRREGULAR XYLEM 1/CESA8

irx3
At5g17420
IRREGULAR XYLEM 3/CESA7/MUR10

irx4
At1g15950
IRREGULAR XYLEM 4/CINNAMOYL COA



REDUCTASE 1

ixr1-1
At5g05170
ISOXABEN RESISTANT 1/CESA3

lxr1-2
At5g05170
ISOXABEN RESISTANT 1/CESA3

lxr2-1
At5g64740
ISOXABEN RESISTANT 2/PROCUSTE1/CESA6

knf
At1g67490
KNOPF

mur1-1
At3g51160
GDP-D_MANNOSE-4,6-DEHYDRATASE

mur2-1
At2g03220
FUCOSYLTRANSFERASE 1

mur3-2
At2g20370
XYLOGLUCAN GALACTOSYLTRANSFERASE

mur4-2
At1g30620
UDP-D-XYLOSE 4-EPIMERASE

mur5-1

MURUS 5

mur6-1

MURUS 6

mur7-1

MURUS 7

mur8-1

MURUS 8

mur9-1

MURUS 9

mur10-2
At5g17420
CESA7/IRX3


mur11-1


At3g59770


SUPPRESSOR OF ACTIN 9


pmr4-1
At4g03550
POWDERY MILDEW RESISTANT 4

pmr5 pmr6-3
At5g58600;
POWDERY MILDEW RESISTANT 5;


At3g54920
POWDERY MILDEW RESISTANT 6

pnt1-1
At5g22130
PEANUT 1

prc1-1
At5g64740
PROCUSTE1/CESA6/IXR2

rhd1-1
At1g64440
ROOT HAIR DEFECTIVE 1/UDPOGLUCOSE 4-



EPIMERASE

rhd3-1
At3g13870
ROOT HAIR DEFECTIVE 3

rsw2-1
At5g49720
RADIAL SWELLING 2/IXR2

rsw3-1
At5g63840
RADIAL SWELLING 3

sos5-1
At3g46550
SALT OVERLY SENSITIVE 5

vtc1-1
At2g39770
VITAMIN C DEFECTIVE 1/GDP-MANNOSE



PYROPHOSPHORYLASE



















































Of the 30 mutants tested, only mur11-1 consistently showed increased saccharification relative to wild type. Map-based cloning of the mur11-1 allele identified a transition mutation (G→A) in a conserved domain of the previously characterized gene, SUPPRESSOR OF ACTIN9 (SAC9), which encodes a phosphoinositide phosphatase ( FIG. 6 ). Table 2 shows the genotypes used in the study.








TABLE 2



Allele
Lesion a
Genomic position b
Amino acid



mur11-1
G → A
1157 bp
R 278 → H


(SEQ ID NO: 6)

(SEQ ID NO: 5)

sac9-3
SALK_058870

pid-100
G → A
974 bp
D 223 → N


(SEQ ID NO: 8)

(SEQ ID NO: 7)

pid-14
SALK_049736

pid-2
CS8063

pin1-1; ttg-1
CS8065

pin1
SALK_047613

arf5-2
SALK_021319

dpe2-100
G → A
1457 bp
W 323 → Stop


(SEQ ID NO: 10)

(SEQ ID NO: 9)

dpe2-101
G → A
3201 bp
R 561 → K


(SEQ ID NO: 12)

(SEQ ID NO: 11)

dpe2-5
SALK_073273

sex4-100
G → A
2194 bp
Splice junction


(SEQ ID NO: 13)

sex4-5
SALK_126784

sex1-100
SALK_077211

isa3-3
CS88929

bam1
SALK_039895

bam2
SALK_020838

bam3
SALK_041214

bam4
SALK_037355



a Type of lesion due to EMS mutagenesis or T-DNA insertion.

b Position of base pair change is given from the start codon of genes isolated from the whs primary screen.








































This result was verified by demonstrating that other mur11 alleles also showed improved saccharification by acid hydrolysis ( FIG. 1( c ) ). Because previous biochemical analysis of sac9 mutants suggests this phosphatase modulates phosphoinositide signaling during stress, the original MUR11 cell wall defects may be a secondary consequence of the mutation. With the finding that mutations in SAC9 gave increased sugar release it was decided to assay loss-of-function alleles of the complete SAC family of genes in Arabidopsis (sac1-sac9). However, no other SAC genes were found that contributed to lignocellulose sugar release, which is perhaps not surprising since SAC9 is only distantly related to the other SAC members of this family ( FIG. 7 )
The scarcity of improved sugar release from the cell wall mutant collection underscored the limited utility of a reverse genetic approach to identify increased saccharification mutants using weak acid hydrolysis. The mutational space was therefore expanded by applying the acid screen to a population of EMS-mutagenized Arabidopsis seedlings ( FIG. 2( a ) ).
The screen was limited to plants that showed no obvious growth or developmental defects, since such defects would compromise the application value of the genes identified. From approximately 23,000 M2 plants representing 32 M1 parental groups, 63 mutants were identified that showed increased saccharification (Table 3). Designated wall hydrolysis sensitive (whs), the mutant lines were sub-categorized into four groups based on the amount of sugar they released per fresh leaf disc.





TABLE 3



Amount of hexose released (μg/fresh leaf disc)








4.5-9
9.1-13
13.1-17
17.1-21



# of mutants








30
21
10
3





whs34
whs14
whs4
whs1


whs35
whs15
whs5
whs2


whs36
whs16
whs6
whs3


whs37
whs17
whs7


whs38
whs18
whs8


whs39
whs19
whs9


whs40
whs20
whs10


whs41
whs21
whs11


whs42
whs22
whs12


whs43
whs23
whs13


whs44
whs24


whs45
whs25


whs46
whs26


whs47
whs27


whs48
whs28


whs49
whs29


whs50
whs30


whs51
whs31


whs52
whs32


whs53
whs33


whs54
mur11-1


whs55


whs56


whs57


whs58


whs59


whs60


whs61


whs62


whs63



























































To determine if any of these mutants showed defects in cell wall sugars, gas chromatographic analysis of alditol acetates was performed to identify changes in monosaccharide composition of the cell wall ( FIG. 2( b ) ). Interestingly many of the whs lines showed increases in rhamnose and fucose compared to wild type samples, which indicated that many of the mutations did perturb cell wall composition. Next, the mutant collection was further studied by enzymatic hydrolysis assays using cellulase and cellobiase, to assay cellulose hydrolysis, cellulase, cellobiase and xylanase, to monitor hemicellulose break down, and a cocktail of cellulase, cellobiase, xylanase and peroxidase which, in addition to cellulose and hemicellulose, degrades lignin ( FIG. 2( c ) ). The presence of starch in the samples was also assayed, as this source of carbon could potentially contribute to an increased sugar release phenotype in these assays. Finally, in addition to the fresh leaf material, an assay was carried out on senesced whole plant tissue hydrolyzed with 0.2 M sulphuric acid, biomass that is more akin to field grown plant material and acid concentrations that are more similar to industrial standards.
Hierarchical clustering of the various assays broadly identified three subcategories. One category consisted of five mutant lines (whs27, whs6, whs4, whs20, whs36) that showed good sugar release in both fresh and senesced tissue acid hydrolysis. A second category consisted of twelve lines (mur11-1, whs1, whs43, whs53, whs14, whs2, whs5, whs21, whs3, whs60, whs9, whs22) which hyper-accumulated starch. Within this grouping, two lines (whs9 and whs22) were of particular interest as they also showed excess sugar release in all enzymatic assays. The remaining mutant lines did not show good saccharification in senesced tissues or in any enzymatic assay and therefore were not further studied.
Example 2: Specific Genes Involved in Starch Metabolism Improve Saccharification
To understand the molecular nature of the mutant category that showed both a high saccharification and increased starch accumulation, map-based cloning of the mutant alleles was performed on three lines (whs1, whs22 and whs9). The whs1 and whs22 lines contained allelic mutations in the DISPROPORTIONATING ENZYME 2 (DPE2) gene, which encodes a glucosyltransferase required for starch degradation, and these lines were subsequently re-designated dpe2-100 and dpe2-101 respectively ( FIG. 6 , Table 2). Subsequent molecular analysis of lines whs3, whs5, whs14, whs21 showed they were siblings of whs1. The whs9 line contained a new allele of STARCH EXCESS 4 (sex4-100), which encodes a glycan phosphatase involved in starch degradation ( FIG. 6 , Table 2).
The identification of these genes was validated by showing that T-DNA knockout insertion alleles in both DPE2 and SEX4 also showed improved sugar release by acid hydrolysis ( FIG. 3( a ) ).
The identification of dpe2 and sex4 in the screens suggested that starch could be a source of acid-dependent sugar release. The contribution of starch to saccharification was determined by treating senesced whole plant tissue with α-amylase, which specifically converts starch to glucose and maltose ( FIG. 3( b ) ). Once tissue was devoid of starch, it was subjected to acid hydrolysis to determine the residual hexose release ( FIG. 3( b ) ). This analysis clearly showed that the improved sugar release observed in both dpe2 and sex4 mutants can be accounted for by their increased starch content. By contrast, the mur11-1 samples showed a higher sugar release than wild type even after a-amylase treatment, suggesting some of the increased saccharification is due to polymers other than starch.
The connection of starch over-accumulation and increased saccharification by acid hydrolysis was further explored by subjecting a collection of well characterized Arabidopsis starch mutants to the acid hydrolysis assay. The analysis included starch-excess 1 (sex1), which is defective in the regulation of starch degradation, isoamylase 3 (isa3), which is defective in a starch debranching enzyme 15, and b-amylase (barn) mutants, which are defective in the breakdown of starch (bam1 through 4) ( FIG. 3( a ) ). Surprisingly, only alleles of mur11, dpe2 and sex4 mutants showed increased sugar release.
Example 3: Inhibiting Polar Auxin Transport Improves Saccharification
Among those lines which showed good sugar release in both fresh and senesced tissue, one line (whs20) in particular stood out because it showed an incompletely penetrant pin-shaped inflorescence phenotype that was reminiscent of mutations that perturb the polar transport of the plant hormone auxin. Subsequent molecular analysis of this line identified a mutation in the PINOID (PID) gene ( FIG. 6 ; Table 2). PID encodes a serine threonine protein kinase that is thought to play a role in the cellular localization of the PIN efflux auxin carrier. Mutations in other genes that result in a pin-shaped phenotype, such as pin1 and mp (also known as arf5), also show an improved saccharification phenotype ( FIG. 4( a ) ). By contrast, other auxin response factor mutants defective in auxin signalling (arf6, 7, 8 and 19), did not show increase sugar release, however, these mutants also do not have the pin inflorescence phenotype. Furthermore, none of the single, double or triple combination of arf mutants tested displayed an increase in cell wall accessibility ( FIG. 8 ).
Finally, maize mutants with barren inflorescence phenotypes were tested. Barren inflorescence2 (bif2) is a co-ortholog of PID in Arabidopsis 20 and barren stalk1 (ba1), a basic helix-loop-helix transcription factor, has been shown to be a downstream target of BIF2 in maize. Consistent with the results from Arabidopsis , both bif2 (SEQ ID NOS: 1 and 2) and bat (SEQ ID NOS: 3 and 4) maize inflorescence mutants show an improved saccharification phenotype ( FIG. 4( b ) ).
The connection between auxin transport and increased sugar release was further probed using a specific inhibitor of auxin transport N-1-naphthylphthalamic acid (NPA). Application of varying concentrations of NPA to wild type Arabidopsis seedlings resulted in a 1.5 to 2 fold increase in the release of sugars relative to untreated plants ( FIG. 4( c ) ). More importantly, the ability to chemically perturb auxin transport allowed the expansion of the analysis to Zea mays (maize). Application of NPA to two different cultivars of maize also resulted in a significant increase in cell wall accessibility ( FIG. 4( d ) ). Together, these results provide strong support that genetic or chemical manipulation of auxin transport increases sugar release. Moreover, it appears that genes and processes identified using Arabidopsis can be transferred to maize and potentially other monocot species dedicated to biofuel production.
Example 4: Screening for Novel Cellulose Synthase (CESA) Alleles
Further genetic screens aimed at identifying resistance to cellulose biosynthetic inhibitors (CBIs) were also conducted. The aim of conducting resistance screens can be to identify potential inhibitor targets. In the case of some CBIs, like isoxaben, resistance screens have been carried out using high concentrations of the inhibitor with the aim of identifying the target protein. Indeed, high resistance to isoxaben is only possible if certain CELLULOSE SYNTHASE (CESA) genes are altered by mutation. An unforeseen consequence of some of the resistance alleles has been to reduce overall cellulose crystallinity, which ultimately leads to overall improved saccharification of starting cell wall material. With this information as a starting point, the inventors sought to identify novel CESA alleles by conducting additional resistance screens, but utilizing much lower CBI concentrations than in the original screens.
EMS mutagenized plants (M2) were screened on 20 nM of two different CBIs, isoxaben or flupoxam. Those plants that showed resistance at this concentration of either CBI were then retested in the M3 generation. In total, 2 million M2 seeds were screened and 12 new CESA alleles were isolated, 3 in CESA1, 8 in CESA3 and 1 in CESA6. All of the new mutant alleles led to single amino acid substitutions, which could not have been predicted a priori. Interestingly, one of these alleles led to an amino acid substitution in the proposed catalytic site of the enzyme (ixr1-4). Table 4 shows a summary of the identified mutant alleles.









TABLE 4







Concentration at


Genetic


which root length is

Allele
Background
Gene
Mutation
50% of wt












wild-type
Ler
—
—
5
nM

wild-type
Col-o
—
—
5
nM

Isoxaben Resistant

ixr1-1 (published)
Col-0
CesA3
G(998)D
>1
μM

ixr1-2 (published)
Col-0
CesA3
T(942)I
500
nM

ixr1-3
Ler
CesA3
G(998)S
100
nM




(SEQ ID NOS:




26 and 27)

ixr1-4
Ler
CesA3
R(806)K
50
nM




(SEQ ID NOS:




28 and 29)

ixr1-5
Ler
CesA3
L(797)F
10
nM




(SEQ ID NOS:




30 and 31)

ixr1-6
Ler
CesA3
S(377)F
50
nM




(SEQ ID NOS:




32 and 33)

ixr1-7
Ler
CesA3
R(276)H
50
nM




(SEQ ID NOS:




34 and 35)

ixr 2-1 (published)
Col-0
CesA6
R(1064)W
50
nM

ixr 2-2
Ler
CesA6
S(1002)F
10
nM




(SEQ ID NOS:




36 and 37)



Flupoxam resistant (Described in http://www.jstor.org/stable/4046145

with recent work in DOI: 10.1111/J.1365-313X.2011.04619.x)








fpx 1-1
Col-o
CesA3
S(1040)L
500
nM




(SEQ ID NOS:




20 and 21)

fpx 1-2
Ler
CesA3
S(1037)F
>1
μM




(SEQ ID NOS:




22 and 23)

fpx 1-3
Ler
CesA3
S(983)F
100
nM




(SEQ ID NOS:




24 and 25)

fpx2-1
Ler
CesA1
G(1013)R
>1
μM




(SEQ ID NOS:




14 and 15)

fpx 2-2
Ler
CesA1
P(1010)L
100-500
nM




(SEQ ID NOS:




16 and 17)

fpx 2-3
Ler
CesA1
G(1009)D
1
μM




(SEQ ID NOS:




18 and 19)








































































The mutants were further characterized by determining their relative cellulose crystallinity, as well as their saccharification profiles. This was accomplished by using an X-ray diffractometer to measure the proportion of crystalline cellulose relative to the proportion of amorphous cellulose in stem tissue ( FIG. 9 ). To determine the saccharification properties of the mutant lines, commercial enzyme cocktails were used to digest cell wall preparations and determine the amount of sugar released ( FIG. 10 ). It is significant that many of these alleles, to a greater or lesser extent, showed reduced cellulose crystallinity and in addition were also more amenable to enzyme hydrolysis ( FIG. 9 and FIG. 10 ). However, some lines with apparently unaltered cellulose crystallinity did show improved hydrolysis (e.g. fpx1-1, fpx1-2, fpx 1-3) or some lines with reduced crystallinity did not show improved hydrolysis (e.g. ixr1-7). This indicates that there isn't a tight correlation between cellulose crystallinity and hydrolysis properties.
The value of screening for CESA alleles using this methodology is twofold. Novel CESA alleles can be easily identified, many of which cause cellulose hydrolysis to improve, in a high-throughput manner. The fact that no a priori assumptions about CESA function and structure are required makes this approach particularly useful. In addition, it should be possible to conduct similar screens on target plants to create modified biomass feedstocks directly without the need for generating transgenic plants. One potential limitation is that the CBI that is used may need to specifically target the CESA complex in that plant. For example, the sensitivity to isoxaben is lower in grasses than it is in broadleaf species, which might indicate that alternative CB's would be required for conducting resistance screens in grasses.
Examples 1-5: Materials and Methods
Plant Materials and Growth Conditions
Arabidopsis thaliana M2 ecotype Columbia seeds mutagenized by ethyl methane sulfonate (EMS) were purchased from Lehle Seeds (Round Rock, Tex.). EMS mutant alleles and T-DNA insertions were provided by the Arabidopsis Biological Resource Centre (Ohio State University, Columbus, USA). Seeds were surface sterilized in 50% bleach, 0.01% Tween™-20 for 5 min, rinsed 5 times with sterile water and stored in the dark at 4° C. for 4 days to synchronize germination. Seeds were plated on 0.5× strength Murashige and Skoog (MS) agar plates and sealed with surgical tape under continuous light at room temperature. The maize mutants, bif2-N2354 (stock #108A) and bal (stock #318B) in the W23/M14 genetic background, were obtained from the Maize Genetics Cooperation Stock Center.
Anthrone Mutant Screen
The M2 generation of EMS-mutagenized Arabidopsis (Col-0) seeds were chilled for 4 days and sowed onto 0.5× MS plates placed vertically under continuous light conditions at room temperature. After 7 days, the seedlings were transferred to soil in 96-well flats. Leaf 3 or 4 was excised from 21 day-old plants using a hole punch and placed abaxial side up in a 96-well plate corresponding to the same coordinates as the flat. Samples were submerged in 200 μl of 1M H 2 SO 4 and incubated at room temperature for 1 hour. A 50 μl aliquot was removed and incubated with 100 μl of 0.2% anthrone in concentrated H 2 SO 4 . The samples were incubated at 100° C. for 5 minutes, cooled and the absorbance was read at 660 nm. Approximately 22,000 seedlings from 32 pools were screened from which 63 wall hydrolysis sensitive (whs) mutants were identified as having an absorbance reading greater than 2 standard deviations from wild type ( FIG. 5 ). whs mutants were retested in the M3 generation.
Enzymatic Digestion
Approximately 0.1-0.2 g of senesced tissue was washed twice with water for 30 min at 80° C. and washed with 70% ethanol at 80° C. for 1 hour. The tissue was rinsed with acetone and oven dried at 60° C. for 2 days. Cellulase from Trichoderma reesi ATCC 26921 and the Cellobiase (Novozyme 188) activities were empirically determined to be 111 FPU/mL and 500 U/mL, respectively. Glucose levels were determined via anthrone assay and cellobiase activity was determined by measuring p-nitro phenol (PNP) absorbance levels at 400 nm. 15 FPU/g of tissue of cellulase and 80 U/g of cellobiase were used on 5 mg of tissue/tube with a total volume of 200 μL in triplicates. The samples were incubated with a final 10× dilution of cellulase and cellobiase at 50° C. for 24 hours and heat inactivated at 100° C. for 5 min. Once cooled on ice, the samples were centrifuged and the supernatant was analyzed for its glucose concentration by the Glucose (HK) Assay Kit (GAHK20-1KT) (Sigma) according to the manufacturer's instructions.
Gas-Liquid Chromatography
Hydrolysis of leaf material and quantification of monosaccharides by gas-liquid chromatography of alditol acetates was carried out as previously described by Reiter el al., 1993. At least 5-20 mg of fresh tissue from 5 plant lines were pooled and extracted three times with chloroform:methanol (1:1) for 30 min. Three technical replicates were performed for each whs mutant. The tissue was washed with 70% ethanol at 70° C. for 1 hour, rinsed with acetone and left to air dry overnight and hydrolyzed in 1M H 2 SO 4 at 120° C. for 1 hour. The released monosaccharides were converted into alditol acetates and quantified by gas chromatography. Relative sugar composition values were calculated as a mol percentage.
Clustering and Heatmap Analysis
Monosaccharide composition of 62 whs mutants (whs35 not determined) and mur11-1 was determined by liquid gas chromatography and calculated as a percent difference relative to wild type ( FIG. 2C ). Cluster 3.0 using the C Clustering Library version 1.49 was used to cluster the values by Average Linkage and centered correlation. Java TreeView 1.1.5r2 was then used to display the data and colour-coded yellow (more than wild type) or blue (less than wild type). Glucose values quantified from the acid hydrolysis and enzymatic assays performed on the 63 whs mutants, excluding the starch staining, were calculated as a percent difference relative to wild type. Mutants with values equal to wild type were given color coded black and mutants with hexose values greater than wild type were color coded yellow. For starch staining, 14 day-old seedlings were stained with IKI and were visually analyzed for the presence of starch in their cotyledons and determined qualitatively.
Amylase Digestion
Five milligrams of tissue was weighed out in triplicate and re-suspended in 0.1 M sodium acetate, pH 5, and incubated at 80° C. for 30 min to gelatinize the starch. The tubes were cooled on ice then 30 μL of 0.1× α-amylase (Sigma A7595, activity: 250 U/mL for 1×) from Bacillus amyloliquefaciens was added. In addition, 15 μL of pullulanase M1 from Klebsiella planticola (Megazyme 42 U/mg) and 15 μL of pullulanase M2 from Bacillus licheniformis (Megazyme 26 U/mg) were added to bring the total liquid volume to 1 mL. The samples were vortexed then placed in an incubator at 37° C. for 16 hours. The samples were spun down at 12,000 g for 10 min and the reducing sugar equivalents were quantified using 0.2% anthrone. It should be noted that the HK Assay did not detect the products of the amylase digestion.
NPA Treatment of Monocot Plants
Polar auxin transport inhibition was carried out as described by Wu & McSteen, 2007. The two maize cultivars, Syngenta hybrid N39-Q1 and Tuxedo Sweet Corn, were grown in a greenhouse at 24° C. with a 12 hour day/night cycle. The plants were grown four weeks before NPA treatment followed by a two week watering regime using 120 μM NPA (ChemService, West Chester, Pa., USA) or DMSO alone (solvent) applied every two days in a volume of 150 mL for each pot. Plants were fertilized once a week with 20-20-20 fertilizer. After 2 weeks of treatment, whole plants were collected and de-stained in chloroform:methanol (1:1 v/v). Acid hydrolysis was performed as described previously.
Genetic and Physical Mapping of Mutants
Genetic mapping was accomplished using an F2 population derived from a cross between the whs mutants (Columbia genotype, Col-0) and Landsberg erecta (Ler). F2 seedlings were scored for wall hydrolysis sensitivity by anthrone screening. Genomic DNA was isolated from individual F2 plants from a mapping population showing the mutant phenotype and assigned to a chromosome using published simple sequence length polymorphism (SSLP) markers. New molecular markers were developed using the Monsanto Col-0 and Ler polymorphism database. The cloned WHS genes were amplified by PCR using X-Taq DNA polymerase with proofreading activity (Takara). Sequencing reactions were performed by The Centre for the Analysis of Genome Evolution and Function (CAGEF) at the University of Toronto. F2 mutants from two independent crosses were used for sequencing and verifying lesions.
The compositions, methods, mutant genes, cells, plants and other materials described in this application may be employed in the production of biomass useful, for example, in production of biofuels such as bioethanol, as well as other materials such as bioplastic, biofoam, biorubber, biocomposite, forestry biofibre, agricultural textile, chemical, monosaccharide, disaccharide, polysaccharide, biocosmetics, and in other feed stock production.
The scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.
REFERENCES


1. Himmel, M. E. et al. Biomass Recalcitrance: Engineering plants and enzymes for biofuels production. Science 315, 804-807 (2007).
2. Carroll A. & Somerville C. Cellulosic biofuels. Annu Rev Plant Biol. 60, 165-82 (2009).
3. Pauly, M. & Keegstra, K. Plant cell wall polymers as precursors for biofuels. Curr. Opin. Plant Sci. 13, 305-312 (2010).
4. Pingali, S. V. et al. Breakdown of cell wall nanostructure in dilute acid pretreated biomass. Biomacromolecules 11, 2329-2335 (2010).
5. Kumar, P. et al. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 48, 3713-3729 (2009).
6. Vanholme, R., Van Acker, R. & Boerjan, W. Potential of Arabidopsis systems biology to advance the biofuel field. Trends in Biotech. 28, 543-547 (2010).
7. Austin et al. Next-Generation Mapping of Arabidopsis Genes. Plant J . Apr. 23. doi: 10.1111/j.1365-313X.2011.04619.x. [Epub ahead of print] (2011).
8. Fry, S. C. The Growing Plant Cell Wall: Chemical and Metabolic Analysis . (The Blackburn Press, Caldwell, N.J., USA, 1988).
9. Reiter, W.-D., Chapple, C. & Somerville, C. R. Mutants of Arabidopsis thaliana with altered cell wall polysaccharide composition. Plant J. 12, 335-45 (1997).
10. Williams, M. E. et al. Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of Ptdlns(4,5)P2 and constitutive expression of the stressresponse pathway. Plant Phys. 138, 686-800 (2005).
11. Reiter, W.-D., Chapple, C. C. S. & Somerville, C. R. Altered growth and cell walls in a fucose-deficient mutant of Arabidopsis. Science 261, 1032-1035 (1993).
12. Chia, T. et al. A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night. Plant J. 37, 853-863 (2004).
13. Kotting, O. et al. STARCH-EXCESS4 is a laforin-like phophoglucan phophatase required for starch degradation in Arabidopsis thaliana. Plant Cell 21, 334-46 (2009).
14. Caspar, T. et al. Mutants of Arabidopsis with altered regulation of starch degradation. Plant Phys. 95, 1181-1188 (1991).
15. Wattebled, F. et al. Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal for anylopectin. Plant Phys. 138, 184-195 (2005).
16. Fulton, D. C. et al. b-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active b-amylases in Arabidopsis chloroplasts. Plant Cell 20, 1040-1058 (2008).
17. Okada, K. et al. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation. Plant Cell 3, 677-684 (1991).
18. Christensen, S. K., Dagenais, N., Chory, J. & Weigel, D. Regulation of auxin response by the protein kinase PINOID. Cell 100, 469-78 (2000).
19. Przemeck, G. K. H. et al. Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200, 229-237 (1996).
20. Wu, X. & McSteen, P. The role of auxin transport during inflorescence development in maize ( Zea mays , Poaceae). Am. J. Bot. 11, 1745-1755 (2007).
21. Skirpan, A., Wu, X. & McSteen, P. Genetic and physical interactions suggest that BARREN STALK1 is a target of BARREN INFLORESCENCE2 in maize inflorescence development. Plant J. 55, 787-797 (2008).
22. Reinhardt, D., Madel, T. & Kuhlemeier, C. Auxin regulates the initiation and radial position of plant lateral organs. Plant Cell 12, 507-518 (2000).
23. Reinhardt, D. et al. Regulation of phyllotaxis by polar auxin transport. Nature 426, 255-260 (2003).
24. Fu, C. et al. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc. Natl. Acad. Sci. USA 108, 3803-8 (2011).
25. Chen F, Dixon R A. Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotech. 25, 759-61 (2007).
26. Li, L et al. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. 100, 4939-44 (2003).
27. Somerville C. R. et al. Toward a Systems Approach to Understanding Plant Cell Walls. Science 306, 2206-2211 (2004).
28. Sánchez-Rodriguez C., Rubio-Somoza I., Sibout R., & Persson S. Phytohormones and the cell wall in Arabidopsis during seedling growth. Trends Plant Sci. 15, 291-301 (2010).
29. Feraru, E. et al. PIN polarity maintenance by the cell wall in Arabidopsis. Curr. Biol. 4, 33-43 (2011).
30. McCourt P. & Desveaux D. Plant chemical genetics. New Phytol. 185, 15-26 (2010).
31. Scheible W R, Eshed R, Richmond T, Delmer D, Somerville C. Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants. Proc Natl Acad Sci USA. 98(18):10079-84 (2001).
32. Harris, D., Stork, J. and Debolt, S. Genetic modification in cellulose-synthase reduces crystallinity and improves biochemical conversion to fermentable sugar. GCB Bioenergy, 1: 51-61(2009).