(en)The present invention relates to promoters that enable gene expression which is both specific to the endosperm and early during the development of the endosperm, as well as nucleic acid molecules encoding basal endosperm transfer cell layer (BETL) proteins.

1.ApplicationNumber: US-201113078905-A
1.PublishNumber: US-2012011621-A1
2.Date Publish: 20120112
3.Inventor: PEREZ PASCUAL
GUTIERREZ-MARCOS JOSE
DICKINSON HUGH

4.Inventor Harmonized: PEREZ PASCUAL(FR)
GUTIERREZ-MARCOS JOSE(GB)
DICKINSON HUGH(GB)

5.Country: US
6.Claims:
(en)The present invention relates to promoters that enable gene expression which is both specific to the endosperm and early during the development of the endosperm, as well as nucleic acid molecules encoding basal endosperm transfer cell layer (BETL) proteins.

7.Description:
(en)REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of application Ser. No. 12/795,126 filed Jun. 8, 2010, which is a division of application Ser. No. 10/578,126 filed Jul. 17, 2006, now U.S. Pat. No. 7,745,697, which is the national stage of application No. PCT/EP2004/052760 filed Nov. 2, 2004, which claims priority of application No. 0329239.4 filed on Nov. 3, 2003 in the European Patent Office. The entire contents of each of these applications are hereby incorporated by reference herein in their entirety.


SUBMISSION OF SEQUENCE LISTING
The Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is 11887 — 24_Seq_List. The size of the text file is 47 KB and the text file was created on Apr. 1, 2011.
The present invention relates to controlling the expression of genes during the development of the endosperm. It concerns in particular nucleic acid molecules which enable expression which is both specific to the endosperm and early during the development of the endosperm, as well as nucleic acid molecules encoding basal endosperm transfer cell layer (BETL) proteins.
The endosperm, a characteristic formation of Angiosperm seeds, is a nutritive tissue for the embryo. The maize endosperm originates with series of free-nuclear divisions, followed by cellularisation and the subsequent formation of a range of functional cellular domains. This tissue is complex in its structure and development, in particular in cereals.
The endosperm is the main storage organ in maize seeds, nourishing the embryo while the seed develops, and providing nutrients to the seedling on germination.
Thus, the uptake of assimilates by the growing endosperm is a critical process in seed development.
The central area of the endosperm consists of large cells with vacuoles, which store the reserves of starch and proteins, whilst the region surrounding the embryo is distinguished by rather small cells, occupied for the major part by cytoplasm.
The Basal Endosperm Transfer Layer (BETL) area is highly specialized to facilitate uptake of solutes during grain development. These transfer cells of the basal endosperm have specialised internal structures adapted to absorb solutes from the maternal pedicel tissue, and translocate these products to the developing endosperm and embryo. Usually, BETL genes are expressed between 8 to 20 days after pollination (DAP).
The international patent application WO 99/50427 reports Basal Endosperm Transfer cell Layer genes and regulatory sequences thereof.
However, there is still a need for regulatory sequences specific to the endosperm.
The authors of the present invention have now isolated novel promoter nucleotide sequences that enable expression of the coding sequences to which they can be linked, which is specific to the endosperm in Angiosperm seeds and which intervenes particularly in the early stage of the development of the endosperm. These novel promoter nucleotide sequences are useful as an alternative to the few promoters described in the literature.
Such promoter sequences are particularly useful for targeting or regulating the expression of genes of interest.
Interestingly, the nucleotide sequences having promoter activity according to the invention have a maternal parent-of-origin pattern of expression so the genes under transcriptional control of these promoters are preferentially expressed through the maternal allele during early endosperm development.
Advantageously, these nucleotide sequences having promoter activity are active as early as 4 DAP, when maternally inherited.
Surprisingly, the authors found that the proteins encoded by the genes related to these promoter sequences are implicated in the mechanism of pathogen resistance and/or endosperm development.
The present invention relates to an isolated nucleic acid molecule having promoter activity specific to the endosperm that comprises a DNA sequence selected from the group consisting of :
(a) a sequence as depicted in any one of SEQ ID No: 1 to 3 (promoter MEG1-1 (pMEG1-1), promoter MEG1-2 (pMEG1-2), and promoter MEG1-3 (pMEG1-3), respectively);
(b) a fragment of a sequence as defined in (a), wherein said sequence has promoter activity specific to the endosperm;
(c) a sequence that has at least 70% sequence identity with a sequence as defined in (a), wherein said sequence has promoter activity specific to the endosperm;
(d) a sequence hybridizing with the complementary strand of a sequence as defined in (a) and/or (b) under stringent conditions, wherein said sequence has promoter activity specific to the endosperm; and
(e) a sequence that comprises a nucleotide sequence which is conserved among at least two of SEQ ID No: 1 to 3.
As used herein, the term “conserved” sequence refers to a nucleotide stretch that shows 100% identity between at least two sequences of reference. Preferably said conserved sequence is found in all of the three sequences SEQ ID No: 1 to 3. A sequence alignment of SEQ ID No: 1 to 3 is shown in FIG. 14 .
According to an embodiment, said conserved sequence comprises 10 contiguous nucleotides from SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3, preferably 15, 20, 25, or 30 contiguous nucleotides. Preferably, the sequence as defined in alternative (e) comprises 5′ ATATAGATAGATATAGCAAATTCACCAAATAATATAG 3′ (SEQ ID No: 57). More preferably said sequence consists in SEQ ID No: 57. However, any conserved sequence, or a combination of conserved sequences, as shown in FIG. 14 is within the scope of the invention.
According to another embodiment, said sequence as defined in alternative (e) comprises a motif of 9 contiguous nucleotides from SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3, wherein nucleotides 1-3 and 7-9 of said motif are conserved among at least two, preferably three, of SEQ ID No: 1 to 3, and nucleotides 4-6 may be variable.
Particularly, the nucleic acid molecule according to the invention enables expression (promoter activity) which is both specific to the endosperm, more particularly to the BETL, and early during the development of the endosperm.
“Promoter activity specific to the endosperm” means, as used in the present invention, that the promoter is predominantly expressed in the endosperm, and preferably exclusively expressed in the endosperm.
“Early during the development of the endosperm” means that expression of the promoter occurs from the very first days after pollination, preferably as early as the fourth day after pollination.
The various nucleotide sequences of the invention can be of artificial origin or not. They may be DNA sequences obtained by screening banks of sequences by means of probes produced on the basis of SEQ ID No: 1, 2, 3, 5, 7, 9, 11, 13, and 15. Such banks can be prepared by conventional techniques of molecular biology, known to persons skilled in the art.
The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or by mixed methods including the chemical or enzymatic modification of sequences obtained by screening banks. According to an embodiment of the invention, a nucleic acid molecule having such a promoter activity consists in SEQ ID No: 1 (pMEG1-1), SEQ ID No: 2 (pMEG1-2), or SEQ ID No: 3 (pMeg1-3).
By reference to these promoter sequences, a “ fragment ” denotes a sequence, particularly a DNA sequence, which has a reduced length with regard to said sequence of reference.
Such a fragment, according to the invention, has promoter activity specific to the endosperm, preferentially specific to the BETL, and contains at least 15, 25, 35, 37, 50, 75, 91, 95, 100, 121, 122, 127, 150, 200, 220, 240, 250, 270, 300, 350, 360 or 365 consecutives nucleotides of the DNA sequence of reference.
A preferred fragment contains the region defined by nucleotides 1 to 121 of SEQ ID No: 1, which corresponds to the sequence shown in SEQ ID No: 4, or contains the region defined by nucleotides 1 to 127 of SEQ ID No: 1, which corresponds to the sequence shown in SEQ ID No: 59.
Are also preferred fragments the regions defined by nucleotides 26 to 120 of SEQ ID No:4 and 29 to 119 of SEQ ID No:4.
A fragment according to the invention may also be obtained by fusion of at least two consecutive regions of a DNA sequence of reference. An example of fusion fragment according to the invention may comprise the region defined by nucleotides 1-121 of SEQ ID No: 1, which is required for pMEG1-1 activation by ZmMRP1, and the region defined by nucleotides 244-370 of SEQ ID No: 1, which contains a TATA box.
It is further possible to make a fusion of the region defined by nucleotides 1-121 of SEQ ID No: 1 (Meg1 promoter region) to any minimal promoter region (such as a −46 CaMV 35S promoter) in order to produce a promoter that is liable to be activated by ZmMRP1 and thus that is BETL-specific, the minimal promoter region being used to replace the region defined by nucleotides 244-370 from MEG1-1 promoter sequence (SEQ ID No: 1) in the fusion fragment defined above.
Such a result is also obtained using fragments 26 to 120 or 29 to 119 of SEQ ID No: 4.
A nucleic acid molecule “hybridizes” to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al., 1989).
Such an hybridizing sequence has promoter activity specific to the endosperm according to the invention, preferentially specific to the BETL, and contains at least 15, 25, 35, 37, 50, 75, 100, 121, 122, 127, 150, 200, 220, 240, 250, 270, 300, 350, 360 or 365 nucleotides. Preferably, said hybridizing sequence may contain no more than 100 or 200 nucleotides.
The invention also encompasses modifications of the DNA sequences as depicted in SEQ ID No: 1 to 3 (pMEG1-1, pMEG1-2, pMEG1-3), or of sequence motifs thereof by, e. g., nucleotide replacements that do not affect the overall structure or binding motif of the nucleic acid molecule having promoter activity specific to the endosperm so that it remains capable of driving endosperm specific expression of a gene, and more particularly, a BETL specific expression of a gene.
“Homologous nucleic acid sequence”, or “homologous DNA sequence”, means any nucleic acid sequence which differs from any of the sequence SEQ ID No: 1 to 3 by a substitution, deletion and/or insertion of one or more nucleotides at positions such that these homologous nucleic acid sequences preserve the specificity property of promoters of sequences SEQ ID No: 1 to 3.
Preferably such a homologous nucleic acid sequence is at least 70% identical to one of the sequences SEQ ID No: 1 to 3, preferably at least 85% identical, more preferably at least 90, 91, 95, 98, 99.9% identical. Also preferably, the degree of identity is defined by comparison with the entire sequence of reference, SEQ ID No: 1, SEQ ID No: 2 or SEQ ID No: 3.
Homology is generally determined using a sequence analysis software (for example, the Sequence Analysis Software package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Similar nucleotide sequences are aligned in order to obtain the maximum degree of homology (i.e. identity). To this end, it may be necessary to artificially introduce gaps in the sequence. Once the optimum alignment has been achieved, the degree of homology (i.e. identity) is established by recording all the positions for which the nucleotides of the two compared sequences are identical, with respect to the total number of positions.
In a preferential manner such a homologous nucleic acid sequence specifically hybridizes to a sequence which is complementary to one of the sequences SEQ ID No: 1 to 3 under stringent conditions. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).
For sequences comprising more than 30 bases, Tm is defined by the equation:
Tm=81.5+0.41 (% G+C )+16.6 Log (concentration in cations)−0.63 (% formamide)−(600/number of bases) (Sambrook et al., 1989).

For sequences shorter than 30 bases, Tm is defined by the equation: Tm=4(G+C)+2(A+T).
Under appropriate stringency conditions, in which non-specific (aspecific) sequences do not hybridize, the temperature of hybridization is approximately between 5 and 30° C., preferably between 5 and 10° C. below Tm and hybridization buffers used are preferably solutions of higher ionic force like a solution 6*SSC for example.
In addition, it is possible to derive essential regulatory elements from the promoters provided herein. Thus, those elements of the promoter sequence responsible for both its function as a promoter and, more importantly, its endosperm specificity, particularly its BETL specificity, can be isolated and incorporated into nucleic acid molecules which, although not falling within the definitions (a) to (e) above, nonetheless still function in an equivalent manner
The present invention also provides a recombinant or isolated nucleic acid molecule comprising, or consisting of, one or more regulatory elements derived from any one of the sequences (a) to (e) and which is capable of driving expression specifically in the endosperm, and preferably at an early stage of the development of the endosperm.
According to an embodiment of the invention, the nucleic acid molecule having promoter activity specific to the endosperm preferentially has a maternal parent-of-origin pattern of expression. As used herein, the term “maternal parent-of-origin pattern of expression” refers to the expression of a gene maternally imprinted. Imprinting is an epigenetic mechanism by which a particular allele is silenced according to its parental origin. This means that genes subjected to genomic imprinting have monoallelic expression, instead of biallelic expression as usual for autosomal genes, which is based on the sex of the transmitting parent.
The isolation of promoters that are regulated via imprinting provides an additional level of control compared to normal, non-imprinted promoters. Accordingly, the level of expression of a gene of interest can advantageously be controlled via the direction of the genetic cross between one plant carrying an imprinted transgene and the other plant lacking the transgene.
Preferably, the nucleic acid molecule having promoter activity specific to the endosperm which has a maternal parent-of-origin pattern of expression consists in SEQ ID No: 1.
SEQ ID No: 62 is also a nucleic acid molecule having promoter activity specific to the endosperm which has a maternal parent-of-origin pattern of expression, and is a part of the invention as any of SEQ ID No: 1 to 3.
The nucleic acid molecules having promoter activity specific to the endosperm according to the invention can be isolated from various plant species, notably Angiosperm plants, Monocotyledons or Dicotyledons and are preferably nucleic acid molecules isolated from a plant selected from the group consisting of maize, teosintes, rice, sorghum, wheat, barley, rye, pea, and sugar cane. Still preferably, the plant is maize.
It is possible for the person skilled in the art to isolate with the help of promoter sequences of the invention, corresponding genes from other species (“orthologous” genes).
This can be done by conventional techniques known in the art, for example, by using a promoter sequence depicted in any one of SEQ ID No: 1 to 3 as a hybridization probe or by designing appropriate PCR primers.
It is preferable to start with coding DNA sequences or Protein sequences via TBLASTN queries. The approach used to isolate rice Meg1 promoters, for example, is to use the Protein sequence of ZmMeg1-1, preferably the most conserved region identified via an alignment of all the Meg proteins, do a TBLASTN with this sequence against Rice ESTs, then use this EST to find the genomic sequence or directly use TBLASTN against the rice genome sequence. The same approach is used to isolate wheat Meg1 promoters.
It is then possible to isolate the corresponding promoter region by conventional techniques and test it for its expression pattern by known techniques (e.g. reporter gene analysis).
Another object of the present invention is a nucleotide construction, referred to as an expression cassette, comprising a nucleic acid molecule having promoter activity specific to the endosperm as defined above, operatively linked to at least one gene of interest.
“Operatively linked” refers to functional linkage between a nucleic acid molecule having promoter activity according to the invention and the gene of interest.
The gene of interest can be of a heterologous origin.
The gene of interest can be placed in the sense or antisense orientation.
According to an embodiment, the gene of interest may be selected from the group consisting of a sequence that encode a peptide or a protein, an antisense RNA sequence, a sense RNA sequence, both a sense and antisense. RNA sequence and/or a ribozyme.
Preferentially, the gene of interest is a sequence that codes for a protein or for a peptide.
The said gene of interest can for example code for a protein involved in the development of the embryo and/or of the endosperm, the determination of seed size and/or quality (e.g. MRP1 or Ferretin (Lobreaux S. et al. 1992)), cell growth (proteins regulating cell division including cytokinin or auxin genes, e.g. ipt (Zhang et al. 1995), the flow of nutrients or nutrient transfer (transporters (Bolchi A. et al. 1999)), proteins involved in fatty acids metabolism. The gene of interest may also encode an enzyme involved in sugar metabolism such as invertases (e.g. incW2 (Taliercio E W et al. 1999)), sucrose synthases (e.g. Sh1), the saccharose phosphate synthase, saccharose synthase, UDP-Glucose pyrophosphorylase, ADP-glucose pyrophosphorylase (Thomas W. Greene et al. 1998), starch branching enzyme (Ming Gao et al. 1997) or the starch synthase (Mary E. Knight et al. 1998). The gene of interest could also code for a hexokinase as the one described by Jang et al. (1997) in order to improve grain filling. The gene of interest may additionally code for a protein that is involved in amino acids transfer, such as a methionine permease or a lysine permease, or a sulphur transporter etc. It can also code for a toxic protein such as Barnase, for a protein activating or inhibiting other genes, such as transcriptional regulators including transactivators modified to act as dominant activators or repressors of transcription (e.g. fusions to the engrailed domain (Poole et al., 1985) or co-repressors for example), or for a protein improving resistance to pathogens (e.g. BAP2, MRP1).
Preferably, said gene of interest encodes a protein selected from:
a protein whose specific expression in the endosperm, and particularly in the BETL, makes it possible to increase nutrient uptake and thus seed size and/or quality; examples of such a protein include an invertase like Incw2 or like Ivr1 (EP 0 442 592), a sucrose synthase like Sh1 (WO 02/067662) or any transporters of sugar and nitrogen or a MRP 1 protein etc;
a protein that improves resistance to pathogens; examples of such a protein include a BAP Protein (Basal Layer Antifungal Protein) (Sema et al., 2001), or anti-fungal peptides, or a MRP1 protein or a protein that encodes an oxalate oxidase (WO 92/15685) or a protein that encodes a chitinase (WO 92/01792 or US 5, 446, 138) or a protein that encodes a glucanase (WO 93/02197) etc.
A protein that “improves resistance to pathogens” or “a protein improving resistance to pathogens” means a protein that, when expressed in a plant or a part of a plant, confers or improves resistance to pathogens to said plant, or part thereof. Said transformed plant has a better resistance to pathogens than the non-transformed plant (wild-type).
The said gene of interest can also be associated with other regulating elements such as transcription termination sequences (terminators). By way of examples of such sequences, it is possible to cite the polyA 35S terminator of the cauliflower mosaic virus (CaMV), described in the article of Franck et al. (1980) and the NOS terminator corresponding to the region in the non-coding 3′ region of the nopaline synthase gene of the Ti-plasmid of the Agrobacterium tumefaciens nopaline strain (Depicker et al. 1992).
Preferably, the terminator used is the 3′CaMV.
According to the invention, the expression cassette, comprising a nucleic acid molecule having promoter activity specific to the endosperm as defined above, operatively linked to at least one gene of interest may further comprise one or several selection marker genes for plants, useful for transformation and selection.
In the present invention, the term “selectable marker”, “selectable gene”, “selectable marker gene”, “selection marker gene”, “marker gene” are used interchangeably.
These selectable markers include, but are not limited to, antibiotic resistance genes, herbicide resistance genes or visible marker genes. Other phenotypic markers are known in the art and may be used in this invention.
A number of selective agents and resistance genes are known in the art. (See, for example, Hauptmann et al., 1988; Dekeyser et al., 1988; Eichholtz et al., 1987; and Meijer et al., 1991).
Notably the selectable marker used can be the bar gene conferring resistance to bialaphos (White et al., 1990), the sulfonamide herbicide Asulam resistance gene, sul (described in WO 98/49316) encoding a type I dihydropterate synthase (DHPS), the //nptll gene conferring resistance to a group of antibiotics including kanamycin, G418, paromomycin and neomycin (Bevan et al., 1983), the hph gene conferring resistance to hygromycin (Gritz et al., 1983), the EPSPS gene conferring tolerance to glyphosate (U.S. Pat. No. 5,188,642), the HPPD gene conferring resistance to isoxazoles (WO 96/38567), the gene encoding for the GUS enzyme, the green fluorescent protein (GFP), expression of which, confers a recognisible physical characteristic to transformed cells, the chloramphenicol transferase gene, expression of which, detoxifies chloramphenicol.
Advantageously, the selectable marker gene is inserted between a promoter and a terminator in a second expression cassette. Said second expression cassette being integrated in the same vector as the expression cassette containing the gene of interest under transcriptional control of a promoter according to the invention.
According to this advantageous embodiment, the marker gene is preferably controlled by a promoter which allows expression in cells, thus allowing selection of cells or tissue containing the marker at any stage of development of the plant. Preferred promoters are the promoter of nopaline synthase gene of Agrobacterium , the promoter derived from the gene which encodes the 35S subunit of cauliflower mosaic virus (CaMV) coat protein, and the rice actin promoter. However, any other suitable second promoter may be used.
Any terminator may be used. Preferred terminators are the 3′CaMV and Nos terminator as previously described.
Advantageously, the expression cassette containing the selectable marker gene is comprised between two Ds elements (transposons) in order for its removal at a later stage by interacting with the Ac transposase. This elimination system is described in Yoder et al. (1993).
For the transformation step, two vectors could be used, the first one comprising the expression cassette containing the gene of interest and the second one comprising the expression cassette containing the selectable marker gene. The same host cell being transformed with these two vectors (co-transformation).
The expression cassettes according to the invention may additionally contain transit peptide sequences. There are numerous examples in the art of transit peptides which may be used to deliver a target protein into a plastid organelle such as the small subunit (SSU) transit peptide of ribulose biphosphate carboxylase.
Other elements like introns and enhancers can also be present in the nucleic sequence of interest in order to improve the expression of the gene of interest.
Among useful introns, the first intron of maize //adhl S can be placed between the promoter and the coding sequence. This intron when included in a gene construct increased the expression of the desired protein in maize cells (Callis et al., 1987). One also can use the 1 st intron of the shrunken 1 gene of the maize (Maas et al., 1991), the 1 st intron of the catalase gene of the bean catalase (CAT-1) (Ohta et al., 1990), the 2 nd intron of the ST-LS1 gene of potato (Vancanneyt et al. 1990), the DSV intron of the yellow dwarf virus of tobacco (Morris et al., 1992), the actin-1 intron (act-1) of rice (McElroy et al., 1990) and intron 1 of triosephosphate isomerase (TPI) (Snowdon et al., 1996).
Preferentially, the intron used in the present invention is the Hsp70 intron or the Sh1 intron.
The expression cassettes may additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Such 5′ leaders are known in the art and include, but are not limited to, picornavirus leaders, for example, the EMCV leader (Encephalomyocarditis 5′ noncoding region) (Elroy-Stein, Fuerest, and Moss B., 1989) ; potyvirus leaders, for example, the TEV leader (Tobacco etch Virus) (Allison et al., 1986) ; the human immunoglobulin heavy-chain binding protein leader (BiP) (Macejack and Sarnow, 1991) ; the untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling and Gehrke, 1987) ; the tobacco mosaic virus leader (TMV) (Gallie et al., 1989) ; and the maize chlorotic mottle virus leader (MCMV) (Lommel et al., 1991). See also, Della-Cioppa et al. (1987). Other methods known to enhance translation can be utilized, for example introns, and the like.
Any expression cassette according to the invention, may further comprises a gene encoding a MRP1 protein, preferably the ZmMRP1 protein.
The ZmMRP1 protein is described in Gomez et al (2002). The nucleic acid sequence and amino acid sequence of ZmMRP1 are shown in SEQ ID No:51 and 52, respectively. However, any protein homologous to MRP1, and particularly to ZmMRP1, that have the same expression pattern can be used.
Such MRP1 protein can be used according to different ways:
MRP1 as anti-fungal protein (resistance to pathogens) operatively linked to MEG1 promoter sequence SEQ ID No: 1 to 3, or a fragment thereof :
A first expression cassette comprising an MRP1 protein or a fragment thereof, preferably the ZmMRP1 protein, operatively linked to a MEG1 promoter, preferably ZmMEG1-1 promoter, and to a terminator and a second expression cassette comprising a marker gene inserted between a promoter and a terminator; these two expression cassettes being inserted in the same vector for transformation or these two cassettes being inserted in two different vectors being co-transformed as previously described.
MRP1 as transactivator for MEG1 promoter:
A first expression cassette comprising a gene of interest, operatively linked to a MEG1 promoter, preferably ZmMEG1-1 promoter, and to a terminator; a second expression cassette comprising a MRP1 protein, preferably the ZmMRP1 protein inserted between a promoter and a terminator ; a third expression cassette comprising a marker gene inserted between a promoter and a terminator; these three expression cassettes being inserted into the same vector for transformation or these three expression cassettes being inserted into three different vectors being co-transformed as previously described.
This second way of use is a binary system of expression, i.e. two plants are crossed, the first one with a gene of interest operatively linked to MEG1 promoter (preferably ZmMEG1-1) and the other with for example a leaf promoter linked to MRP1. The result is that in the hybrid the expression of the gene of interest is not only present in the BETL but also in the leaf.
In preparing the expression cassettes, the various DNA sequences or fragments may be manipulated, so as to provide DNA sequences or fragments in the proper orientation and, as appropriate, in the proper reading frame. Towards this end, adapters or linkers may be employed to join the DNA fragments and/or other manipulations may be required to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, ligation, PCR, or the like may be employed, where nucleotide insertions, deletions or substitutions, for example transitions and transversions, may be involved. These techniques are well known by those skilled in the art.
Another object of the invention is any nucleotide vector referred to as an expression vector, such as a plasmid, which can be used for transforming host cells, characterized in that it contains at least an expression cassette as defined above. The construction of expression vectors for the transformation is within the capability of one skilled in the art following standard techniques.
The decision as to whether to use a vector, or which vector to use, is guided by the method of transformation selected, and by the host cell selected.
Where a naked nucleic acid introduction method is used, then the vector can be the minimal nucleic acid sequences necessary to confer the desired phenotype, without the need for additional sequences.
Possible vectors include the Ti plasmid vectors, shuttle vectors designed merely to maximally yield high numbers of copies, episomal vectors containing minimal sequences necessary for ultimate replication once transformation has occured, transposon vectors, including the possibility of RNA forms of the gene sequences. The selection of vectors and methods to construct them are commonly known to persons of ordinary skill in the art and are described in general technical references (Mullis, K B (1987), Methods in Enzymology ).
For other transformation methods requiring a vector, selection of an appropriate vector is relatively simple, as the constraints are minimal. The apparent minimal traits of the vector are that the desired nucleic acid sequence be introduced in a relatively intact state. Thus, any vector which produces a plant carrying the introduced DNA sequence should be sufficient. Also, any vector which introduces a substantially intact RNA which can ultimately be converted into a stably maintained DNA sequence should be acceptable.
However, any additional attached vector sequences which confer resistance to degradation of the nucleic acid fragment to be introduced, which assists in the process of genomic integration or provides a means to easily select for those cells or plants which are actually, in fact, transformed are advantageous and greatly decrease the difficulty of selecting useable transgenic plants.
The vector can exist, for example, in the form of a phage, a plasmid or a cosmid. The construction of such expression vectors for transformation is well known in the art and uses standard techniques. Mention may be made of the methods described by Sambrook et al. (1989).
Another object of the invention is a host cell, containing at least an expression vector as described above.
The decision as to whether to use a host cell, or which host cell to use, is guided by the method of transformation.
The host cell can be any prokaryotic or eukaryotic cell. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, bio-safety and costs. Useful hosts include bacteria such as E. coli sp. or Agrobacterium . A plant host cell, may be also used, notably an Angiosperm plant cell, Monocotyledon as Dicotyledon plant cell, particularly a cereal or oily plant cell, selected in particular from the group consisting of maize, wheat, barley, rice, rape and sunflower, preferentially maize.
More particularly, the host cell used in carrying out the invention is Agrobacterium tumefaciens , according to the method described in the article of An et al., 1986, or Agrobacterium rhizogenes , according to the method described in the article of Jouanin et al., 1987.
The invention also concerns a transgenic plant or part of a transgenic plant, in particular fruit, seed, grain or pollen, comprising such a cell or generated from such a cell. Where the plant contains endogenously a MEG1 promoter or gene according to the invention, it will be understood that the transgenic plant according to the invention comprises an additional “exogenous” gene MEG1 gene or promoter, for instance integrated by transgenese.
A whole plant can be regenerated from a single transformed plant cell. Thus, in a further aspect the present invention provides transgenic plants (or parts of them) including nucleic acid sequences in accordance with the invention. The regeneration can proceed by known methods.
The seeds which grow, by fertilization, from this plant also contain this transgene in their genome.
Advantageously, the transgenic plant obtained can produce grains with a larger endosperm in comparison with a non-transformed plant, particularly grains with starch, oil contents or protein contents which are modified in comparison with a non-transformed plant.
A plant or part of a plant according to the invention could be a plant or a part of it from various species, notably an Angiosperm, Monocotyledons as Dicotyledons, preferably a cereal or oily plant, selected in particular from the group consisting of maize, rice, wheat, barley, rape and sunflower, preferentially maize.
As used herein, the term “oily plant” denotes a plant that is capable of producing oil, and preferably that is cultivated for oil production. The hybrid plants obtained by crossing plants according to the invention also form part of the invention.
An other object of the invention is a method of obtaining a plant having improved agronomic qualities and/or improved resistance to pathogen, comprising the steps consisting of :
(a) transforming at least one plant cell by means of a at least a vector as defined previously ;
(b) cultivating the cell(s) thus transformed so as to generate a plant containing in its genome at least an expression cassette according to the invention, whereby said plant has improved agronomic qualities and/or improved resistance to pathogen.
According to the invention, “improved agronomic qualities” means improved agronomic qualities and/or improved nutritional qualities, notably yield, food or industrial qualities of a plant or a part thereof Seed size, yield, seed number, seed composition are considered as elements conferring improved agronomic qualities to a plant as compared to a non-transformed plant (wild-type).
According to the invention, “improved resistance to pathogens” means that the transformed plant have a better resistance to pathogens than the non-transformed plant (wild-type).
The transformation of vegetable cells can be achieved by any one of the techniques known to one skilled in the art.
It is possible to cite in particular the methods of direct transfer of genes such as direct micro-injection into plant embryoids (Neuhaus et coll. 1997), vacuum infiltration (Bechtold at al. 1993) or electroporation (Chupeau et coll., 1989) or direct precipitation by means of PEG (Schocher et coll., 1986) or the bombardment by gun of particules covered with the plasmidic DNA of interest (Fromm Met al., 1990).
It is also possible to infect the plant with a bacterial strain, in particular Agrobacterium . According to one embodiment of the method of the invention, the vegetable cells are transformed by a vector according to the invention, the said cell host being able to infect the said vegetable cells by allowing the integration, in the genome of the latter, of the nucleotide sequences of interest initially contained in the above-mentioned vector genome. Advantageously, the above-mentioned cell host used is Agrobacterium tumefaciens , in particular according to the method described in the article by An et al., (1986), or Agrobacterium rhizogene , in particular according to the method described in the article by Guerche et al. (1987).
For example, the transformation of vegetable cells can be achieved by the transfer of the T region of the tumour-inducing extra-chromosome circular plasmid of Agrobacterium tumefaciens , using a binary system (Watson et al., 1994). To do this, two vectors are constructed. In one of these vectors the T region has been eliminated by deletion, with exception of the right and left borders, a marker gene being inserted between them to allow selection in the plant cells. The other partner of the binary system is an auxiliary plasmid Ti, a modified plasmid which no longer has any T region but still contains the virulence genes vir necessary to the transformation of the vegetable cell.
According to a preferred mode, it is possible to use the method described by Ishida et al. (1996) for the transformation of Monocotyledons.
According to another protocol, the transformation is achieved according to the method described by Finer et al.(1992) using the tungsten or gold particle gun.
Selection :
The engineered plant material may be selected or screened for transformants (those that have incorporated or integrated the introduced nucleotide construction(s)). Such selection and screening methodologies are well known to those skilled in the art. The selection and screening method is chosen depending on the marker gene used.
An isolated transformant may then be regenerated into a plant.
Regeneration :
Normally, regeneration is involved in obtaining a whole plant from the transformation process. The term “regeneration” as used herein, means growing a whole plant cell, a group of plant cells, a plant part or a plant piece (for example, from a protoplast, callus, or tissue part).
Methods of regenerating whole plants from plant cells are known in the art, and the method of obtaining transformed and regenerated plants is not critical to this invention.
In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification, of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing appropriate plant hormones in accordance with known methods and shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques.
The invention further relates to the use of at least an expression cassette as previously defined, for obtaining a transgenic plant exhibiting improved agronomic qualities and/or improved resistance to pathogen.
The agronomic quality of a plant is improved by acting in particular on the size of the embryo or of the endosperm and/or its development.
This is because an early specific action on the development of the tissues of the embryo and of the endosperm can be sought : according to the relative size of one or other it would be possible to obtain seeds or fruits with a higher starch (large endosperm) and/or oil (large embryo) content, via the use respectively of stimulator genes (hormone of the cellular cycle for example) or inhibitor genes (toxic protein or transcription inhibitor for example). A reduction in BETL size would create a miniature type seed which could have a reduced endosperm/embryo ratio. The most likely effect of increasing BETL activity/size would be to increase endosperm size. This may translate into a bigger embryo also.
By way of example, the use of genes coding for hormones (cytokinins, auxins) under the control of the promoters described according to the invention, would make it possible to modify the process of cellularisation and thus the development of the endosperm.
An effect on the accumulation of nutrients in the embryo and endosperm can also be sought, by using for example, as genes of interest, genes coding for transporters of nutrients (sugar in particular), or genes coding for inhibitors of these transporters, leading to differential accumulation of nutrients in the endosperm or embryo.
The invention also concerns the use of the transgenic plants obtained according to the invention, or parts of these plants, in particular seeds, grains, and fruits for preparing derived products, in particular food products.
The invention relates to seeds obtained from a plant transformed with a nucleic acid sequence according to the invention (SEQ ID No: 1-4, 5, 7, 9, 11, 13, 15 and 58).
The products obtained, whether it be seeds with a higher oil content, flours of seeds or grains with a higher starch, protein or oil content, also come within the scope of the invention.
The invention also provides any composition for human or animal food prepared from the said obtained products.
The invention further provides an isolated nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein, which comprises a sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a protein consisting of an amino acid sequence as depicted in SEQ ID No: 6, 8, 10, 53, 12, 14 or 16 (ZmMEG1-1, ZmMEG1-2, ZmMEG1-3 (first and second ORF), ZmMEG1-4, ZmMEG1-5, and ZmMEG1-6, respectively) and variants thereof; (b) a nucleotide sequence as depicted in SEQ ID No: 5, 7, 9, 11, 13, 15, or 58 (cDNA of ZmMEG1-1, ZmMEG1-2, ZmMEG1-3, ZmMEG1-4, ZmMEG1-5, and ZmMEG1-6, and genomic sequence of ZmMEG1-1, respectively); (c) a nucleotide sequence hybridizing under stringent conditions with the complementary strand of a nucleic acid molecule as defined in (a) or (b); (d) a sequence encoding a fragment of a protein encoded by a sequence as defined in any one of (a) to (c).

Preferably, the protein encoded by the nucleic acid molecule according to the invention is specific to the BETL.
“Specific to the BETL” means, as used in the present invention, that the protein is expressed predominantly in the BETL, and more particularly, exclusively in the BETL.
Preferentially, the amino acid sequence encoded by a nucleotide sequence according consists in SEQ ID No: 6.
Also preferably, the sequence encoding a fragment of a BETL protein according to the invention comprises a nucleic acid sequence encoding the PCKDNKCYCCIGGRTH sequence (SEQ ID No:54). This fragment was found to be conserved among all maize MEG1 proteins (ZmMEG1-1, ZmMEG1-2, ZmMEG1-3 (first and second ORF), ZmMEG1-4, ZmMEG1-5, and ZmMEG1-6) but also in related proteins from barley and wheat.
Preferentially, the nucleic acid molecules comprising a nucleotide sequence according to the invention consists in any one of SEQ ID No: 5, 7, 9, 11, 13 or 15, more preferentially consists in SEQ ID No: 5. Additionally said nucleic acid molecules may comprise the genomic sequence of Zm MEG1-1 shown in SEQ ID No: 58.
As used herein “variants” means that the sequence differs in one or more positions in comparison with these sequences SEQ ID No: 6, 8, 10, 53, 12, 14 or 16 as long as they encode a protein expressed in the BETL.
Such molecules comprise those which are variants of the BETL protein according to the invention and differ, for example, by way of amino acid and/or nucleotide deletion(s), insertion(s), substitution(s), addition(s) and/or recombination(s) or any other modification(s) known in the art either alone or in combination from the above described amino acid sequences or their underlying nucleotide sequence(s). Methods for introducing such modifications in the nucleic acid molecules according to the invention are well-known to the person skilled in the art. The invention also relates to nucleic acid molecules the sequence of which differs from the nucleotide sequence of any of the above-described nucleic acid molecules due to the degeneracy of the genetic code.
The invention further relates to a protein encoded by said nucleic acid molecules. More specifically, the invention provides a plant basal endosperm transfer cell layer (BETL) protein or an biologically active fragment thereof encoded by a nucleic acid molecule as defined above. Preferably, a protein according to the invention may comprise, or consist in, an amino acid sequence selected from the group consiting of SEQ ID No: 6, 8, 10, 53, 12, 14 and 16. Still preferably said protein comprises, or consists in, SEQ ID No:6. Additionally, a preferred BETL protein comprises amino acid sequence shown in SEQ ID No: 54.
The proteins encoded by the various variants of the above-described nucleic acid molecules share specific common characteristics, such as biological activity, molecular weight, conformation, etc., as well as physical properties, such as electrophoretic mobility, chromatographic behavior, sedimentation coefficients, pH optimum, temperature optimum, stability, solubility, spectroscopic properties, etc.
Hybridization stringent conditions are defined as described above.
A “fragment ” of a BETL protein or a nucleic acid molecule means a sequence, nucleotide sequence or amino acid sequence, which has a reduced length with regard to the nucleotide sequence or amino acid sequence of reference.
A preferred BETL protein fragment according to the invention comprises, or consists in, the amino acid sequence PCKDNKCYCCIGGRTH (SEQ ID No:54).
All such fragments and variants of the protein of the invention are included within the scope of the present invention, as long as the essential characteristic biological properties remain unaffected in kind, that is the novel nucleic acid molecules of the invention include all nucleotide sequences encoding proteins or peptides which have at least a part of the primary structural conformation for one or more epitopes capable of reacting with antibodies to BETL proteins which are encodable by a nucleic acid molecule as set forth above.
The nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein according to the invention can be isolated from various plant species, notably Angiosperm plants, Monocotyledons as Dicotyledons and are preferably nucleic acid molecules isolated from a plant selected from the group consisting of maize, teosintes, wheat, barley, rye, pea, rice, sorghum, and sugar cane. Preferably said plant is maize.
Another object of the present invention is a nucleotide construction, referred to as an expression cassette, comprising a nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein according to the invention, operatively linked to regulatory elements allowing the expression in prokaryotic and/or eukaryotic host cells. Regulatory elements are notably 5′ and 3′ regulatory sequences.
“Operatively linked” refers to functional linkage between the 5′ and 3′ regulatory sequences and the controlled nucleic acid sequence.
The 5′ regulatory sequences are notably promoters.
Any suitable promoter could be used. It could be for example a tissue-specific promoter such as a root-specific promoter, a leaf-specific promoter, a seed-specific, etc. Numerous tissue-specific promoters are described in the literature and any one of them can be used. Preferably, the promoter is a pathogen inducible promoter. Such promoters include those from pathogenesis-related protein, which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-1,3 glucanase, chitinase,etc.
Examples of promoters useful for plant transformation include the 35S promoter or the 19S promoter (Kay et al., 1987), the pCRV promoter (Depigny-This et al., 1992), the ubiquitin 1 promoter of maize (Christensen et al., 1996), the regulatory sequences of the T-DNA of Agrobacterium tumefaciens , including mannopine synthase, nopaline synthase, octopine synthase, the promoters regulated during seed development such as the HMWG promoter (High Molecular Weight Glutenin) of wheat (Anderson O. D. et al., 1989, Roberts et al., 1989), the waxy, zein or bronze promoters of maize, a promoter that is inducible by pathogens. The promoter may be a promoter as depicted in any of SEQ ID No: 1 to 3.
Preferentially, the promoter used is a pathogen inducible promoter or alternatively a constitutive promoter used to control the desired level of disease control (resistance to pathogen) in the plant.
The 3′ regulatory sequence is notably terminators.
Among the terminators useful for plant transformation within the framework of the present invention, the ones which can be used are the polyA 35S terminator of the cauliflower mosaic virus (CaMV), described in the article of Franck et al. (1980), the NOS terminator corresponding to the region in the non coding 3′ region of the nopaline synthase gene of the Ti-plasmid of Agrobacterium tumefaciens nopaline strain (Depicker et al. 1992), the histone terminator (EP 0 633 317), and the tml terminator.
Preferentially the terminator is the 3′Nos or 3′CaMV terminator.
Any other element, as previously described (intron, enhancer, transit peptide, . . . ) may be comprised in the expression cassette.
According to the invention, the expression cassette, comprising a nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein as defined above, operatively linked to regulatory elements may further comprises one or several selection marker gene for plants, useful for transformation and selection.
The selectable marker genes useful have been described previously.
Another object of the invention is any nucleotide vector referred to as an expression vector, such as a plasmid, which can be used for transforming host cells, characterized in that it contains at least an expression cassette comprising a nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein, as defined above.
Examples of useful expression vector have been described above.
Another object of the invention is a host cell, containing at least an expression vector as described above.
Examples of useful host cells have been described above.
The invention also relates to a transgenic plant, or a part of a transgenic plant (leaves, plant cell, plant tissue, grain, fruit, seed, . . . ) comprising a cell as described, notably comprising stably integrated into the genome a nucleic acid molecule encoding a plant basal endosperm transfer cell layer (BETL) protein, operatively linked to regulatory elements allowing transcription and/or expression of the nucleic acid molecule in plant cells.
A plant or part of a plant (plant cell, plant tissue, grain, seed, leaves, . . . ) according to the invention could be a plant or a part of it from various species, notably an Angiosperm, Monocotyledons or Dicotyledons, preferably a cereal or oily plant, selected in particular from the group consisting of maize, rice, wheat, barley, rape, and sunflower, preferentially maize.
In yet another aspect, the invention also relates to harvestable parts and to propagation material of the transgenic plants according to the invention which either contain transgenic plant cells expressing a nucleic acid molecule according to the invention or which contain cells which show a reduced level of the described protein. Harvestable parts can be in principle any useful parts of a plant, for example, leaves, stems, fruit, seeds, roots etc. Propagation material inclues, for example, seeds, fruits, cuttings, seedlings, tubers, rootstocks etc.
The invention further relates to a plant basal endosperm transfer cell layer (BETL) protein or an immunologically or biologically active fragment thereof encodable by a nucleic acid molecule according to the invention.
The invention also relates to an antibody specifically recognizing a BETL protein according to the invention or a fragment, such as SEQ ID No: 54, or epitope thereof
These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Techniques for producing such antibodies are classical methods well known by the one skilled in the art.
Another object of the invention is a method for improving plant pathogen resistance, comprising the steps consisting of:
a) transforming at least a plant cell by means of at least a vector as defined previously; b) cultivating the cell(s) thus transformed so as to generate a plant containing in its genome at least an expression cassette according to the invention, whereby a plant with improved pathogen resistance is obtained.

Pathogens of the invention include, but are not limited to, viruses or viroids, bacteria, insects, nematodes, fungi, and the like. For example, Fusarium species (notably Fusarium moniliforme, Fusarium graminearum ), Sclerotinia sclerotiorum, Phoma, Corn root worm, Aphis gossypii, etc. For maize, this would include especially ear mold fungal pathogens, such as Fusarium monoliforme.
Biological activity of the BETL proteins according to the invention can be assayed by any method known in the art. Assays to measure for example antimicrobial activity and the developmental pathways and defense responses that are influenced by the BETL protein are well known in the art.
Assays that measure antipathogenic activity (resistance to pathogen activity) are commonly known in the art, as are methods to quantitate disease resistance in plants following pathogen infection. Such techniques notably include, measuring over time, the average lesion diameter, the pathogen biomass, and the overall percentage of decayed plant tissues.
Antimicrobial and antipathogenic compositions are also included in the scope of the invention. The compositions of the present invention can be in a form suitable for direct application to the plant in need thereof or as a concentrate of a primary composition, which requires dilution with a suitable quantity of water or other diluent before application.
Additionally, the compositions can be used in formulations used for their antimicrobial and antipathogenic activities (resistance to pathogens). The proteins of the invention can be formulated with an acceptable carrier into a pesticidal composition(s) for example, a suspension, a solution, an emulsion, a dusting powder, a dispersible granule, a wettable powder, and an emulsifiable concentrate, an aerosol, an impregnated granule, an adjuvant, a coatable paste, and also encapsulations in, for example, polymer substances.
The methodologies for transformation, selection, regeneration, are known to one skilled in the art and have been previously described.
Another object of the invention is a method for improving the agronomic quality of a plant, comprising the steps consisting of :
a) transforming at least a plant cell by means of at least a vector as defined previously; b) cultivating the cell(s) thus transformed so as to generate a plant containing in its genome at least an expression cassette according to the invention, whereby a plant with improved agronomic quality is obtained.

More particularly, said plant exhibits an increased seed size as improved agronomic quality.
The overexpression of MEG1 protein into a plant result in a increased seed size. When overexpressing MEG1 protein, any promoter could be used in the expression cassette.
Preferably the promoter is chosen amongst a BETL specific promoter such as any one of the promoters according to the present invention, or the BETL2 promoter (WO 99/50427).
The invention relates to a nucleic acid construct comprising a) a BETL specific promoter, operatively linked to b) a nucleic acid coding for a MEG1 protein as depicted in SEQ ID NO: 6, 8, 10, 12, 14 or 16 or a variant of SEQ ID NO: 6, 8, 10, 12, 14 or 16. In the nucleic acid constructs, the promoter can be a BETL specific promoter that is not an imprinted promoter. A preferred BETL specific promoter is the BETL9 promoter as depicted in SEQ ID No: 69. A preferred MEG1 protein is SEQ ID No: 8, or a variant of SEQ ID No: 8 as depicted in SEQ ID No: 65. A preferred a nucleic acid coding for a MEG1 protein is represented by SEQ ID No: 66. A preferred nucleic acid construct comprises SEQ ID No: 70 or SEQ ID No: 71. The nucleic acid construct of SEQ ID No: 70 comprises the BETL-9 promoter of SEQ ID No: 69 and the optimized MEG1 sequence of SEQ ID No: 66. The nucleic acid construct of SEQ ID No: 71 comprises the BETL-9 promoter of SEQ ID No: 69 and the optimized MEG1 sequence with c-Myc tag of SEQ ID No:68.
Another aspect of the invention provides vectors containing the nucleic acid construct comprising a) a BETL specific promoter, operatively linked to b) a nucleic acid coding for a MEG1 protein as depicted in SEQ ID NO: 6, 8, 10, 12, 14 or 16 or a variant of SEQ ID NO: 6, 8, 10, 12, 14 or 16, host cells containing at least the nucleic acid construct, and transgenic plants or parts of transgenic plants comprising the host cells. The vectors can further comprise a selection marker gene for plants. Preferably, the plant or part of a plant is a cereal, more preferably a cereal selected from the group consisting of maize, rice, wheat, and barley.
A further aspect of the invention provides a method of obtaining a plant having increased seed size, comprising:
a) transforming at least one plant cell by means of at least a vector containing a nucleic acid construct comprising a) a BETL specific promoter, operatively linked to b) a nucleic acid coding for a MEG1 protein as depicted in SEQ ID NO: 6, 8, 10, 12, 14 or 16 or a variant of SEQ ID NO: 6, 8, 10, 12, 14 or 16; and
b) cultivating the cell(s) thus transformed so as to generate a plant containing in its genome at least the nucleic acid construct of step a), whereby a plant having increased seed size as compared to a non-transformed plant is obtained.
Moreover, the present invention relates to a kit comprising at least one of the aforementioned nucleic acid molecules, probes, conserved sequences, vectors, proteins, compositions or antibodies of the invention. The kit, or its components, according to the invention can be used in plant cell and plant tissue cultures, for example to detect expression levels of the transgene. The kit and its application are particularly useful to screen for antipathogenic effects in fungal cultures. The kit of the invention and its components are expected to be very useful in breeding new varieties of, for example, plants which display improved properties such as nutritional value or disease resistance (resistance to pathogens).



The present invention will be further understood in view of the annexed figures and following examples.
FIGS. 1 and 2 represent Allelic Message Display (AMD) analysis of ZmMEG1-1.
FIG. 1 is an autoradiograph of an AMD gel showing a maternal pattern of expression for ZmMEG1-1 (arrows). 1, F2 selfed; 2, Mo17 selfed; 3, F2×Mo17; 4, Mo 17×F2; 5, A69Y selfed; 6, F2 selfed; 7, F2×A69Y; 8, A69Y×F2.
FIG. 2 depicts an allele-specific RT-PCR analysis of ZmMEG1-1 in the endosperm. Top, ZmMEG1-1 maternal allele is monoallelic at 4 DAP (single band) and biallelic at 12 DAP (double band). Bottom, monoallelic expression of Meg1 in interploidy crosses at 4 DAP. The parental inbred lines used were F2, A69Y (69) and tetraploid W23 (4x).
FIG. 3 represents the amino acid alignment of putative proteins with a ZmMEG1-1 domain. Arrows indicate the position of conserved cysteines and a circle indicates the position of conserved tyrosines putatively involved in the anchoring to the cell wall.
FIGS. 4 to 7 represent ZmMEG1-1 protein characterization, and expression analysis of ZmMEG1-1 and five related cDNAs.
FIG. 4 : RT-PCR analysis was carried out with different RNA samples isolated from a range of tissues and amplified with gene specific primers (see oligonucleotide table 1). Dark and light box coloration indicate high and intermediate signal respectively, and a white box indicates absence of signal.
FIG. 5 depicts the In situ hybridization analysis of ZmMEG1-1 in developing seeds. Left, 4 DAP; Right 12 DAP.
FIG. 6 shows Immunolocalization of ZmMEG1-1 protein. Left, 12 DAP BETL region; Right, cell wall localization of ZmMEG1-1 in BETL cells.
FIG. 7 is a Western analysis of proteins isolated from 10 DAP endosperms after subcellular fractionation.
1, cytoplasmic fraction I; 2, cytoplasmic fraction II; 3, cell wall fraction I; 4, cell wall fraction II. Arrows indicate the presence of different ZmMEG1-1 proteins. Em, embryo; En, endosperm; ESR, embryo surrounding region; PC, pedicel.
FIG. 8 indicates that ZmMEG1-1 is a member of a small gene family of which parental alleles are differentially methylated in the endosperm ZmMEG1-1 methylation analysis in embryo (1-4) and endosperm (5-8) at 6 DAP.
1, W22 self; 2, A69Y self; 3, W22×A69Y; 4, A69Y×W22; 5, W22 self; 6, A69Y self; 7, W22×A69Y; 8, A69Y×W22. Arrows indicate the presence of differentially methylated genomic fragments in the endosperm detected with a ZmMEG1-1 gene specific probe.
FIG. 9 represents the result of a sequence alignment of ZmMeg1-1 and ZmMeg1-2 with BAP proteins.
FIG. 10 represents analysis of the ZmMEG1-1 promoter region, Left, a minimal promoter sequence of ZmMEG1-1 is conserved amongst several ZmMEG1-1 promoters from the F2 inbred line. Right, these promoters showed high conservation of the four regions also present in other BETL-specific genes (named box I to IV). White boxes, putative TATA motifs; boxes I to IV are the conserved sequences amongst promoter regions of several known BETL-specific genes.
FIG. 11 represents the differential expression of pZmMEG1-1:GUS in maize endosperm when inherited maternally and paternally. (A-J) sagittal sections of kernels stained for the GUS reporter, where (A, C, E, G and I) are seeds from an A188::pZmMEG1-1:GUS×A188 cross; (B, D, F, H and J) are seeds from the reciprocal A188 x A188::pZmMEG1-1:GUS cross. Note the presence of GUS signal in (A) and (C) at early stages of endosperm development but absent when paternally transmitted (B and D). (A and B) 4 DAP; (C and D) 6 DAP; (E and F) 10 DAP; (G and H) 15 DAP; (I and J) 20 DAP.
FIG. 12 is an analysis of the minimal promoter region of ZmMEG1-1. Left, Schematic representation of the promoter-deletion constructs employed in a co-bombardement experiment with a p35S:MRP1 construct. Right, results obtained from the co-bombardment assay. Plus signs indicate the presence of GUS staining (transactivation); Minus signs denote absence of GUS staining (lack of transactivation).
FIG. 13 is a diagrammatic representation of a crossing strategy enabling to obtain crops with improved value.
FIG. 14 depicts a pairwise sequence alignment of MEG1 (SEQ ID No: 1=promoter MEG 1-1), MEG2 (SEQ ID No: 2=promoter MEG 1-2), and MEG3 (SEQ ID No: 3=promoter MEG 1-3) performed with CLUSTAL W (1.74). The nucleotides shown in bold correspond to SEQ ID No: 57. Underlined nucleotides correspond to conserved regions between BETL specific genes (megl, betl's and DD1s), and shaded nucleotides correspond to conserved region between the above genes and ESRs. Boxed nucleotides correspond to TATA boxes.
FIG. 15 shows that MEG1 is localized to the ETT where it is required for transfer cell differentiation. a, TEM immunogold detection of MEG1 in plasma membrane (arrowheads) and cell walls (CW) of three adjoining transfer cells in an 8 dap wild-type endosperm. MT, mitochondrion, CWI, cell wall ingrowths. Scale bar=200 μm. b, Semi-thin sections of a 10 dap wild-type endosperm and Meg1 RNAi endosperm displaying abnormal transfer cells with limited cell wall ingrowths (arrows) ETT, endosperm transfer tissue, PC, placento-chalazal; scale bar, 5 μm.
FIG. 16 shows that Meg1 regulates seed size and ETT development. a, Longitudinal sections of wild-type and sibling Meg1 RNAi mature seeds. Red shading denotes embryo area, green shading denotes endosperm area, white arrowheads indicate length of filial:maternal interface (FMI). b, bar chart showing embryo area, endosperm area, total seed area, and FMI length in wild-type and Meg1 RNAi mature longitudinally sectioned kernels (unpaired Student's t test, P<0.0001). c, relative expression levels of ETT-specific transcripts in wild-type and in Meg1 RNAi lines. d, Immunodetection of INCW1/2 in wild-type and in a Meg1 RNAi kernel displaying comparatively reduced localisation. Scale bars: a, 5 mm; d, 10 μm.
FIG. 17 shows that Meg1 influences cell wall invertase activity and sugar homeostasis in maize seeds. a, In vivo soluble and b, cell wall bound acid invertase activities assayed in 15 dap longitudinally sectioned wild-type and Meg1 RNAi seeds. c, Hexose/sucrose sugar ratios in wild-type and Meg1 RNAi basal seeds, and d, in whole seeds. Error bars depict SD from a minimum of three biological repeats (unpaired Student's t test, *P<0.05, **P<0.01, ***P<0.0001). Scale bars: a, b, 2 mm.
FIG. 18 shows that Meg1 influences total sugar content in developing seeds. Sugar content in wild-type and Meg1 missexpression seeds shown for a, glucose, b, fructose, c, sucrose. d, Total hexose/sucrose ratios in developing wild-type and Meg1 missexpression seeds. Error bars depict SD from a minimum of four biological repeats (unpaired Student's t test, *P<0.05, **P<0.01, ***P<0.001).
FIG. 19 shows synMeg1 dosage analysis. a, Functional complementation of Meg1 RNAi line M60 with pBet9>synMeg1 and partial complementation with pMeg1>synMeg1. b, Western blot analysis of MEG1 and synMEG1 proteins in four independent transgenic lines. c, Relative GFP signal from seeds segregating for pBet9>synMeg1; no copies (−−/−), 1 copy (−−/+), 2 copies (++/−), 3 copies (++/+);(E) Relative fluorescence units of GFP per mg per ml of soluble protein from wild-type endosperms and endosperms with different doses of pBet9>synMeg1. Scale bars: a, 1 cm; c, 5 mm.
FIG. 20 shows that Meg1 is required for correct seed development. a-c, Meg1 downregulation causes reduced seed size and weight (unpaired Student's t test, ***P <0.0001). d,Endosperm transfer tissue (ETT) development in wild-type in Meg1 RNAi seeds; PC, placento-chalazal maternal tissue. e, Transfer cell-specific immunodetection of MRP1 in wild-type and in Meg1 RNAi endosperms; EM, embryo. Scale bars: a, b, 1 cm; d, e, 10 μm; f, g, 50 μm.
FIG. 21 shows that Meg1 regulates sugar partitioning within the developing seed. a, In vitro activity of cell wall acid invertases. b-d, Sugar content in wild-type and in Meg1 RNAi seeds. Error bars depict SD from a minimum of three biological replicas (unpaired Student's t test, *P<0.05, **P<0.01, ***P>0.001).
FIG. 22 shows that Meg1 is sufficient for transfer cell development. a-c, Macroscopic effects of Meg1 missexpression on seed size and weight (unpaired Student's t test, ***P<0.0001). d, Presence of ectopic transfer cells (ETCs) in Meg1 missexpression lines, magnified in e. f, Immunodetection of MRP1 in ectopic transfer cells. g, ETC formation indicated by B-glucuronidase activity of pBet1::uidA (blue staining and arrowhead) in cultured seeds from dex-inducible Meg1 missexpression lines. dic, days in culture. Scale bars: a, b, 1 cm; d, f, 50 μm; e, 10 μm; g, 1 mm.
FIG. 23 shows the dosage effects of non-imprinted pBet9>synMeg1 in the endosperm. a, Summary of dosage effects of pBet9>synMeg1 observed in mature seeds. One-way ANOVA tests revealed significant differences in dry seed weight (gm), embryo area, endosperm area, and transfer tissue interface (TTI) for pBet9>synMeg1 dosage series in the endosperm, where ***P<0.0001. Where *P<0.05 and **P<0.01, post hoc Tukey HSD tests revealed a significant difference between sample means when comparing parental transmission of pBet9>synMeg1 with wild-type (no transmission). A minimum of 534 seeds were analysed per seed trait. b, Immunodetection of MRP1 in 10 dap wild-type endosperm and in a 3-dose pBet9>synMeg1++/+ endosperm, displaying an extended ETT (indicated by arrowheads). Scale bars, 10 μm.



EXAMPLES
The invention will now be described by the way of the following examples, which should not be construed as in any way limiting the scope of the invention.
Several oligonucleotides and primers could be used to amplify or isolate the sequences or sequence fragments according to the present invention. The oligonucleotides and primers described are not limiting. The man skilled in the art knows how to design an oligonucleotide, or a specific oligonucleotide, based on a sequence.
Example 1
Plant Material and Growth Conditions
Maize ( Zea mays L.) diploid inbred lines W22, F2, B73 and Mo17, and tetraploid W23 (COOP Maize Stock Centre) were glasshouse grown under the following regime: 16 h day length (supplemented with metal halide lamps at 250 mmols, when required) at 22-28° C. during the day, and at 16-20° C. at night. Humidity levels were set at approximately 40-50% daytime and 60-70% at night. All lines were selfed and reciprocally crossed, and kernels were harvested at 6-24 days after pollination (DAP). Embryos and endosperms were isolated and pooled, then frozen in liquid nitrogen and stored at −80° C.
Example 2
Identification and Characterization of the ZmMeg1 Gene Family
A genomic screen designed to identify endosperm transcripts showing parent-of-origin patterns of expression was carried out by allelic message display (AMD) (Gutierrez-Marcos et al., 2003).
Allelic Message Display (AMD):
Total endosperm RNA was extracted from selfed and reciprocally-crossed inbred lines and used for AMD-PCR according to the protocol of Hagiwara et al. (1997).
Reverse transcribed RNA was used as source material for PCR (HIEROGLYPH kit; Genomix-Beckman), labeling with [alpha 33P]-dATP. A combination of 240 primers was used to carry out PCR reactions and products were analyzed in a semi-automatic Genomix LR DNA sequencing system. Following exposure to film (Biomax M R, Kodak), candidate bands were excised from the gel and amplified using PCR.
This resulted in the presence of several fragments showing monoallelic maternal expression, one of which was termed Zea mays maternally-expressed-gene1 (ZmMEG1) ( FIG. 1 ).
Identification of ZmMEG1 cDNAs and Expression Analysis.
2.1) Isolation of Meg1 cDNA and Related Sequences:
Full length cDNAs were obtained after screening a 7 DAP maize endosperm cDNA library (Hueros et al., 1999b). Among the 500,000 plaques screened, five cDNA clones were identified and sequenced. This identified the full-length cDNA, ZmMEG1-1, plus three other similar, but not identical, cDNAs which we termed ZmMEG1-2, -3, and -4. After screening the maize genome database, a further two ESTs showing partial similarity to the C-terminus of the translated MEG1-1 protein sequence were identified and denominated ZmMEG1-5, and -6.
2.2) Mapping:
The ZmMEG1 gene family was mapped to the short arm of maize chromosome 7, between markers csul3 and bng11200, by restriction fragment length polymorphism (RFLP) analysis using a population of immortalized F2 lines.
2.3) Southern Analysis:
The diversity of the ZmMEG1 family was revealed in the F2 inbred line by Southern analysis and BAC screening, this analysis indicated that the ZmMEG1 gene family comprises from two to four gene clusters with a few copies present in every cluster.
For Southern blotting, DNA was digested with the restriction enzyme XhoI, the hybridization solution was 5×SSPE/5× Denhardt's/0.5% SDS, and the blots were probed with full length Meg1-1 coding region (SEQ ID No: 5).
Southern analysis under high stringency conditions (hybridization 65° C. and wash for 10 minutes at 65° C. with 0.1×SSPE/0.5% SDS) revealed that the ZmMEG1 gene family comprises between 5-8 copies present in most maize inbred lines, teosintes and other grasses (rice, sorghum, sugar cane).
Southern analysis under low stringency conditions (hybridization 50° C. and wash for 10 minutes at 65° C. with 2×SSPE/0.5% SDS) also revealed bands in wheat and barley.
2.4) Expression Pattern
The expression pattern of ZmMEG1-1 and the other ZmMEG1-like cDNAs (ZmMEG1-2, ZmMEG1-3, ZmMEG1-4, ZmMEG1-5, ZmMEG1-6) was assessed by reverse transcription-polymerase chain reaction (RT-PCR) using gene-specific oligonucleotide pairs shown in Table 1 below.





TABLE 1



List of oligonucleotides used for RT-PCR analysis.






Gene
Forward
Reverse
PCR size

Name
Oligonucleotide
Oligonucleotide
(bp)



MEG1-1
TGCTGCTCATGCGCA
TTAGAAGCAKGCATGWC
165


TGGGGCTG,
TACACTSAGCC,



SEQ ID No: 23
SEQ ID No: 24




MEG1-2
ATGCACATGGGAAGG
TTAGAAGCAKGCATGWC
193


GTCATGTC,
TACACTSAGCC,



SEQ ID No: 25
SEQ ID No: 26




MEG1-3
GCATAGCAGGAGTGG
GAAGCAGGCATGACTAC
292


AGGGC,
ACTC,



SEQ ID No: 27
SEQ ID No: 28




MEG1-4
TGGCCAATGTCGCCT
TTAGAAGCAKGCATGWC
320


CCGAG,
TACACTSAGCC,



SEQ ID No: 29
SEQ ID No: 30




MEG1-5
ATGGCTGGCTATGGT
GTGCAGTTTGCAGGTAAG
150


GTTGATG,
CCC,



SEQ ID No: 31
SEQ ID No: 32




MEG1-6
TGTACGCCTGACTTG
TTAGAAGCAKGCATGWC
181


GCTAGCAACC,
TACACTSAGCC,



SEQ ID No: 33
SEQ ID No: 34




GST
GCAACGTACCGTACC
ACGCTGCATTCAATTACC



TTTCCGA,
GGGAAG,



SEQ ID No: 35
SEQ ID No: 36




GUS
GGGCCAACAGTTCCT
CCCCGTTGACTGCCTCTT



GATTAACC,
CG,



SEQ ID No: 55
SEQ ID No: 56




















































The expression pattern is shown in FIG. 4 .
Specific ZmMeg1-1 alleles showed 99.9% identity in several inbred lines (A188, B73, Mo17, F2, W22 and tetraploid W23) and were detected using cleaved and amplified polymorphic sequences (Neff et al., 1998).
To distinguish between ZmMeg1-1 alleles, RT-PCR analysis was performed employing MEG1 (5′-TGCTGCTCATGCGCATGGGGCTG-3′, SEQ ID No:17) and MEG1HpaI (5′-TTGTATATAAAAACAGTGATGTTAA-3′, SEQ ID No: 18) primers to produced a DNA fragment that after HpaI digestion generated fragments of 177-bp for F2 and 198-bp for A69Y or W23.
The gluthatione synthasel gene was used as a control for a biallelic parent-of-origin expression.
ZmMEG1-1 transcript was only present in endosperm samples between 4 and 20 DAP indicating that its expression is endosperm-specific. ZmMEG1-2 and ZmMEG1-4 also showed a similar pattern of expression, whereas ZmMEG1-3 was expressed in endosperm as well as in anther and pollen samples. By contrast, ZmMEG1-5 and ZmMEG1-6 were found to be expressed in most tissues tested.
Example 3
Parent-of-Origin Pattern of Expression of ZmMEG1-1
3.1) ZmMeg1-1 has a Maternal Parent-of-Origin Pattern of Expression
To confirm the maternal parent-of-origin expression pattern of ZmMEG1-1, a polymorphism was detected in the 3′UTR of the gene, between F2 and A69Y, and W23 and other inbred lines, which allowed us to design oligonucleotides for allele-specific RT-PCR.
The results revealed exclusive expression of the ZmMEG1-1 maternal allele at early stages of endosperm development (4 DAP) ( FIG. 2 ). Surprisingly, it has been found that at later stages of endosperm development (12 DAP) ZmMEG1-1 expression became biallelic ( FIG. 2 ).
To investigate the effects of altering the maternal to paternal genomic ratio in the endosperm on ZmMEG1-1 expression, reciprocal crosses between diploid and tetraploid inbred lines were performed. Allele-specific RT-PCR showed that the ZmMEG1-1 expression pattern remained unaltered ( FIG. 2 ).
3 . 2 ) ZmMeg1-1 Alleles are Differentially Methylated in the Endosperm
Since ZmMEG14 is subjected to a parent-of-origin pattern of expression in the endosperm it can be predicted that changes in methylation might occur between the different parental alleles.
The inventors have tested this hypothesis by examining the methylation status of ZmMEG1-1 parental alleles using methylation-sensitive restriction enzyme digestion and Southern analysis with a gene specific probe on genomic DNA obtained from embryo and endosperm samples.
Differences in the methylation status of parental ZmMEG1-1 alleles were apparent in the endosperm alone; ZmMEG1-1 maternal alleles appeared demethylated whereas paternal alleles were methylated ( FIG. 8 ).
These results strongly suggest a correlation between methylation status of a given ZmMEG1-1 allele in the endosperm and a parent-of-origin pattern of expression.
Example 4
ZmMeg1-1 Protein Characterisation
4.1) ZmMeg1-1 Encodes a Basal Endosperm Transfer Cell Wall Protein
The full length ZmMEG1-1 cDNA contained an open reading frame of 88 amino acids ( FIG. 3 ), encoding a predicted 9794 Da protein with a pI of 7.5. The ZmMEG1-1 polypeptide contains a hydrophobic N-terminal region with characteristics of a 27 amino acid signal peptide (von Heijne, 1986). Further analysis identified a putative cleavage site between His-26 and Glu-27, thus producing a polypeptide containing 61 amino acids, with a predicted molecular mass of 6730 Da and p16.2.
An interesting feature of the ZmMEG1 polypeptide is the presence of a highly conserved cysteine-rich domain in the C-terminal portion, also present in the ZmMEG1-likes (ZmMEG1-2, -3, -4, -5, and -6) ( FIG. 3 ). This conserved region contains six cysteines and two tyrosines, and resembles other cysteine domains found in several plant proteins with hypothetical cell-wall localization (de Oliveira et al., 1990; Domingo et al., 1999).
In situ hybridization of ZmMEG1-1 was performed on kernel sections at various stages of endosperm development with a gene specific probe kernels according to a published method (Jackson, 1991), with minor modifications DB (Costa et al., 2003). ( FIG. 5 ).
No signal was detected with the sense probe, however, the antisense probe only gave signal in the transfer cells from 4 DAP and showed maximum expression at around 6-10 DAP ( FIG. 5 ), clearly indicating that ZmMEG1-1 is exclusively expressed in the basal transfer region of the endosperm.
4.2) ZmMEG1-1 Protein Localization:
To determine the localization of ZmMEG1-1 protein in maize endosperm, a polyclonal antiserum was raised by using a synthetic peptide designed to the N-terminus of the putatively processed ZmMEG1-1 polypeptide.
Immunolocalization
Polyclonal antiserum was raised in rabbit against a synthetic peptide (NAPAEEGILREKRAQC, SEQ ID No: 19) and affinity purified with an immobilized peptide using a Sulpholink coupling gel system (Pierce, UK). Maize kernels were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer pH 7.2, for 12-24 h depending on the tissue volume. Samples were dehydrated in an ethanol series and wax embedded. Sections were de-paraffinized and blocked in 1% BSA in PBS (10 mM sodium phosphate, 150 mM NaCl pH 7.4) for 30 min at room temperature, and incubated overnight with anti-MEG1 antiserum or pre-immune serum (both diluted 1:500). The immunoreactions was detected using alkaline phosphatase-coupled secondary antibody (Sigma; diluted 1:1000) and NBT/BCIP as substrate.
Immunolocalization was performed using the purified antiserum, which detected ZmMEG1-1 protein adjacent to the cell wall ingrowths of basal endosperm transfer cells ( FIG. 6 ).
To obtain biochemical evidence of protein localization to the cell wall, protein extracts from 10 DAP endosperms were fractionated as described by Sema et al. (2001) and analyzed by immunoblotting ( FIG. 7 ).
Subcellular Fractionation of Proteins
Fractionation of subcellular components of endosperms (F2 inbred) was performed as described by Serna et al. (2001). Proteins were fractionated by SDS-PAGE electrophoresis according to (Laemmli, 1970), and electroblotted onto a polyvinylidine difluoride (PVDF) membrane. Proteins were detected using an enhanced chemiluminescence method (ECL, Amersham, UK).
Several proteins with predicted molecular masses that ranged from 6 to 50 kDa were present in the cytoplasmic fractions, two of which corresponded to ca. 9-10 kDa and ca. 6-7 kDa. Interestingly, the ca. 6-7 kDa proteins gave the greatest signal intensity in the cell wall preparation, thus providing evidence for cell wall localization of the putative cleaved ZmMEG1-1 polypeptide ( FIG. 7 ). Three other proteins with molecular masses between 2.5 to 3.5 kDa were observed in the cell wall fraction.
4.3) ZmMEG1-1 Role:
The BETL and BAP families comprise the largest basal transfer layer-specific protein families found to date. These small proteins are secreted into the pedicel region or are localized to the cell wall and are strongly suggested to be implicated in antifungal attack and pathogen defence (Hueros et al., 1995; Serna et al., 2001).
As known from the art (Hueros et al. 2002), the BETL-specific genes isolated to date share some structural features that strongly suggest that they play a role in defense against pathogen entry into the developing seed. They encode small, Cys-rich secreted peptides that accumulate only transiently during endosperm development.
Amino acid sequence analysis of ZmMeg1-1 revealed a cysteine-rich motif in the C-terminal region comprising six cysteine residues and two tyrosine residues: C-(X)4-CYCC-(X)8-Y-(X)4-C-(X)3-C (SEQ ID No: 20), which was conserved in the other ZmMEG1-like proteins (ZmMEG1-2, ZmMEG1-3, ZmMEG1-4, ZmMEG1-5 and ZmMEG1-6) ( FIG. 3 ) and showed high similarity to many cysteine-rich domains identified in other plant proteins (Hueros et al., 1995; Domingo et al., 1999; Hueros et al., 1999b; Serna et al., 2001) (Schopfer et al., 1999). Cysteine-rich domains are believed to be required for the maintainance of particular protein conformations, which result in the exposure of interdigitated side chain residues (Berg and Shi, 1996), such as tyrosine residues found in ZmMEG1. Importantly, it has been reported that exposure of tyrosine residues facilitate the binding of synthetic-proteins to the cell wall (reviewed in Cassab, 1998).
Since ZmMEG1-1 protein localization is proximal to the maternal-filial interface, it is strongly expected that it may have a defensin-like function, as reported for BETL1,3 (Hueros et al., 1995; Hueros et al., 1999a) and ZmES proteins (Cordts et al., 2001).
Furthermore the modification of MEG1 protein level modifies seed size as the decrease of MEG1 protein level induces a reduced seed size (see below).
An alignement of MEG1-1 and MEG1-2 amino acid sequence with that of BAPs proteins has been performed and the result is shown in FIG. 9 .
Two Megl constructs (expression cassettes) to determine the effect of Megl gene overexpression in maize have been made:
Maize Transformation:
Embryogenic type II calli were transformed with the construct and regenerated as described by Bonello et al., 2000.
The following constructs have been obtained by classical molecular biology methods.
Construct A: p35S-HSP70intron-Meg1 genomic-3′CaMV
34 maize transformants have been generated, some plants exhibit improved resistance to the pathogens.
Construct B: p35S-HSP70intron-Meg1 cDNA-3′CaMV
36 maize transformants have been generated, some plants exhibit improved resistance to the pathogens.
Construct C p35S Meg1-RNAi-3′OCS
A Meg1 construct (expression cassette) to determine the effect of Meg1 downregulation has been made.
Vector pGUSART, was obtained by subcloning an EcoRV fragment (789 to 1820) of the GUS gene into pART7 (Gleave A.P (1992) Plant Mol. Biol. 20:1203-1207).
A fragment of the Meg1-1 cDNA sequence (151 to 380) was amplified by PCR using two oligonucleotides:







meg1RNAi.FOR (ClaI-EcoRI)


(SEQ ID No: 63)


5′-ATCGATGAATTC-GCTCAAGGGTTTCTTCCATG-3′





meg1RNAi.REV (BamHI-XhoI)


(SEQ ID No: 64)


5′-GGATCCTCGAG-CCTCTAGTATCGGTCTGAC-3′














The PCR product was subcloned into pGEM-T easy and sequenced. After digestion with BamHI and ClaI, and EcoRI and XhoI the fragments were subcloned into pGUSART to generate a 3′-5′ and 5′-3′ construct flanking the deleted portion of the GUS gene capable of produce a duplex formation.
24 maize transformants have been generated, some plants exhibit reduced resistance to the pathogens.
In particular, 4 out of the 24 Meg1 RNAi plants exhibited a segregation in seed size on the cob. Kernels that inherited the RNAi transgene were found to be smaller than wild type segregant kernels (figure not shown). Thus inhibition of Meg1 expression reduces seed size.
Example 5
ZmMEG1-1 Promoter Isolation and Sequence Characterization
5.1) Meg1 Genomic DNA Isolation
A Meg1.1 probe was used to screen an F2 BAC library (O'Sullivan et al., 2001), resulting in six genomic ZmMeg1-1 fragments that were subcloned into pBluescript KSII (Stratagene,UK) and sequenced. Sequence analysis revealed the existence of at least four different copies of ZmMEG1-1 in the F2 inbred line, all of which showed subtle differences in their promoter region, yet highly conserved exon and intron regions. A minimal region of the promoter (ca. 370 bp) was found to be conserved across other promoters belonging to known basal endosperm transfer cell-specific genes (Hueros et al., 1999b; Sevilla-Lecoq et al., 2003) ( FIG. 10 ).
5 . 2 ) Reporter Cconstruct Preparation:
A minimal promoter region of ZmMEG1-1 was isolated by PCR using two specific oligonucleotides (5′-ACACCTCAAATAGATATGGATATAG-3′, SEQ ID No: 60, and 5′-GTCGCAAGAAATGTTAAGGAACTCC-3′, SEQ ID No: 61) and subcloned into pGEM-T easy vector (Promega, Madison, USA).
After digestion with EcoRI and ClaI, the 371 bp fragment was fused to the β-glucoronidase (GUS) encoding sequence and the nopaline synthase (NOS) terminator of the pSLJ4K1 vector (Jones et al., 1992) to generate the transcriptional fusion vector denominated pZmMEG1-1-GUS.
5.3) Maize Transformation and Expression Analysis:
To test whether the minimal promoter region of ZmMEG1-1 was able to confer transfer cell-specific gene expression we stably transformed maize plants with the pZmMEG1-1-GUS transcriptional fusion construct.
Maize Transformation:
Embryogenic type II calli were transformed with the construct and regenerated as described by Bonello et al., 2000.
Five independent transgenic lines were generated (0757-1C, 0757-2E, 0757-2F, 0757-2D, 0757-2L) and selected on the basis of high levels of β-glucuronidase expression, as detected by histochemical staining of kernels. Plants were genotyped for β-glucuronidase (GUS) transcriptional fusions via PCR using GUS-specific oligonucleotides (SEQ ID No: 55 and 56). Histochemical analysis of transgeneic lines was performed according to (Jefferson et al., 1987; Costa et al., 2003). Briefly, fixed kernels were dehydrated to 70% ethanol before analysis and digitally imaged.
GUS staining was only ever observed in endosperm tissue, and not in any other plant tissues tested.
These lines were subsequently backcrossed with pollen from an A188 standard inbred for four consecutive generations to confirm the stable pattern of expression in the endosperm.
Two of the independent transgenic lines (0757-2E and 0757-2D) were randomly selected and reciprocally outcrossed with wild-type A188 plants to test for changes in patterns of transgene expression based on their parental mode of inheritance.
For each line, around 200-280 kernels at different developmental stages were isolated from four plants and histochemically stained for GUS. For each time point analyzed, around 50-70 kernels were isolated from four plants, and histochemically stained for GUS, as mentioned above.
Interestingly, we found differential temporal GUS expression when transmitted either maternally or paternally.
When pZmMEG1-1-GUS plants were either selfed or outcrossed as females, GUS staining was present in transfer cells from 4 DAP, when the endosperm becomes fully cellular, and remaining until 20 DAP. A maximum level of GUS staining was attained at 10-12 DAP.
However, when pZmMEG1-1-GUS plants were outcrossed as males (i.e. transmitted paternally through pollen), histochemical GUS activity was only ever detected after 10 DAP, when it was at its highest, and the declined in expression by 20 DAP. The apparent delay in expression of the paternally inherited transgene was confirmed at the transcriptional level by Northern analysis (data not shown).
Taken together, the data demonstrate that a minimal region of MEG1-1 promoter is able to confer different temporal transfer cell-specific expression of uidA reporter gene depending on parental origin.
So, expression of pZmMEG1:GUS in BETL cells is dependent on parental inheritance.
Example 6
Transactivation of the ZnMEG1-1 Promoter by ZmMRP1
It has been recently found that a transfer cell-specific transcription factor, ZmMRP1, is able to transactivate expression of several BETL promoters (Gomez et al., 2002).
To investigate whether ZmMRP 1 was also capable of transactivating ZmMEG1-1, etiolated maize leaves were cobombarded with the pZmMEG1-1-GUS construct and a 35S enhanced version of the ZmMRP1 protein (p35S-MRP1 translational fusion).
The data showed that pZmMEG1-GUS was significantly transactivated by ZmMRP1 ( FIG. 11 ), indicating that ZmMRP1 is sufficient for activation of ZmMEG1-1 in leaf tissues and is probably sufficient to activate ZmMEG1-1 expression in endosperm transfer cells.
The same results are obtained with the MEG1 promoter according to SEQ ID No: 62.
Example 7
Promoter Deletion Analysis
To define the region essential for transactivation by ZmMRP1, we generated a deletion series of the conserved ZmMEG1-1 promoter region and fused these to the uidA reporter gene.
The serial deletion analysis was performed by PCR amplification of different regions of the ZmMEG1-1 promoter, using multiple oligonucleotide pairs (see table 2 below) and then subcloning into pGEM-T easy vector (Promega, Madison, USA).





TABLE 2



List of oligonucleotides used in

deletion analysis.





Con-



struct
Forward
Reverse

name
Oligonucleotide
Oligonucleotide



pMEG1-
ACACCTCAAATAGATATG
GTTATCTATTCTATTCTATCAT

GUS
GATATA,
ATCTATC,


SEQ ID No: 37
SEQ ID No: 38



pMEG1-
GATATAGATATATAGAAG
GTTATCTATTCTATTCTATCAT

31/
AGATATAGATGG,
ATCTATC,

30GUS
SEQ ID No: 39
SEQ ID No: 40



pMEG1-
AGATAGATATGATAGAAT
GTTATCTATTCTATTCTATCAT

32/
AGATAGATAAC,
ATCTATC,

30GUS
SEQ ID No: 41
SEQ ID No: 42



pMEG1-
ATTTTGTCTAAAGAGACTA
GTTATCTATTCTATTCTATCAT

33/
AATCACTGC,
ATCTATC,

30GUS
SEQ ID No: 43
SEQ ID No: 44



pMEG1-
ACACCTCAAATAGATATG
CCAATTCACTGGGTTATCTATT

30/
GATATA,
CTATTCTATCATATCT,

31GUS
SEQ ID No: 45
SEQ ID No: 46



pMEG1-
ACACCTCAAATAGATATG
CCAATTCACTGGCCATCTATAT

30/
GATATA,
CTTCTATATATCTATA,

32GUS
SEQ ID No: 47
SEQ ID No: 48



pMEG1-
ACACCTCAAATAGATATG
CCAATTCACTGGCCCCTTGTTA

30/
GATATA,
TATCTATATCTATACC,

33GUS
SEQ ID No: 49
SEQ ID No: 50


















































Constructs were digested with EcoRI and BstXI and fragments were subcloned into the pZmMEG1-1-GUS construct pre-digested with EcoRI and BstXI. By following this approach the putative TATA boxes identified in the ZmMEG1-1 promoter remained intact.
The ZmMRP-1 coding region was isolated by PCR using oligonucleotides MRP.FOR (5′GGATCCATGAATCCCAACTTCAACAGTG3′, SEQ ID No:21) and MRP.REV (5′GAATTCTTATCGGTTATATATCTGGCTCTCC3′, SEQ ID No:22). PCR fragments were subcloned in pGEM-T easy (Promega, Madison, USA), digested with BamHI/EcoRI and the 327 bp fragment was subcloned into the plasmid described by Pang et al. (1996) to generate p35S:HSP70::MRP1. Plasmid DNA was isolated by the QIAprep midi kit (Qiagen, Germany) and coated onto tungsten (M10) particles according to (Klein et al., 1992).
For transient transformation, Hi-II maize seeds were surface sterilized and germinated in the dark. Etiolated leaves (2 cm wide) were sectioned into 1-2 cm long pieces and co-bombarded with ZmMEG1-1 promoter deletion and ZmMRP1 constructs using a BioRad Biolistic PDS-1000/He device. Gold particles (0.6 nm, from BioRad, UK) were coated with the DNA plasmid mixture including 2.5 g of p35S:MRP1 derived plasmid and 2.5 g of ZmMEG1-1 promoter deletion. Tissues were positioned 6 cm from the microcarrier stopping screen itself located 5 cm below the 6.2 Mpa rupture disc.
After bombardment, samples were incubated for 24 h at 26° C. in the dark on solid medium (Murashige and Skoog, 1962)containing 100 mg/l myo-inositol, 2 g/l glutamine, 30 g/l sucrose, and Murashige and Skoog vitamins (Sigma, UK).
A minimum of three independent experiments were conducted for each promoter deletion made.
Histochemical detection of GUS expression was performed by staining the leaf discs according to (Jefferson, 1989) with modifications. Leaf pieces were stained in a medium containing 0.5 mg/ml Xglucuronide (Clonetech, Palo Alto, Calif.), 0.5 mM phosphate buffer, pH 7.0, 0.1% triton X-100 and 20% (v/v) methanol.
It has been found that removal of the distalmost portion of the promoter (−370 to −240 region) disabled transactivation of pZmMEG1 GUS by ZmMRP1.
To determine whether this minimal −370 to −250 promoter region ( FIG. 12 ) was sufficient to allow transactivation by ZmMRP1, either alone or in combination with other regions within the conserved ZmMEG1-1 promoter, deletions were performed in the reverse orientation.
From this analysis, the −370 to −250 minimal promoter region in combination with the putative TATA box region (−127 to 1) was deemed sufficient to confer transactivation of the ZmMEG1-1 promoter by ZmMRP1.
Taken together, these results conclusively demonstrate that ZmMRP1 activates ZmMEG1-1 expression by transactivation of the −370 to −250 conserved domain also present in promoters of many BETL-specific genes.
The same results are obtained with the −345 to −251 region (95 nucleotides in length) that corresponds to nucleotides +26 to +120 of SEQ ID No:4 and with the fragment from nucleotide +29 to +119 of SEQ ID No:4 (91 nucleotides in length).
Example 8
Expression in the BETL Area of Genes of Interest
SEQ ID No: 1 and SEQ ID No: 62 have 90% identity and 6.7% of gaps after alignment on the basis of SEQ ID No: 1. This differences correspond to deletions, insertions, and base replacements that do not affect the functionality of the sequence so that same results are obtained with SEQ ID No: 1 and SEQ ID No: 62.
A MEG1 promoter according to the invention is useful to express from an early stage of BETL development proteins or RNA that result in desirable changes in seed development or quality. Such proteins or RNA may for example increase the size of the BETL layer and/or increase its capacity to import nutrients into the developing seed. Alternatively such proteins or RNA may improve resistance to pathogens and thus better protect the developing seed from transfer of pathogens from maternal tissues resulting in seeds (and progeny plants) of higher quality and vigour.
As an example of the first utility Meg1 promoter (ZmMEG1-1) is linked to the cell wall invertase incW2 and as an example of the second utility Meg1 promoter (ZmMEG1-1) is linked to the anti-fungal protein BAP2.
8.1) Overexpression of IncW2 in the BETL
IncW2 is naturally expressed in the BETL. Its importance in the determination of seed size was demonstrated via the analysis of IncW2 mutants which exhibit a miniature kernel phenotype (Cheng et al (1996) Plant Cell June; 8(6):971-983)).
Meg1-1 promoter is fused to the coding region of IncW2 and cloned into a binary vector for agrobacterial mediated transformation of maize. Analysis of the transformed plants indicates that some plants overexpress IncW2 and this expression is earlier than normal in seed development. These plants possess seeds that are larger than segregant seeds that lack the transgene which have normal levels of IncW2.
The same results are obtained with a construct comprising the MEG1 promoter as defined in SEQ ID No: 62.
8.2) Overexpression of BAP2 in the BETL
BAP2 is naturally expressed in the BETL. Its importance in resistance of the developing seed to fungal attack was strongly suggested in studies where the isolated protein demonstrated significant anti-fungal activity in vitro (Sema et al (2001)).
Meg1-1 promoter is fused to the coding region of BAP2 and cloned into a binary vector for agrobacterial mediated transformation of maize. Analysis of the transformed plants indicates that some plants overexpress BAP2 and this expression is earlier than normal in seed development. These plants possess seeds that have improved resistance to pathogens compared to segregant seeds that lack the transgene which have normal levels of BAP2.
The same results are obtained with a construct comprising the MEG1 promoter as defined in SEQ ID No: 62.
Example 9
Use of Meg1 Promoter to Improve the Crop Value
The Meg1-1 promoter is active earlier in the BETL if inherited from the female than from the male. Early overexpression of proteins in the BETL layer leading to a desirable kernel phenotype in some cases can disrupt the normal development of the kernel such that the germination of the kernel is reduced. For example early overexpression of the transactivator MRP1 (Gomez et al (2002)) in the BETL leads to seeds with a hypertrophied BETL layer. Since the BETL layer is important for nutrient flow into the developing endosperm, this hypertropy leads to the production of a larger endosperm and a larger seed. However in some genetic backgrounds or crops this hypertrophy gives a larger endosperm at the expense of embryo development. Thus although the resulting seed has a higher endosperm to embryo ratio and thus a higher value, the MPRP1 transgene is not utilisable due to poor seed germination.
Overexpression from an imprinted promoter, particularly such as ZmMEG1-1, overcomes this problem.
Maize plants were transformed with a construct comprising in the sens orientation the MEG1 promoter (as defined in SEQ ID No: 62), the ZmMRP1 cDNA, and the AtSac66 terminator (Jenkins et al. 1999).
Homozygous pMEG1:MRP1 plants are used as pollen donors in crosses to wild-type plants (poor germination of these plants is acceptable at the multiplication stage). All the hybrid progeny of this cross will have normal kernels since the late paternal expression of MRP1 has little effect on BETL size. The hybrid progeny of this cross will have 50% of kernels with increased endosperm size, enough to significantly improve the crop value ( FIG. 13 ).
The same results are obtained with a construct comprising the MEG1 promoter as defined in SEQ ID No: 1 (pMEG1-1).
Example 10
Maternal Control of Nutrient Allocation in Plant Seeds by Genomic Imprinting
Genomic imprinting occurs in flowering plants and eutherian mammals, resulting in the preferential uniparental expression of alleles from the same gene. Functional studies of mammalian imprinted genes have highlighted the antagonistic roles of imprinted alleles in the placenta to control the distribution of nutrients from mother to foetus (Constancia et al., 2002; Vrana et al, 2001). Such findings have prompted several theories implicating imprinted genes in a parental tug-of-war over resource allocation to the embryo (Moore et al., 1991; and Wilkins and Haig, 2003). However, despite the recent identification of over 200 putative imprinted genes in the placental-like seed endosperm (Gehring et al, 2009; Hsieh et al., 2009; and Hsieh et al., 2011), only few have been functionally characterised (Raissig et al), and none to date have been involved in regulating the maternal influx of nutrients to the offspring. Here we describe the functional characterisation of Meg1 and show that it acts as a positive regulator for the development of a specialised group of nutrient transfer cells located at the seed filial:maternal interface. Consistent with these findings, we also show that Meg1 influences sugar partitioning within the seed. Further, we provide genetic evidence demonstrating that MEG1 acts in a dosage-dependent manner to regulate endosperm transfer tissue (ETT) formation, thereby affecting the nutrient transfer capacity of the endosperm and, ultimately, seed biomass. Thus akin to the situation in mammals, Meg1 represents the first example of an imprinted gene that controls maternal resource allocation to the offspring in plants.
Materials and Methods
Methods Summary
Meg1 RNAi and Meg1 missexpression lines were generated by biolistic transformation of maize embryonic calli, evaluated by qRT-PCR and selected on the basis of altered Meg1 expression. Seed phenotypes were analysed by standard histochemical staining techniques and by immunodetection. Invertase enzyme activity and sugar content of seeds were measured by colorimetric methods. Dex-inducible Meg1 missexpression and synth Meg1 overexpression lines with the imprinted Meg1 promoter and non-imprinted Bet9 promoter were generated by Agrobacterium tumefaciens transformation and selected on the basis of expression of a visual reporter (GUS and GFP respectively).
Plant Growth
The maize cultivar A188 was used in all experiments. Plants were grown at 28° C. day/20° C. night in a 16 □h light/8 □h dark cycle with a light intensity of 230 μE m-2 s-1. Seeds were germinated in three-inch diameter pots containing peat-based soil and grown for three weeks before transferring to 40 L pots to flowering. Controlled pollinations were conducted manually and seeds were harvested 40 days after pollination (dap).
Plant Material
A Meg1-RNAi vector was generated by PCR amplification of a 156 bp fragment of the Meg1 transcript (Table 3) followed by digestion with BamHI and BglII prior to subcloning into anpe35S::A-uidA-S vector to generate a 3′-5′ and 5′-3′ construct flanking a portion of the uidA gene capable of producing a duplex formation (Chuang et al., 2000). To generate the MEG1 missexpression construct, the complete Meg1 open reading frame was amplified by PCR (Table 3), digested with BamHI and EcoRI and ligated into pmon30049 vector behind the e35S promoter (Pang et al. 1996). Embryogenic type II maize calli were transformed with the pe35S::Meg1 construct by biolistic bombardment and regenerated as described by Bonello et al., 2000. Transgenic plants were selected on media containing 5 mg/L PPT during the entire tissue culture period and regenerated plantlets were transferred to soil and the integrity and copy number of the transgene was determined by Southern hybridization and qPCR. To identify lines suitable for Meg1 functional analysis, we grew an average of 10 plants from each independent transgenic line (40-45 lines per construct) and selected those showing both altered levels of Meg1 expression and segregating discernable seed phenotypes.
To generate the dexamethasone-inducible Meg1 construct, we PCR amplified the Meg1 open reading frame engineered with attB ends to allow recombination into the entry clone pDONR207 (Invitrogen). The resulting clone (pDONR_Meg1) was inserted by recombination into a maize optimized pOp6/LhGR vector (based on the plant binary vector pTF1011 described in Frame et al., 2002). The resulting plasmid was transferred into Agrobacterium tumefaciens strain EH101 (pSB1) by electroporation and used for plant transformation as described in Ishida et al., 1996. To evaluate lines suitable for analysis we grew an average of 10 plants from each of the 20-40 independent transgenic lines generated and selected lines that showed ectopic MEG1 expression when exposed to irrigation with 20 μM dexamethasone as described by Samalova et al., 2005. Dexamethasone induction of Meg1 was carried out in 20 h post-fertilisation ovules generated by crossing pBET1::uidA lines with DEX-Meg1 plants. Fertilised ovules were cultured in MS media supplemented with 20 μM dexamethasone for 24 h and then transferred to a dexamethasone-free media for further culture (2, 4 and 6 days).
To generate the imprinted and non-imprinted Meg1 constructs (pMeg1> syn Meg1 and pBet9> syn Meg1, respectively), we designed a synthetic Meg1 ( syn Meg1) sequence (SEQ ID No. 66) with codon usage optimized for maize, which shared minimal homology to endogenous Meg1 at the nucleotide level, and contained a single Myc-tag at the carboxyl terminus (SEQ ID No: 65-MEG-1 protein variant of SEQ ID No:8; SEQ ID No: 67-MEG-1 protein of SEQ ID No:65 with c-Myc tag ; SEQ ID No. 68-optimized maize sequence with c-Myc tag). syn Meg1 was synthesized by Genescript and cloned into a pDONR207 entry vector (Invitrogen). The resulting construct (pDONR —syn Meg1) was then cloned either behind the Meg1 promoter forming pBIOS1751, or behind the BETL9 promoter (SEQ ID No: 69), forming pBIOS1752, using binary destination vectors pBIOS1027 and pBIOS960, respectively. These vectors were modified from plant binary vector pSB12 as described in Komai et al., 1996, to also contain a selectable marker gene and a GFP-encoding gene under the constitutive promoter pCsVMV (Verdaguer et al. 1998), which allows visualisation of transgene segregation in mature seeds. The resulting plasmids were transferred into Agrobacterium tumefaciens strain LBA4404 (pSB1), which was used to transform A188 plants, as described by Ishida et al, 1996. Single copy insertion events were selected via qPCR analysis for the selectable marker gene and verified by PCR and expression of the GFP reporter in subsequent generations. To evaluate lines suitable for analysis, we grew an average of 10 plants each from 25 independent transgenic lines per construct, which were used in reciprocal crosses with Meg1-RNAi line M60 to assess the phenotypic complementation of the reduced seed phenotypes.





TABLE 3



Sense and antisense oligonucleotide sequences used for qRT-PCR to

verify Meg1 expression and syn Meg1 copy number are listed together

with expected size of amplicon.







Gene
Size




Amplicon
(bp)
FW Primers (5′-3′)
RV Primers (5′-3′)
Purpose



MEG1_RNAi
156
ATCGATGATGAATTCG
GGATCCTCGAGCCTC
RNAi



CTCAAGGGTTTCTTAAA
TAGTATCGGTCTGAC,




TG, SEQ ID No: 72
SEQ ID No: 73




MEG1_OX
357
ATGCATCCAACGCGTT
TTAGAAGCATGCATG
Overexpression



GGGAGC,
ACTACACTG,




SEQ ID No: 74
SEQ ID No: 75




MEG1_DEX
411
GGACAAGTTTGTACAA
GGACCACTTTGTACA
DEX inducible



AAAAGCAGGCTATGCA
AGAAAGCTGGGTTTA




TCCAACGCGTTGGGAG
GAAGCATGCATGACT




C, SEQ ID No: 76
ACACTG,





SEQ ID No: 77




BAR
76
GCACCATCGTCAACCA
GTCCACTCCTGCGGT
Copy number



CTACA,
TCC,




SEQ ID No: 78
SEQ ID No: 79




BAR_Probe
N/A
FAM-5′CACGGTCAA
N/A
Copy Number



CTTCCGTAC-MGB-





NFQ*,





SEQ ID No: 80





PEPC-1
119
AAGGCGATCCATTCCT
GGGCGTCACCTTGAA
Copy number



GAAG,
GTTG3,




SEQ ID No: 81
SEQ ID No: 82




PEPC_Probe
N/A
VIC-5′TACATCAC
N/A
Copy number



CACCCTGAAC3′-MGB-





NFQ*, SEQ ID No: 83





















































GFP Quantification
Transgene dosage was determined in dry seeds by quantifying the relative GFP abundance in endosperms containing 0, 1, 2 and 3 copies of syn Meg1 transgenes. Briefly, endosperms from mature imbibed seeds were dissected and homogenized in four times (w/v) ice-cold extraction buffer (30 mM Tris-Cl pH 8.0, 10 mM EDTA, 10 mM NaCl, 5 mM dithiothreitol) to isolate soluble protein extracts. Total protein concentrations were calculated against a BSA standard curve using Bradford Reagent (Bio-rad Laboratories, Hercules, Calif.) and readings were taken at A 595 nm in a Tecan GENios Microplate Reader. GFP fluorescence was quantified in triplicate using 96-well flat-bottom black polystyrene plates (Greiner Bio-One Ltd) and relative fluorescence measurements were taken with 485 nm excitation and 535 nm emission. Copy number of the transgene was also independently verified by qPCR (below).
Quantitative Real Time PCR
For qPCR, genomic DNA was extracted using DNeasy Plant Mini kit (Qiagen). qPCR reactions were performed TaqMan Universal PCR Mastermix, no AmpErase UNG from Applied Biosystems on a 7900 HT Real-Time PCR system (Applied Biosystem). Each sample was assayed in quadruplicate and analyzed with the SDS Software 2.3 (Applied Biosystem) and quantification was performed using the 2-DeltaDeltaCt method (Livak et al., 2001). For qRT-PCR, RNA was isolated using TRIzol reagent (Invitrogen). cDNA was reverse-transcribed using Superscript III RT (Invitrogen). qRT-PCR reactions were performed using Tay polymerase (Invitrogen) and SYBR green (Molecular Probes) on a BIO-RAD iCycler thermocycler. Gene-specific signals were normalized relative to FKBP-66 expression. Oligonucleotides for qPCR and qRT-PCR are listed in Table 4.





TABLE 4



List of sense and antisense oligonucleotide sequences used for qRT-PCR.

Size of expected amplicon and target expression domain of the markers

are also indicated. Asterisks indicate the position of the intron.







Gene
size


Expression

Amplicon
(bp)
FW Primers (5′-3′)
RV Primers (5′-3′)
domain



MEG-1
105
TGCTCATGCGCATGG
CCCAATGCAACAGTAGC
Transfer cells



G*GCTG,
ACTTG,




SEQ ID No: 84
SEQ ID No: 85




INCW-2
114
GACCCTACCAA*GTC
CGACCGGTCGA*TCAGGC
Transfer cells



GTCCCTGA,
TTC,




SEQ ID No: 86
SEQ ID No: 87




MRP-1
120
GACTACAGATGAGC
GCATGGCTAGAGATCTGC
Transfer cells



ACAG*GAATTTC,
A,




SEQ ID No: 88
SEQ ID No: 89




BETL-1
93
CAGCACAATCGTCG
TTCTTGGGTTTCCCGATG
Transfer cells



CGCTT,
C*AGC,




SEQ ID No: 90
SEQ ID No: 91




BETL-2
115
TGCACGCACAACAA
AGCATGGCCCGTCGTCAT
Transfer cells



GTG*GGC,
T,




SEQ ID No: 92
SEQ ID No: 93




BETL-10
76
TCCTTGTGGCCTATC
GCTCATGCATGGGCCGTG
Transfer cells



GT*GCG,
AT,




SEQ ID No: 94
SEQ ID No: 95




TCRR-1
106
ATTGGAATTCTTAGA
CGATTC*CTTCACTTCCCT
Transfer cells



TGCG*AAC,
AA,




SEQ ID No: 96
SEQ ID No: 97




BETL-9
110
CAAGGAAGTAGAGA
TAAGAGGAACTGTATAA
Transfer cells



GAAGGTATG,
C*TCCCG,




SEQ ID No: 98
SEQ ID No: 99




AL-9
283
CTATGTTTGCCATAG
GCTGGAACCTTGTAGC*T
Aleurone



GCTCTCATGC,
TCCG,




SEQ ID No: 100
SEQ ID No: 101




ESR-6
90
GCCATAACCATGCC
TGCAGACGCATCCATTC*
Embryo



GTCCT,
CGA,
surrounding



SEQ ID No: 102
SEQ ID No: 103
region



FKBP-66
117
GGGTGCTGTTGTTGA
GCAATAA*CTTCCTCTTC
Ubiquitous



AG*TCA,
ATCG,




SEQ ID No: 104
SEQ ID No: 105


































































Phenotypic Analysis
Seed weight and dimension measurements were conducted on dry mature maize seeds. All other measurements were conducted on mature seeds imbibed overnight, which were bisected longitudinally with a razor blade along the central embryonic axis. ImageJ (NIH) was used to calculate embryo and endosperm areas, as well as transfer tissue interface lengths.
For histological analyses, 10 dap seeds were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.2, for 24 h at 4° C. Samples were dehydrated in an ethanol series and embedded in paraffin wax. For phenotypic analysis, 10-micrometer thick sections were treated with 0.5% periodic acid and stained with Schiff's reagent (Sigma, UK) and viewed using an Olympus BX50 compound microscope.
Immunolocalization
Polyclonal antiserum was raised in rabbit against a MEG1 synthetic peptide (H2N-APAEEGILREKRAQC-CONH2) (SEQ ID No: 106) or MRP1 synthetic peptides (H2N-CHMPNVVPNQRRHAVK-CONH2 (SEQ ID No: 107) and H2N-SSIAPPATEESQIYNR-CONH2 (SEQ ID No: 108)). Anti-MEG1 peptide antiserum was affinity purified with an immobilized peptide using a Sulpholink coupling gel system (Pierce, Rockford, Ill.).
For standard tissue immunolocalization, 10 micrometer sections of 10 dap wax embedded seeds were deparaffinized and blocked in 1% BSA in PBS (10 mM sodium phosphate and 150 mM NaCl, pH 7.4) for 30 min at room temperature and incubated overnight with purified anti-MEG1 antibody (diluted 1:100), anti-MRP1 (diluted 1:2000) or mouse anti-INCW1/2 (diluted 1:3000) (Carlson and Chourey, 1999). The immunoreactions were detected using goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Sigma, St. Louis, USA; diluted 1:50) with TBS-1% BSA, and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Sigma, Pooler, UK) as detection substrate.
For TEM immunodetection, 10 dap dissected endosperm samples were fixed for 12 h at room temperature in a solution of 2.5% (v/v) glutaraldehyde and 2% (w/v) paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2). Samples were dehydrated in an ethanol series, and embedded stepwise in medium grade LR White resin (Agar Scientific, Stansted, UK) over 1 week. Polymerization was under UV light for 24 h at 20° C. and for another 24 h at 0° C. For light microscopy, sections approximately 1 μm thick were cut and collected on Poly-lysine coated slides, and stained with 0.1% (w/v) toluidine blue O. For immunogold labeling, ultrathin sections (80 nm) were prepared using a diamond knife cutter and collected on formvar-coated 300-mesh nickel grids (Agar Scientific, UK). Sections were first blocked for 30 min in TRIS (20 mM TRIS, 15 mM NaN 3 , 225 mM NaCl), pH6.9, supplemented with 1% BSA (TRIS-BSA). They were then treated with 0.1% Tween20 in TRIS-BSA and 0.02 M glycine in TRIS-BSA, each for 15 min, washed three times in TRIS-BSA, and incubated in a 1:100 dilution of rabbit anti-MEG1 polyclonal antibody at 4° C. overnight. After washing in TRIS-BSA, sections were treated with a 1:20 dilution (in TRIS-BSA) of goat anti-rabbit secondary antibody conjugated to 10 nm gold (British BioCell International, Cardiff, UK) for 1.5 h at room temperature. Sections were washed thoroughly in TRIS-BSA, then in and ultrapure water and dried on filter paper. All sections were stained with 2% uranyl acetate in 70% ethanol followed by Reynold's lead citrate, and examined with a JEOL JEM-1010 (JEOL UK, Hertfordshire, UK) transmission electron microscope at 80 kV.
Enzymatic Assays
Invertase (EC 3.2.1.26) assays were performed using methodology previously described in Cheng et al., 1996, with minor modifications. Briefly, three whole seeds were homogenized in liquid nitrogen and resuspended in five volumes of ice-cold extraction buffer (50 mM Tris-Maleate pH 7.0, 1 mM dithiothreitol). The homogenate was centrifuged at 14,000 g for 10 min and the pellet was washed three times in extraction buffer followed by a 30 min incubation on ice with extraction buffer containing 1 M NaCl. The sample was subsequently centrifuged and the supernatant containing the cell wall-bound invertase fraction was dialyzed overnight against extraction buffer. Total protein concentration in enzyme extracts were calculated against a BSA standard curve using Bradford Reagent (Bio-rad Laboratories, Hercules, Calif.). Five to fifty microlitres of dialysed insoluble enzyme preparation was incubated with 2% (w/v) sucrose at 37° C. for 15 min before transfer to Somagyi's reagent and heated to 100° C. for 10 min. Nelson's reagent was added to the reaction mix and reducing sugars were measured at A 595 nm Samples were replicated in triplicate and quantified by comparison with a glucose standard curve.
Colorimetric assays to measure glucose, fructose and sucrose content of whole seeds or dissected seed bottoms were performed using established methods as described in LeClere et al. (2010) with minor modifications. Briefly, frozen samples were placed in a 1.5 ml screw cap centrifuge tube containing 2 mm glass beads and homogenized for 60 sec in a Silamat dental shaker (Ivoclar Vivadent). Soluble sugars were extracted with 80% ethanol (in a ratio of 10 mg ground tissue to 1.5 ml of 80% ethanol) at 85° C. over three wash and centrifugation cycles. Supernatants were stored at −20C. Ten to forty microliters of soluble sugar extract was analyzed in an 80 μl reaction. Assays were performed on 96-well flat-bottom clear polystyrene plates (Greiner Bio-One Ltd) and read at A 485 nm. Glucose assay reactions contained 20 mM HEPES, 47 μM MgSO4, 952 μM ATP, 0.125 mg ml −1 NAD, 1.25 units ml −1 hexokinase (Sigma, UK), 0.4 units ml −1 Glc-6-P dehydrogenase (Sigma, UK), 0.4 mg ml −1 p-iodonitrotetrazolium, 0.1 mg ml −1 phenazine methosulfate, and 0.6% BRIJ 35. Fructose and sucrose assays also included 4 units ml −1 phosphoglucose isomerase (Sigma, UK), and an additional 75 units ml −1 invertase (Sigma, UK) was used in sucrose assays only. All samples were replicated in triplicate and quantified by comparison with a standard curve.
Western Blot Analysis
Proteins were separated on a Novex 16% Tricine gel (Invitrogen) and electroblotted onto a polyvinylidine difluoride membrane. Proteins were immunodetected using anti-MEG1 antibody (1:2000 dilution) and an enhanced chemiluminescence method (Amersham, Buckinghamshire, UK).
Statistical Analyses
Unpaired student's t tests were performed using GraphPad InStat, version 3.0a (San Diego, USA). All tests were two-tailed with significance set at P≦0.05. One-way analysis of variance (ANOVA) followed by post hoc analysis using Tukey's test was used to determine significant differences in seed size and weights between wild-type and varying syn Meg1 dosage for pBet9> syn Meg1 and pMeg1> syn Meg1 lines. Correlation coefficient values generated by Excel were calculated from plotted means of embryo area, endosperm area, TTI lengths and dry seed weights for different doses of pBet9> syn Meg1.
MEG1 Localizes to the Plasma Cell Membrane and Extracellular Matrix of the ETT of Maize Cells
Similar to the chorionic villi of mammalian placentas, specialised transfer cells form in plant seeds to facilitate maternal nutrient uptake and partitioning (Pate and Gunning, 1972). In maize and other crop plants, these transfer cells are considered key determinants of sink strength and seed biomass yield (Cheng et al., 1996; Pugh et al.; and Thompson et al., 2001). However little is known of the molecular mechanisms regulating their development and function. Intriguingly, Meg1 is the only imprinted gene to show restricted expression in seed transfer cells. Meg1 encodes a small cysteine-rich peptide, which localises to the plasma cell membrane and extracellular matrix of the ETT of maize seeds ( FIG. 15 a ). Intriguingly, MEG1 belongs to a group of recently identified secreted cysteine-rich peptide molecules that regulate cell differentiation processes during plant development (Marshall et al.; Hara et al., 2007; and Sugano et al.).
Meg1 Influences Development of the Specialized ETT
We therefore postulated that Meg1 might influence development of the specialised ETT, and therefore sought to determine Meg1 function during seed development. In the absence of Meg1 knockout mutants, we generated transgenic maize lines expressing Meg1 hairpin RNA-mediated interference (RNAi) constructs under the control of a cauliflower mosaic virus (CamV) 35S promoter. Resulting plants produced female inflorescences with 48.4% (n=1699) seeds segregating for a small seed phenotype ( FIGS. 20 a, b ) and a 26.4% to 44.3% reduction in seed weight (unpaired Student's t test, P<0.0001)( FIG. 20 c ). Cross sections of mature seeds revealed Meg1RNAi seeds to contain significantly smaller embryos and endosperms, and a reduced maternal:transfer tissue interface (TTI) ( FIGS. 16 a, b ), thus suggesting a compromised ETT.
To determine whether the Meg1 knockdown seed phenotype was due to ETT defects, we first performed q-PCR analysis, which revealed Meg1 and other ETT-specific transcripts to be greatly reduced in these seeds ( FIG. 16 c ). Consistent with our findings, microscopic examination of Meg1 knockdown seeds revealed irregularities in the organisation and differentiation of the ETT, such that ETT length and depth was restricted; at most comprising one disrupted layer of cells containing fewer cell wall ingrowths compared to wild-type ( FIG. 20 d - g and FIGS. 15 b,c ). We next investigated whether ETT cell identity and function was perturbed in mutant kernels by determining localisation of a pivotal MYB-like transcriptional regulator of ETT differentiation, MRP1 (Gomez et al., 2009), and of cell wall invertases INCW1 and INCW2 which account for the majority of apoplastic cleavage of sucrose to hexose sugars within the maize seed (Chourey et al., 2006). Consistent with our histological observations, ETT-specific proteins MRP1 and INCW1/2 exhibited reduced and patchy localisation confined to the basal most cells of the endosperm in Meg1 RNA1 seeds ( FIGS. 20 h, i and FIG. 16 a ). Collectively, our data suggest that Meg1 is necessary for ETT development.
Meg1 Influences Sugar Partitioning within the Seed
Sugar assimilation and partitioning is a limiting factor of seed growth in most crops (Pugh et al.; LeClere et al., 2010; Maitz et al., 2000; Radchuk, 2006; and Wang et al., 2008). Therefore we determined how Meg1 downregulation might affect these metabolic processes by assaying the enzymatic activity of total soluble and cell wall bound invertases in seeds ( FIG. 21 a ; FIGS. 17 a,b ). Consistent with our immunolocalisation data, Meg1 knockdown seeds exhibited a dramatic (35.2% to 70.1%) reduction in cell wall invertase activity only (unpaired Student's t test, P<0.0001) ( FIG. 21 a ; FIG. 17 b ).
A recent study of the miniature1 (mn1) mutant, which is impaired in INCW2 activity (Gomez et al., 2009), revealed sugar partitioning to be altered locally within the basal endosperm (LeClere et al, 2010). We therefore measured sugar levels in meg1 RNAi seeds to determine whether Meg1 downregulation also altered localised sugar partitioning. In accordance with our invertase measurements, hexose sugars were 9.4% to 43.5% lower in Meg1 RNAi seeds from 8-20 days after pollination (dap), while sucrose content was considerably higher, exhibiting a 13.8% to 82.7% increase from 12 dap onwards ( FIG. 21 b - d ). The significant (18.4% to 52.8%) reduction in hexose/sucrose ratios observed in Meg1 knockdown seeds ( FIG. 17 c ) were also consistent with those reported for mn1 mutants (LeClere et al., 2010). Hence our data suggest that Meg1 is required for correct transfer cell function and sugar homeostasis during seed development.
Meg1 is a Positive Regulator of ETT Development
To gain further insight into the role of Meg1 ETT function and development, we generated transgenic maize lines expressing Meg1 under a CaMV 35S promoter, which produced a discernable seed phenotype only. Meg1 missexpression seeds were slightly smaller and lighter ( FIG. 22 a - c ), and possessed slight perturbations with ETT organisation ( FIGS. 22 c, f ). In addition, these seeds contained discrete clusters of ectopic transfer cells (ETCs), which were often situated in the abgerminal epidermis of the endosperm ( FIGS. 22 c,d ). Moreover, we were able to detect MRP1 and INCW1/2 localisation in these ETCs, which confirmed their transfer cell identity ( FIG. 22 ). Consistent with these findings, total seed hexose/sucrose ratios were 31.8% to 168.1% higher in these lines than in wild-type and contrasted with those in Meg1 knockdown seeds ( FIG. 22 and FIGS. 18 d, e ), suggesting that Meg1 expression is sufficient for ETT development and function.
To better define the impact that timing of Meg1 missexpression has on ETC formation, we generated dexamethasone (dex)-inducible Meg1 transgenic lines. Upon induction, we observed dramatic ectopic expression of an ETT-specific reporter (pBet1::uidA) around the endosperm epidermis only ( FIG. 22 g - i ). Notably, Meg1 induction during early stages of seed development (20 hr post-fertilisation) dramatically impacted on ETC formation, giving rise to large ETC sectors in the peripheral endosperm, thus reinforcing the model for the early and atypical lineage-dependent development of this tissue-type in plants (Costa et al., 2003). Collectively, our missexpression data identify Meg1 as a positive regulator of ETT development.
Dosage-related phenotypes have been observed for some mammalian imprints, which have provided deeper insights into their critical functions during placental and embryonic development (da Rocha et al, 2009; John, 2010). We therefore sought to investigate the phenotypic consequences of altering Meg1 dosage specifically in the ETT during seed development. To this aim, we generated transgenic maize expressing a synthetic Meg1 gene ( syn Meg1) (SEQ ID No: 66) under the control of its endogenous imprinted promoter (pMeg1> syn Meg1) or under a non-imprinted promoter (pBet9> syn Meg1) (SEQ ID No: 69), which drive expression in the ETT. Functional syn Meg1 lines were selected based on presence of syn MEG1 protein expression, and genetic complementation of Meg1 RNAi lines as indicated by the phenotypic reversion of seed progeny to wild-type. We used these functional syn Meg1 lines to generate an endosperm dosage series by self-pollinating or outcrossing with wild-type plants, and we independently verified endosperm genotype by q-PCR and by relative expression of a visible reporter.
In most cases, we found that increased dosage of syn Meg1 led to significant increases in seed size and dry seed weight compared with wild-type (Table 5 and Table 6). This was particularly evident when pBet9> syn Meg1 was transmitted biparentally in the endosperm (++/+, Table 5). Consistent with these findings, greater expansion of the ETT and increased TTI length (+15.8%, n=266) were also observed as a result of augmented MEG1 dosage, revealing a linear correlation (correlation coefficient of R 2 =0.9517 to 0.9870) between these seed traits and MEG1 activity. Notably, no significant phenotypic difference was observed in seeds when pMeg1> syn Meg1 was transmitted paternally or biparentally (see Table 5 and Table 6), reflecting the early paternal allelic silencing conferred by pMeg1. Collectively, our data indicate that MEG1 acts in a dosage-dependent manner during early endosperm development to positively regulate ETT formation and seed form.
TABLE 5 Phenotypic analysis of non-imprinted pBet9 > syn Meg1 dosage series in endosperm. Endosperm Dry seed weight Embryo area Endosperm area TTI length genotype (gm) *** (cm 2 ) *** (cm 2 ) *** (cm) *** Biparental 0.2547 ** ± 0.0159 0.2169 ** ± 0.0193 0.2520 ** ± 0.0203 0.2764 ** ± 0.0195 transmission (++/+) Maternal 0.2401 ** ± 0.0148 0.2112 ** ± 0.0194 0.2385 ** ± 0.0191 0.2582 ** ± 0.0180 transmission (++/−) Paternal 0.2309 * ± 0.0153 0.2065 ± 0.0172 0.2320 ** ± 0.0193 0.2542 ** ± 0.0170 transmission (−−/+) WT (−−/−) 0.2239 ± 0.0167 0.1985 ± 0.0172 0.2201 ± 0.0178 0.2386 ± 0.0181
qOne-way ANOVA tests revealed significant differences in dry seed weight (gm), embryo area, endosperm area, and transfer tissue interface (TTI) for pBet9> syn Meg1 dosage series in the endosperm, where ***P<0.0001. Where *P<0.05 and **P<0.01, post hoc Tukey HSD tests revealed a significant difference between sample means when comparing parental transmission of pBet9> syn Meg1 with wild-type (no transmission). A minimum of 534 seeds were analysed per measurement parameter.

TABLE 6 Phenotypic analysis of imprinted pMeg1 > syn Meg1 dosage series in endosperm. Endosperm Dry seed weight Embryo area Endosperm area TTI length genotype (gm) *** (cm 2 ) *** (cm 2 ) *** (cm) *** Biparental 0.2588 ** ± 0.0172 0.2091 ** ± 0.0198 0.2295 ** ± 0.0231 0.2411 ** ± 0.0189 transmission (++/+) Maternal 0.2502 ** ± 0.0191 0.2049 ** ± 0.211 0.2285 ** ± 0.0215 0.2398 ** ± 0.0168 transmission (++/−) Paternal 0.2342 ** ± 0.0134 0.1918 ± 0.0212 0.2158 ± 0.0234 0.2211 ± 0.0178 transmission (−−/+) WT (−−/−) 0.2312 ± 0.0199 0.1881 ± 0.0201 0.2128 ± 0.0230 0.2234 ± 0.0180
One-way ANOVA tests revealed differences in dry seed weight (gm), embryo area, endosperm area and transfer tissue interface (TTI) for pMeg1> syn Meg1 dosage series in the endosperm, where ***P<0.0001. Where post hoc Tukey HSD tests revealed a significant difference in sample means when comparing parental transmission of pMeg1> syn Meg1 with wild-type (no transmission), **P<0.01. A minimum of 510 seeds were analysed per measurement parameter.

Our study has identified a paternally imprinted gene that is necessary and sufficient for ETT development in maize. By manipulating Meg1 expression, we demonstrated that MEG1 acts as a dosage-dependent positive regulator of ETT development when expressed in the correct temporospatial manner. These findings are consistent with several experimental findings and hypotheses put forward indicative of the dosage sensitivity of imprinted genes (John, 2010; Dilkes and Comai, 2004; Birchler, 1993). Significantly, our data provide the first compelling evidence in plants for the involvement of an imprinted gene to modulate maternal resource allocation to the offspring. In principle, our findings can be considered consistent with the parental conflict model (Moore and Haig, 1991; Wilkins and Haig, 2003; and Haig and Westoby, 1989), as paternal imprinting of Meg1 imposes a restriction on the formation of ETT, thus limiting nutrient distribution to the developing embryo. However, Meg1 more greatly conforms to the coadaptation theory (Wolf and Hager, 2006), suggesting that natural selection strongly favours paternal imprinting at loci important for placenta function in order to achieve an optimal balance of maternal nutrient supply and filial demand, thus leading to greater offspring fitness. Under such circumstances, genomic imprinting in crops would serve the purpose of retaining maternal control over seed biomass, while acting as a potential barrier to genetic improvement.
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