# Patent

## Title
NAC from wheat for increasing grain protein content

## Abstract

The present invention provides wheat NAC nucleic acids and proteins that modulate grain protein content and senescence in plants. Overexpression of a NAC coding sequence can accelerate senescence and increase grain protein content and inhibition of expression can delay senescence. The invention also provides methods of using the nucleic acids to produce transgenic plants with altered grain protein content or senescence.

## Background
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support under Grant (or Contract) No. 2001-52100-11293 awarded by the USDA-IFAFS, No. 97-36200-5272 awarded by the USDA-Fund for Rural America, and No. US-3224-01R and No. US-3573-04C awarded by BARD, The United States-Israel Binational Agricultural Research and Development Fund. The US and Israel Governments have certain rights in the invention.


FIELD OF THE INVENTION
This invention is in the field of plant breeding and plant molecular biology. In particular, this invention relates to non-naturally occurring plants with altered grain protein content and other improved characteristics.
BACKGROUND OF THE INVENTION
High grain protein content (GPC) is one of the most important factors determining pasta and breadmaking quality, and is also important to human nutrition. In addition, high protein determines premium prices for wheat in many regions of the world, making high grain protein content a primary target for hard common wheats and durum wheat breeding-programs. The emphasis on end-use quality in the current export markets has increased the value of this trait.
In spite of the economic importance of high grain protein content, genetic improvement of this trait by conventional breeding has been slow because of the complex genetic system governing this trait, the high influence of the environment and the existence of a negative correlation between GPC and yield. There is thus a need for the identification of genetic determinants of high grain protein content.
BRIEF SUMMARY OF THE INVENTION
In order to meet these needs, the present invention is directed to the sequence of the NAC genes and their use to modulate grain protein in small grain cereals.
Transgenic modifications of this gene can be used to modify the amount of protein in the grain, to alter the senescence process, and to modify the length of grain maturation and grain filling periods in wheat and barley. The promoter of the WNAC-1 gene can be used to express any type of introduced genes in leaves after anthesis.
The current invention describes a non-naturally occurring plant comprising a recombinant nucleic acid encoding a plant WNAC-1 protein, where said nucleic acid hybridizes to the nucleic acid sequence depicted in SEQ ID NO:1 or its complement under hybridization conditions that include at least one wash in 0.1×SSC and 0.1% SDS at 60-65° C. for thirty minutes, or where said nucleic acid is more than 70%, more than 80%, more than 90%, more than 95% identical or substantially identical to the nucleic acid sequence depicted in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15 or 17, or when the nucleic acid encodes a polypeptide which is substantially identical to the amino acid sequence depicted in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16 or 18, or where the nucleic acid encodes a polypeptide which is at least 70%, at least 80%, at least 90%, at least 95%, or greater than 95% identical to the amino acid sequence depicted in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, or 18, or where the nucleic acid is operably linked to a promoter.
Another aspect of the invention is a non-naturally occurring plant comprising a recombinant nucleic acid encoding a plant WNAC-1 protein, where the plant is selected from the group consisting of wheat, rice, barley, rye, oats, rice maize, sorghum and forage grasses, and seeds from the transgenic plants described.
A further aspect of the invention is methods of detecting the presence or absence of a WNAC-1 allele in a plant or plant part comprising detecting the WNAC-1 gene or gene product by PCR and/or the use of a microarray, or hybridization or sequencing, or detecting WNAC-1 protein levels, structure or activity.
Another aspect of the present invention is an antibody which recognizes a WNAC-1 protein.
Another aspect of the current invention is a non-naturally occurring plant with an increased grain protein content compared to its level in a control, naturally occurring plant. The grain protein content can be increased by increasing the mRNA or protein levels encoded by a functional WNAC-1 gene (for example, SEQ ID NOS:1 and 2) in the non-naturally occurring plant compared to the mRNA or protein levels encoded by the functional WNAC-1 gene in a control, naturally occurring plant. The mRNA or protein levels can be increased by introduction of a recombinant WNAC-1 gene or a portion thereof, by mutagenesis, or by introduction of a WNAC-1 allele from a wild relative which is not present in the cultivated crop.
Another aspect of the current invention is a non-naturally occurring plant with a decreased grain protein content compared to its level in a control, naturally occurring plant. The grain protein content can be decreased by decreasing the mRNA or protein levels encoded by a functional WNAC-1 gene (for example, SEQ ID NOS:1 and 2) in the non-naturally occurring plant compared to the mRNA or protein levels encoded by the functional WNAC-1 gene in a control, naturally occurring plant. The mRNA or protein levels can be decreased by introduction of an antisense recombinant WNAC-1 gene, by RNA interference, or by mutagenesis.
Another aspect of the invention is a non-naturally occurring plant exhibiting delayed timing of senescence onset compared to the timing of senescence onset in a control, naturally occurring plant where the timing of senescence onset is delayed in said non-naturally occurring plant by reducing the in RNA or protein levels encoded by a functional WNAC-1 gene in said non-naturally occurring plant compared to the mRNA or protein levels encoded by the functional WNAC-1 gene in the control, naturally occurring plant. The mRNA or protein levels can be reduced by expression of the WNAC-1 gene or a portion thereof in the antisense orientation, or by RNA interference, or by mutagenesis.
A further aspect of the present invention is a method of increasing the rate of senescence in a plant by increasing the mRNA or protein levels encoded by a functional WNAC-1 allele, or increasing the activity of a WNAC-1 protein.
Another aspect of the invention is a method of decreasing the rate of senescence in a plant by reducing the mRNA or protein levels encoded by a functional WNAC-1 allele, or decreasing the activity of a WNAC-1 protein.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts genetic and physical maps of the Gpc-B1 region in wheat. Top: Rice BAC AP004061. Middle: High resolution genetic map. The gray area represents the 0.2 cM region of the genetic map including the Gpc-B1 locus. Bottom: Physical map. Xuhw markers were developed at the University of Haifa and Xucw markers at the University of California at Davis.
FIG. 2 depicts a comparison of the UCW87 region (SEQ ID NOS:19-27) including the polymorphic amino acid with the most similar rice and Arabidopsis proteins.
FIG. 3 depicts RT-PCR results. Top: amplification using ACTIN primers (control). Below: amplification using genome specific primers for WNAC-A1, WNAC-B1, WNAC-B2.1 & 3) RSL152 with DIC allele, rest LDN allele. 1-2) Flag leaves at beginning of spike emergence (Feekes scale 10). 3-4) Flag leaves 10 days after anthesis at medium milk stage (Feekes scale 11.1). 5) Flag leaf at ripening (Feekes scale 11.4). 6-7) Beginning of emergence of the flag leaf (Feekes scale 8), 6) yellowing leaf, 7) young green leaf 8) Water control.
FIG. 4 depicts a phylogenetic analysis of rice NAC proteins and wheat WNAC-B1. Following the red clade, WNAC-B1 clusters alongside of OsNAC. The closest homologue of OsNAC in rice, OsNAC-A, clusters on a separate branch. Analysis done with neighbor-joining method in MEGA3. The numbers beside the branches represent bootstrap values based on 250 replications.
FIG. 5 depicts the expression profile of OsNAC gene in Rice: Top: RT-PCR amplification using Rice-Actin primers (control). Below: amplification using genome specific primers for OsNAC. 1) leaves from seedling stage (Feekes scale 1.0). 2) leaves from tillering stage (Feekes scale 3.0). 3) leaves from stem elongation stage (Feekes scale 7.0). 4) Flag leaves at beginning of spike emergence (Feekes scale 10). 5) Flag leaf at ripening (Feekes scale 11.4). 6) rice genomic DNA control. 7) Water control.
FIG. 6 depicts: a) Grain protein content of RSLs (209, 129, 300 and 121) and control lines (LDN and 65). RSLs 209 and 129 were similar to the low protein control LDN and were assigned the WNAC-B1b low protein allele (black bars), whereas RSLs 300 and 121 were similar to the high protein control RSL 65 and were assigned the WNAC-B1a high protein allele (grey bars). b) Chlorophyll content profile (SPAD units) from 13 to 35 DAA. Grey line with open diamonds represents lines with the WNAC-B1a allele and black line with solid squares represents lines with the WNAC-B1b allele. Error bars are standard errors of the means.
FIG. 7 depicts the effect of the WNAC-B1a allele on the chlorophyll content profile of isogenic lines of hexaploid wheat Anza (a), RSI5 (b), UC1041 (c) and tetraploid wheat Kofa (d). Chlorophyll content is measured in relative SPAD units from 20-25 DA until complete yellowing (50-60 DAA). Black lines with solid squares represent the original recurrent parent and grey lines with open diamonds represent the corresponding isogenic line with the WNAC-B1a allele. Significant differences (P<0.05) between isogenic pairs are represented by arrows on the corresponding date. Error bars are standard errors of the means.
FIG. 8 depicts the effect of WNAC-B1 on the moisture content of grains from isogenic lines of hexaploid wheat Anza (a), RSI5 (b) and tetraploid wheat Kofa (c). Black lines with solid squares represent the original recurrent parent and grey lines with open diamonds represent the corresponding isogenic line with the WNAC-B1a allele. Significant differences (P<0.05) between isogenic pairs are represented by arrows on the corresponding date. Error bars are standard errors of the means.
FIG. 9 depicts the effects of altering the expression levels of WNAC on different senescence phenotypes. a) Chlorophyll content profile (SPAD units). Black line with solid squares represents positive transgenic lines with reduced WNAC expression (n=25) and the gray line with open diamonds represents control lines (n=10). b) DAA for complete yellowing of peduncle. c) DAA for the main spike to be completely dry. In b) and c) black bars represent positive transgenic lines with reduced WNAC expression and the striped patterned bars represent control lines. Error bars are standard errors of the means.
FIG. 10 depicts spike, flag leaf and peduncle color comparisons between positive and negative (control) plants at 34 and 70 DAA for event L19-54. Left panel, positive plant has green flag leaf, peduncle and immature grains compared to a completely senescence control plant. Right panel, at 70 DAA, the positive plant still has a green peduncle and flag leaf is dry but green. Spike (not shown) is completely dry.
FIG. 11 depicts the effect of WNAC-B1 on protein, Zn and Fe content in the grain of RSLs (28, 121, 14 and 77) and control lines (LDN and DIC). RSLs 14 and 77 were similar to the low protein control LDN and were assigned the WNAC-B1 b low protein allele (black bars), whereas RSLs 28 and 121 were similar to the high protein control DIC and were assigned the WNAC-B1a high protein allele (striped pattern bars).
FIG. 12 depicts transcription levels of WNAC-A1, WNAC.-B1, and WNAC-B2 in wheat leaves at different stages of development using real-time reverse transcription PCR (quantitative).
FIG. 13 depicts the vector map of pMCG161



SEQUENCE LISTING
SEQ ID NO:1. WNAC-B1a allele BAC 916017, chromosome 6BS. This gene corresponds to the Gpc-B1 locus. Genomic DNA sequence from the functional Triticum turgidum var. dicoccoides allele. The 5′UTR was determined by 5′ race and the 3′ UTR predicted by presence of a putative TAATAAATA polyadenilation signal. The sequence includes 1000-bp upstream from the start codon and 1000-bp downstream from the stop codon.
SEQ ID NO:2. Predicted protein for the WNAC-B1 a allele from the functional Triticum turgidum var. dicoccoides allele (SEQ ID NO:1). Conserved NAC domains are identified.
SEQ ID NO:3. WNAC-B1b allele, chromosome arm 6BS. Genomic DNA sequence from the Triticum turgidum var. durum non functional allele with a 1-bp frame-shift mutation.
SEQ ID NO:4. Predicted protein for the WNAC-B1b non-functional allele (SEQ ID NO:3, assuming conserved exon structure). No conserved NAC domains nor significant BLASTP results.
SEQ ID NO:5. WNAC-A1. Genomic DNA sequence from the Triticum turgidum var. durum variety Langdon from the A genome (chromosome arm 6AS).
SEQ ID NO:6. Predicted protein for the WNAC-A1 gene from the Triticum turgidum var. durum from the A genome (SEQ ID NO:5). Conserved NAC domains are indicated.
SEQ ID NO: 7. WNAC-D1. Genomic DNA sequence from the D genome (Triticum tauschii).
SEQ ID NO:8. Predicted protein for the WNAC-D1 gene from the D genome, Triticum tauschii (SEQ ID NO:7). Conserved NAC domains are indicated.
SEQ ID NO:9. WNAC-B2 allele. Genomic DNA sequence from the Triticum turgidum var. durum allele from chromosome arm 2BS.
SEQ ID NO:10. Predicted protein for the WNAC-B2 allele (SEQ ID NO:9) from Triticum turgidum var. durum chromosome arm 2BS. Conserved NAC domains are indicated.
SEQ ID NO:11. HvNAC-H1 allele. Genomic DNA sequence from the Hordeum vulgare cv. Optic allele from chromosome 6.
SEQ ID NO:12. Predicted protein for the HvNAC-H1 allele (SEQ ID NO:11) from Hordeum vulgare cv. Optic allele from chromosome 6. Conserved NAC domains are indicated.
SEQ ID NO:13. HvNAC-H2 allele. Genomic DNA sequence from the Hordeum vulgare cv. Morex allele.
SEQ ID NO:14. Predicted protein for the HvNAC-H2 allele (SEQ ID NO:13) from Hordeum vulgare cv. Morex allele. Conserved NAC domains are indicated.
SEQ ID NO:15. UCW87 BAC 409D13, chromosome arm 6BS. Genomic DNA sequence from Triticum turgidum var. dicoccoides. 
SEQ ID NO:16. Predicted protein for the UCW87 gene from chromosome arm 6BS from the Triticum turgidum var. dicoccoides allele (SEQ ID NO: 15). The conserved Rhamnogalacturonate lyase domain is indicated.
SEQ ID NO:17. UCW87, chromosome arm 6BS. Genomic DNA sequence from the Triticum turgidum var. durum allele.
SEQ ID NO:18. Predicted protein for the UCW87 gene from chromosome arm 6BS from the Triticum turgidum var. durum allele (SEQ ID NO: 17). The conserved Rhamnogalacturonate lyase domain is indicated. Only one amino acid substitution (N by K) was observed between the DIC and LDN alleles.
SEQ ID NO:28. Oryza sativa OsNAC rice homologue of WNAC-B1.
SEQ ID NO:29. Predicted protein of Oryza sativa OsNAC gene (SEQ ID NO:29). Conserved NAC domains are indicated.
SEQ ID NO:30. Zea mays ZmNAC maize homologue of WNAC-B1.
SEQ ID NO:31. Predicted protein of Zea mays ZmNAC gene (SEQ ID NO:30). Conserved NAC domains are indicated.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is based, at least in part, on the characterization of the high grain protein content locus Gpc-B1 in wheat. A frame shift mutation in the B genome copy of this gene located on chromosome arm 6BS is associated with the recessive gpc-B1 allele for low grain protein content. This frame shift mutation is linked to the low grain protein content in a segregating population of 3000 gametes and this non-functional mutation is the prevalent allele in the cultivated tetraploid wheats.
The WNAC-1 gene is a transcription factor with a NAC (NAM/ATAF1/CUC2) domain and is associated with leaf senescence. Transcription of this gene starts in the flag leaves at the time of anthesis and increases dramatically during senescence. The presence of the functional Gpc-B1 allele is associated with an acceleration of the senescence process, with an increase in the concentration of free amino acids and soluble protein in the flag leaves, a more efficient remobilization of nitrogen from the vegetative tissues to the grain, and significantly higher grain protein content. The cloning and characterization of this gene has provided means to clone homologues in barley (HvNAC), rice (OsNAC) and maize (ZmNAC). Natural allelic variants from these genes can be used to increase or decrease protein content in the grain of small grain cereals such as wheat, barley, oats, triticale, and sorghum, as well as rice, maize and forage grasses.
This invention includes also the natural allelic variation of NAC genes and their use to develop molecular markers for the different Gpc-B1 alleles. These markers can be used in breeding programs aimed to increase or decrease grain protein content in cereals. This invention also covers the use of antibodies against the different protein variants of the NAC genes as a diagnostic tool for cereal breeding programs. Antibody recognition can be implemented in commercial kits for easy detection of alternative Gpc-B1 alleles.
Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.
Throughout this disclosure, various publications, patents and published patent specifications are referenced. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of plant breeding, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY [(F. M. Ausubel, et al., eds., (1987)]; PLANT BREEDING: PRINCIPLES AND PROSPECTS (Plant Breeding, Vol 1) M. D. Hayward, N. O. Bosemark, I. Romagosa; Chapman & Hall, (1993.); Coligan, Dunn, Ploegh, Speicher and Wingfeld, eds. (1995) CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc.); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)], Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE [R. I. Freshney, ed. (1987)].
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Lewin, Genes V, published by Oxford University Press, 1994 (SBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (SBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology, a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Ausubel et al. (1987) Current Protocols in Molecular Biology, Green Publishing; Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.
In order to facilitate review of the various embodiments of the invention, the following definitions are provided:
NAC proteins and genes: An NAC protein or polypeptide is a protein encoded by the NAC gene, as described herein. The NAC genes of the invention encode transcription factors which comprise an NAC domain. Exemplary conserved NAC domains of NAC proteins of the invention are identified in SEQ ID NOS:2, 6, 8, 10, 12, 29, 31. Plants with a functional NAC gene (as a non-limiting example, the WNAC-B1a allele in wheat, SEQ ID NO:1) exhibit an acceleration in the senescence process, exhibited by higher levels of soluble proteins and amino acids in the flag leaves relative to plants with a non-functional WNAC-1 gene (in wheat, the WNAC-B1b gene, SEQ ID NO:3, is an example). Soluble proteins are the results of degradation of structural or storage proteins into a form which can be transported to the grain. In particular, transgenic plants with a with increased or enhanced NAC gene expression show increased grain protein content relative to identical plants lacking the transgene.
The present invention may be practiced using nucleic acid sequences that encode full length NAC, proteins as well as NAC derived proteins that retain NAC activity. NAC derived proteins which retain NAC biological activity include fragments of NAC proteins disclosed here, generated either by chemical (e.g. enzymatic) digestion or genetic engineering means; chemically functionalized protein molecules obtained starting with the exemplified protein or nucleic acid sequences, and protein sequence variants, for example allelic variants and mutational variants, such as those produced by in vitro mutagenesis techniques, such as gene shuffling (Stemmer W P. Nature. 1994 Aug. 4; 370(6488):389-91 (1994a); Stemmer W P. Proc Natl Acad Sci USA. 1994a October 25; 91(22):10747-51 (1994b)). Thus, the term “NAC protein” encompasses full-length NAC proteins, as well as such NAC derived proteins that retain NAC activity.
Representative but non-limiting NAC sequences useful in the invention include the Triticum turgidum var. dicoccoides WNAC-B1 a DNA and protein sequences (SEQ ID NOS:1 and 2).
Also encompassed within the definition of NAC sequences include the Triticum turgidum var. durum WNAC-1B1b non-functional allele DNA and protein sequences (SEQ ID NOS:3 and 4).
Also encompassed within the definition of NAC sequences include the Triticum turgidum var. durum variety Langdon WNAC-A1 DNA and protein sequences (SEQ ID NOS:5 and 6).
Also encompassed within the definition of NAC sequences include the Triticum tauschii WNAC-D1 DNA and protein sequences (SEQ ID NOS:7 and 8).
Also encompassed within the definition of NAC sequences include the Triticum turgidum var. durum WNAC-B2 DNA and protein sequences (SEQ ID NOS:9 and 10).
Also encompassed within the definition of NAC sequences include the Hordeum vulgare cv. Optic HvNAC-H1 DNA and protein sequences (SEQ ID NOS:11 and 12).
Also encompassed within the definition of NAC sequences include the Hordeum vulgare cv. Morex HvNAC-H2 DNA and protein sequences (SEQ ID NOS:13 and 14).
Also encompassed within the definition of NAC sequences include the Oryza sativa OsNAC DNA and protein sequences (SEQ ID NOS:28 and 29).
Also encompassed within the definition of NAC sequences include the Zea mays ZmNAC DNA and protein sequences (SEQ ID NOS:30 and 31).
NAC Promoter: An NAC promoter is a promoter for an NAC gene of the invention. NAC promoters are generally found 5′ to the NAC protein coding sequence and regulate expression of the NAC gene. Such sequences can be synthesized chemically or they can be isolated from plants. Representative plants from which NAC promoters can be isolated include wheat (spring and winter), barley, rice, and maize.
Expression cassette: A nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. Antisense or sense constructs that are not or cannot be translated are expressly included by this definition. In the case of both expression of transgenes and inhibition of endogenous genes (e.g., by antisense, or sense suppression) one of skill will recognize that the inserted polynucleotide sequence need not be identical and may be “substantially identical” to a sequence of the gene from which it was derived.
Sequence Identity: As noted above, sequence variants of the sequences disclosed here are encompassed by the present invention. Either all or portions of the NAC nucleic acids or proteins or of the invention may vary from the disclosed sequences and still retain the functions disclosed here. Thus, nucleic acids or proteins of the invention may have substantial identity (as defined below) to particular domains (e.g., NAC domains) and retain the functions described here.
The similarity between two nucleic acid sequences or two amino acid sequences is expressed in terms of sequence identity (or, for proteins, also in terms of sequence similarity). Sequence identity is frequently measured in terms of percentage identity; the higher the percentage, the more similar the two sequences are. As described herein, homologs and variants of the NAC nucleic acid molecules may be used in the present invention. Homologs and variants of these nucleic acid molecules will possess a relatively high degree of sequence identity when aligned using standard methods. Such homologs and variants will hybridize under high stringency conditions to one another.
Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in: Smith, T. F. and Waterman, M. S., J. Mol. Biol., 147:195-197 (1981); Needleman, S. B. and Wunsch, C. D., J. Mol. Biol., 48:443-453 (1970); Pearson, W. R., Lipman, D. J., Proc Natl Acad Sci U.S.A., 85:2444-8 (1988); Higgins, D. G. and Sharp, P. M., Gene 73(1):237-44 (1988); Higgins, D. G. and Sharp, P. M., Comput Appl Biosci. 5(2):151-3 (1989); Corpet, F., Nucleic Acids Res., 16:10881-10890 (1988); Huang X. et al., Comput. Appl. Biosci., 8(2):155-65 (1992); and Pearson W R., Methods Mol. Biol., 24:307-31 (1994). Altschul, S. F. et al. (1994) Nat. Genet. 6(2):119-29 presents a detailed consideration of sequence alignment methods and homology calculations.
The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Bio. 215:403-410) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. It can be accessed at the NCBI Website. A description of how to determine sequence identity using this program is available at the NCBI website.
Homologs of the disclosed protein and nucleic acid sequences are typically characterized by possession of at least 40% sequence identity counted over the full length alignment with the nucleic acid or amino acid sequence of the disclosed sequence using the NCBI Blast 2.0, gapped blastp or blastn set to default parameters. For example, using blastp, the adjustable parameters are preferably set with the following values: overlap span 1, overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
Nucleic acids or proteins which are “substantially identical” to a reference sequence will show increasing percentage identities when assessed by these methods. Such nucleic acids and proteins will typically show at least about 70%, 75%, 80%, 90% or 95% sequence identity to a reference sequence.
Polypeptides that are “substantially similar” share sequences as noted above except that residue positions which are not identical may differ by conservative amino acid changes. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the figures, it is understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in the figures as discussed below, will be determined using the number of amino acids in the longer sequence, in one embodiment. In percent identity calculations relative weight is not assigned to various manifestations of sequence variation, such as, insertions, deletions, substitutions, etc.
In one embodiment, only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of “0”, which obviates the need for a weighted scale or parameters as described herein for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the “shorter” sequence in the aligned region and multiplying by 100. The “longer” sequence is the one having the most actual residues in the aligned region.
As will be appreciated by those skilled in the art, the sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleotides, frameshifts, unknown nucleotides, or other types of sequencing errors in any of the sequences; however, the correct sequences will fall within the homology and stringency definitions herein.
High Stringency: Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other, or a third nucleic acid, under stringent conditions. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, stringent conditions will be those in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least about 60° C. or 65° C. For the purposes of this disclosure, stringent conditions for hybridizations are those which include at least one wash in 0.2×SSC at 63° C. for 20 minutes, or equivalent conditions. Moderately stringent conditions include at least one wash (usually 2) in 0.2×SSC at a temperature of at least about 50° C., usually about 55° C., for 20 minutes, or equivalent conditions. High stringency conditions also refer to hybridization to filter-bound DNA in 5×SSC, 2% sodium dodecyl sulfate (SDS), 100 ug/ml single stranded DNA at 55-65° C., and washing in 0.1×SSC and 0.1% SDS at 60-65° C.
Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transformed host cell. A vector may include one or more nucleic acid sequences that permit it to replicate in one or more host cells, such as origin(s) of replication. A vector may also include one or more selectable marker genes and other genetic elements known in the art.
Transformed: A transformed cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transformation encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, plant or animal cell, including transfection with viral vectors, transformation by Agrobacterium, with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration and includes transient as well as stable transformants.
Isolated: An “isolated” biological component (such as a nucleic acid or protein or organelle) has been substantially separated or purified away from other biological components in the cell or the organism in which the component naturally occurs, i.e., other chromosomal and extra chromosomal DNA and RNA, proteins and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term embraces nucleic acids including chemically synthesized nucleic acids and also embraces proteins prepared by recombinant expression in vitro or in a host cell and recombinant nucleic acids as defined below.
Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a protein coding sequence if the promoter affects the transcription or expression of the protein coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary, join two protein coding regions in the same reading frame. With respect to polypeptides, two polypeptide sequences may be operably linked by covalent linkage, such as through peptide bonds or disulfide bonds.
Recombinant: By “recombinant nucleic acid” herein is meant a nucleic acid that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of nucleic acids, e.g., by genetic engineering techniques, such as by the manipulation of at least one nucleic acid by a restriction enzyme, ligase, recombinase, and/or a polymerase. Once introduced into a host cell, a recombinant nucleic acid is replicated by the host cell, however, the recombinant nucleic acid once replicated in the cell remains a recombinant nucleic acid for purposes of this invention. By “recombinant protein” herein is meant a protein produced by a method employing a recombinant nucleic acid. As outlined above “recombinant nucleic acids” and “recombinant proteins” also are “isolated” as described above.
Antibody production: Methods of producing antibodies which specifically recognize a protein of interest are well known in the art. Protocols for preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp. 55-120.
Non-naturally Occurring Plant: A non-naturally occurring plant is a plant that does not occur in nature without human intervention. Non-naturally occurring plants include transgenic plants and plants produced by non transgenic means such as plant breeding.
Transgenic plant: As used herein, this term refers to a plant or tree that contains recombinant genetic material not normally found in plants or trees of this type and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation. Thus, a plant that is grown from a plant cell into which recombinant DNA is introduced by transformation is a transgenic plant, as are all offspring of that plant that contain the introduced transgene (whether produced sexually or asexually). It is understood that the term transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, flowers, leaves, roots, fruit, pollen, stems etc.
Orthologue: Two nucleotide or amino acid sequences are orthologues of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species, sub species, or cultivars. Orthologous sequences are also homologous sequences. Orthologous sequences hybridize to one another under high-stringency conditions. The term “polynucleotide”, “oligonucleotide”, or “nucleic acid” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. The terms “polynucleotide” and “nucleotide” as used herein are used interchangeably. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. A “fragment” or “segment” of a nucleic acid is a small piece of that nucleic acid.
Vectors
The NAC sequences of the invention may be cloned into a suitable vector. Expression vectors are well known in the art and provide a means to transfer and express an exogenous nucleic acid molecule into a host cell. Thus, an expression vector contains, for example, transcription start and stop sites such as a TATA sequence and a poly-A signal sequence, as well as a translation start site such as a ribosome binding site and a stop codon, if not present in the coding sequence. A vector can be a cloning vector or an expression vector and provides a means to transfer an exogenous nucleic acid molecule into a host cell, which can be a prokaryotic or eukaryotic cell. Such vectors include plasmids, cosmids, phage vectors and viral vectors. Various vectors and methods for introducing such vectors into a cell are described, for example, by Sambrook et al. 1989.
The invention also provides an expression cassette containing a promoter operably linked to a protein coding sequence or to a sequence that inhibits expression of a target endogenous gene. For instance, antisense or sense suppression technology can be conveniently used. To accomplish antisense suppression, a nucleic acid segment from the desired gene is cloned and operably linked to a promoter such that the antisense strand of RNA will be transcribed. The expression cassette is then transformed into plants and the antisense strand of RNA is produced. In plant cells, it has been suggested that antisense RNA inhibits gene expression by preventing the accumulation of mRNA which encodes the protein of interest, see, e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA, 85:8805 8809 (1988), and Hiatt et al., U.S. Pat. No. 4,801,340. In some embodiments, small interfering RNAs are used. The phenomenon of RNA interference is described and discussed, e.g., in Bass, Nature 411:428-29 (2001); Elbahir et al., Nature 411:494-98 (2001); and Fire et al., Nature 391:806-11 (1998), where methods of making interfering RNA also are discussed. The siRNA inhibitors are less than 100 base pairs, typically 30 bps or shorter, and are made by approaches known in the art. Introduction of expression cassettes in which a nucleic acid is configured in the sense orientation with respect to the promoter has also been shown to be an effective means by which to block the transcription of target genes. For an example of the use of this method to modulate expression of endogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990), and U.S. Pat. Nos. 5,034,323, 5,231,020, and 5,283,184.
In the constructs of the invention, each component is operably linked to the next. For example, where the construct comprises the NAC promoter, and protein encoding sequence, preferably, the NAC protein, the NAC promoter is operably linked to the 5′ end of the NAC protein encoding sequence or open reading frame.
The NAC coding sequence may be from wheat or other NAC protein coding sequences as defined herein. The protein coding sequence linked to the NAC promoter may be an NAC protein coding sequence or another heterologous protein. The heterologous proteins which find use in the invention include those that provide resistance to plant pests, facilitate translocation of nutrients, provide resistance to stresses typical of the summer: heat and dehydration, etc.
The constructs of the invention may be introduced into transgenic plants. A number of recombinant vectors suitable for stable transformation of plant cells or for the establishment of transgenic plants have been described including those described in Weissbach and Weissbach (1989), and Gelvin et al. (1990). Typically, plant transformation vectors include one or more open reading frames (ORFs) under the transcriptional control of 5′ and 3′ regulatory sequences and a dominant selectable marker with 5′ and 3′ regulatory sequences. Dominant selectable marker genes that allow for the ready selection of transformants include those encoding antibiotic resistance genes (e.g., resistance to hygromycin, kanamycin, bleomycin, G418, streptomycin or spectinomycin) and herbicide resistance genes (e.g, phosphinothricin acetyltransferase).
Standard molecular biology methods, such as the polymerase chain reaction, restriction enzyme digestion, and/or ligation may be employed to produce these constructs.
Transgenic Plants
Standard molecular biology methods and plant transformation techniques can be used to produce transgenic plants that produce plants having a recombinant NAC promoter or gene.
Introduction of the selected construct into plants is typically achieved using standard transformation techniques. The basic approach is to: (a) clone the construct into a transformation vector, which (b) is then introduced into plant cells by one of a number of techniques (e.g., electroporation, microparticle bombardment, Agrobacterium infection); (c) identify the transformed plant cells; (d) regenerate whole plants from the identified plant cells, and (e) select progeny plants containing the introduced construct.
Preferably all or part of the transformation vector will stably integrate into the genome of the plant cell. That part of the transformation vector which integrates into the plant cell and which contains the introduced recombinant sequence may be referred to as the recombinant expression cassette.
Selection of progeny plants containing the introduced transgene may be made based upon the detection of the recombinant NAC allele in transgenic plants, or upon enhanced resistance to a chemical agent (such as an antibiotic) as a result of the inclusion of a dominant selectable marker gene incorporated into the transformation vector.
Successful examples of the modification of plant characteristics by transformation with cloned nucleic acid sequences are replete in the technical and scientific literature. Selected examples, which serve to illustrate the knowledge in this field of technology include: U.S. Pat. No. 5,571,706 (“Plant Virus Resistance Gene and Methods”); U.S. Pat. No. 5,677,175 (“Plant Pathogen Induced Proteins”); U.S. Pat. No. 5,510,471 (“Chimeric Gene for the Transformation of Plants”); U.S. Pat. No. 5,750,386 (“Pathogen-Resistant Transgenic Plants”); U.S. Pat. No. 5,597,945 (“Plants Genetically Enhanced for Disease Resistance”); U.S. Pat. No. 5,589,615 (“Process for the Production of Transgenic Plants with Increased Nutritional Value Via the Expression of Modified 2S Storage Albumins”); U.S. Pat. No. 5,750,871 (“Transformation and Foreign Gene Expression in Brassica Species”); U.S. Pat. No. 5,268,526 (“Overexpression of Phytochrome in Transgenic Plants”); U.S. Pat. No. 5,780,708 (“Fertile Transgenic Corn Plants”); U.S. Pat. No. 5,538,880 (“Method for Preparing Fertile Transgenic Corn Plants”); U.S. Pat. No. 5,773,269 (“Fertile Transgenic Oat Plants”); U.S. Pat. No. 5,736,369 (“Method for Producing Transgenic Cereal Plants”); U.S. Pat. No. 5,610,049 (“Methods for Stable Transformation of Wheat”); U.S. Pat. No. 6,235,529 (“Compositions and Methods for Plant Transformation and Regeneration”) all of which are hereby incorporated by reference in their entirety. These examples include descriptions of transformation vector selection, transformation techniques and the construction of constructs designed to express an introduced transgene.
The transgene-expressing constructs of the present invention may be usefully expressed in a wide range of grain producing higher plants where an increased grain protein content is useful. The invention is expected to be particularly applicable to monocotyledonous cereal plants including barley, wheat, rye, triticale, sorghum, oat, rice, maize and forage grasses.
Methods for the transformation and regeneration of monocotyledonous plant cells are known, and the appropriate transformation technique will be determined by the practitioner. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome-mediated transformation; polyethylene glycol (PEG-mediated transformation); transformation using viruses; micro-injection of plant cells; micro-projectile bombardment of plant cells; vacuum infiltration; and Agrobacterium-mediated transformation. Typical procedures for transforming and regenerating plants are described in the patent documents listed above.
Following transformation, transformants are preferably selected using a dominant selectable marker. Typically, such a marker will confer antibiotic or herbicide resistance on the seedlings of transformed plants, and selection of transformants can be accomplished by exposing the seedlings to appropriate concentrations of the antibiotic or herbicide. After transformed plants are selected and grown the plant can be assayed for expression of recombinant proteins, increased grain protein content, using a near-infrared reflectance equipment (Technicon InfraAnalyzer model 400), micro-Kjeldahl (Bradford 1976) or other protein assays well known to those of skill in the art. Similarly, onset of senescence can be detected by assaying for chlorophyll content using standard procedures described for example in Jing, H C et al., The Plant Journal, 32:51-63 (2002) and Woo, H R et al., The Plant Journal, 31: 331-340 (2002)
Uses of the Non-Naturally Occurring Plants of the Invention
The non-naturally occurring plants of the invention have many uses, including increased or decreased grain protein content and delay or acceleration in the onset of senescence. Non-naturally occurring plants include transgenic plants and plants produced by non transgenic means such as plant breeding.
The non-naturally occurring plants of the invention are useful in that they exhibit altered grain protein content. As defined herein, altered grain protein content means that the transgenic plant will have higher or lower grain protein content than a comparable non-transgenic plant.
Other transgenic plants of the invention are useful in that they exhibit an altered rate of senescence. As defined herein, an altered rate of senescence means that the transgenic plant will senesce at a slower or higher rate than a comparable non-transgenic plant.
The protein coding sequence linked to the NAC promoter may be also any heterologous protein. Heterologous proteins useful in the invention include proteins encoded by polynucleotides from any source, natural or synthetic. Suitable coding regions encode animal RNAs or polypeptides, as well as variants, fragments and derivatives thereof. The encoded products may be recovered for use outside the host plant cell (e.g., therapeutically active products) or they may alter the phenotype of the host plant cell (e.g., conferring disease resistance, the ability to survive or grow in the presence of particular substrates). Examples of such coding regions include polynucleotides derived from vertebrates, such as mammalian coding regions for RNAs (e.g., small interfering RNAs (siRNAs) anti-sense RNAs, ribozymes, and chimeric RNAs having ribozyme structure and activity) or polypeptides (e.g., human polypeptide coding regions). Other coding regions useful in the inventive methods are derived from invertebrates (e.g., insects), plants (e.g., crop plants), and other life forms such as yeast, fungi and bacteria. The heterologous proteins which find particular use in the invention include those that provide resistance to plant pests, facilitate translocation of nutrients, provide resistance to stresses typical of the summer: heat and dehydration, etc. Such protein sequences are available in the literature and known to those of skill in the art. Representative proteins of interest are described and disclosed in Plant Biochemistry and Molecular Biology, 2nd Edition, Peter Lea and Richard C. Leegood editors; Plant Molecular Biology Second Edition; D. Grierson, S. N. Covey John Innes Institute, Norwich, U K Kluwer Academic Publishers, Dordrecht April 1991 and Biochemistry & Molecular Biology of Plants, edited, Bob B. Buchanan, Wilhelm Gruissem, and Russell L. Jones; John Wiley and Sons, Publishers, 2001, all of which are hereby incorporated by reference in their entirety.
Plants Produced by Plant Breeding
Results presented here demonstrated that the allelic variation at the NAC gene is responsible for the variation of GPC and senescence in wheat. Therefore, allelic variation at the NAC gene can be used as a molecular marker for GPC and senescence in marker assisted selection programs.
These markers can be used to transfer different NAC alleles into different germplasm by marker-assisted selection. They can also be used to determine the different haplotypes present in this region in the cultivated wheats and to establish a classification of the different haplotypes. This characterization will be useful to determine the value of the different haplotypes for grain protein content.
This invention will be better understood by reference to the following non-limiting examples.
EXAMPLE 1
Identification of Gene Responsible for High Grain Protein Content in Wheat
Background Efforts to improve GPC without selecting for low yield can be accelerated by the identification of the genes that affect GPC and the direct selection of the alleles with positive effects.
A promising source of high GPC was detected in a survey of wild populations of tetraploid Triticum turgidum ssp. dicoccoides (accession FA-15-3) referred as dicoccoides or DIC hereafter (Avivi, L., Indian Soc. Geinet. Plant Breed., New Delhi, pp. 372-380 (1978)). Cantrell, R. G., and L. R. Joppa., Crop Science 31:645-649 (1991) developed complete sets of disomic substitution lines from each of the chromosomes of this dicoccoides accession into the tetraploid durum cultivar Langdon (LDN). The substitution of the complete chromosome 6B of DIC into LDN [LDN(DIC-6B)] showed the highest protein yield. This significant increase in GPC was not associated with a significant decrease in grain yield or kernel weight (Cantrell, R. G., and L. R. Joppa., Crop Science 31:645-649 (1991)). Moreover, the increase in protein content in LDN(DIC-6B) was associated with an increase in the quality of the pasta products (Joppa, L. R., Crop Science, 31:1513-1517 (1991)). When introduced into adapted durum germplasm, the DIC allele was also associated with an increase in protein content (15 g/Kg) with non significant effects on protein quality, plant height, or heading date (Chee, P., Crop Science 41:295-301 (2001)).
The evaluation of a mapping population of recombinant substitution lines (RSLs) from the cross between LDN(DIC-6B)×LDN, showed that the increase in GPC in LDN(DIC-6B) was associated with a quantitative trait locus (QTL) located on the short arm of chromosome 6B (Joppa, L. R., Crop Science, 37:1586-1589 (1997)). This QTL, designated QGpc.ndsu.6Bb, accounted for approximately 66% of variation in GPC in this cross, suggesting that a single gene or a closely linked group of genes was segregating in this population.
Other QTL studies have shown significant effects on GPC associated with markers located in similar regions of the short arms of homeologous group 6. The Xpsr167 marker on chromosome arm 6AS (R2=18.4%) and the Nor-B2 marker on chromosome arm 6BS (R2=6.5%) were significantly associated with GPC in a cross between durum wheat cultivar ‘Messapia’ and a wild dicoccoides accession MG4343 (Blanco, A., et al., Plant Molecular Biology 48:615-623 (2002)). A significant QTL for GPC on chromosome arm 6AS in hexaploid wheat was found to be associated with AFLP marker XE38M60200 (R2=17.1%) (Sourdille, P., et al., Theoretical & Applied Genetics 106:530-538 (2003)). Finally, QTLs for high-GPC were reported in barley chromosome 6HS associated with GPC (Mickelson, S., et al., Journal of Experimental Botany 54:801-812 (2003); See, D., et al., Crop Science, 42:680-685 (2002)). These studies suggest that a major gene affecting GPC in the Triticeae is probably located in the short arms of the chromosomes from homoeologous group 6.
Development of PCR markers for the high GPC gene into tetraploid and hexaploid wheat cultivars (Khan, I. A., et al., Crop Science 40: 518-524 (2000)) Two sets of PCR primers were designed to amplify regions of the non-transcribed spacer of the XNor-B2 locus. This locus was selected because it mapped on the peak of the QTL for GPC. The first pair of allele specific primers produced an amplification product only when the DIC XNor-B2 allele was present. The second pair of primers amplified fragment(s) of similar length in the different genotypes that after digestion with the restriction enzyme Bam HI allowed differentiation of the DIC allele. Four microsatellites markers were mapped on the short arm of chromosome 6B at both sides of the QTL peak and two on the long arm. These PCR markers together with 10 RFLP markers showed that the hexaploid cultivar Glupro, selected for high GPC, carries a distal segment of chromosome 6BL and a proximal segment of 6BS from dicoccoides accession FA15-3 encompassing the segment with highest LOD score for the GPC QTL. These results demonstrated that the introgression of the DIC allele into common wheat varieties also produced high GPC levels.
Construction and characterization of a BAC library of tetraploid recombinant substitution line of durum wheat cultivar ‘Langdon’ carrying a 30 cM segment of chromosome 6BS from DIC carrying the allele for high grain protein content (Cenci, A., Theor Appl Genet 107:931-939 (2003)) A total of 516,096 clones were organized in 1344 384-well plates and blotted into 28 high-density filters. The average insert size of 500 randomly selected BAC clones was 131-kb, resulting in a coverage of 5.1× genome equivalents for each of the two genomes, and a 99.4% probability of recovering any gene from each of the two genomes of durum wheat. Six known copy number probes were used to validate this theoretical coverage and gave an estimated coverage of 5.8× genome equivalents. The main objective for the construction of this BAC library was the positional cloning of the GPC gene for high grain protein content.
Detailed mapping of the GPC gene (Olmos S., et al., Theor. Appl. Genet. 107:1243-1251 (2003)) A larger set of secondary recombinant substitution lines with recombination events within the QGpc.ndsu.6Bb QTL region and field trials with large number of replications to map the source of the variation in GPC as a single Mendelian locus designated Gpc-B1 (GPC-B1 gene). This locus was mapped within a 2.7-cM region encompassed by restriction fragment length polymorphism (RFLP) loci Xcdo365 and Xucw67.
The precise mapping of the Gpc-B1 locus was followed by a study on the microcolinearity between rice and wheat in the Gpc-B1 region (Distelfeld A. et al., Functional and Integrative Genomics. 4: 59-66 (2004)). This study facilitated the use of rice as a stepping stone for the development of new markers and for the construction of a high-density map of the region. This approach enabled to narrow down the mapping of the Gpc-B1 locus to a 0.3-cM region flanked by PCR markers for loci Xucw79 and Xucw71, which corresponds to a 64-kb region in rice genome located within Nipponbare BAC AP004061.
Physiological study comparing the tetraploid recombinant substitution line #68 (RSL#68) carrying the Gpc-B1 allele from high GPC (DIC) and the near isogenic line Langdon with the allele for low GPC. Immediately after anthesis RSL#68 had higher soluble protein and amino acids concentrations in the flag leaf than the isogenic line Langdon. At maturity, both lines presented a similar above ground biomass and grain yield. However, RSL#68 showed a higher total N content in ears, grain, and chaff than Langdon; N harvest index was also higher due to a lower straw N concentration and higher grain N concentration. It was concluded that the presence of the DIC Gpc-B1 allele resulted in a more efficient N remobilization from the leaves to the ears during grain filling.
Using the tetraploid wheat BAC library and the high-density map we constructed a complete physical map of the Gpc-B1 region including the allele from DIC for high GPC (Distelfeld A. et al., New Phlytologist, 169:753-763 (2006)). The chromosome walk was initiated with the closest proximal marker (Xucw71) and was completed with the identification of overlapping BACs 409D13 and 916017 that formed a contig of approximately 250-kb that includes Xuhw84. The Xuhw89 locus corresponding to the distal end of BAC clone 916017 was mapped 0.1 cM distal to the Gpc-B1 locus. Based on these results it was concluded that the GPC-B1 gene is located within the 250-kb physical contig delimited by loci Xucw71 and Xuhw89. A high-throughput PCR marker was developed for the Xuhw89 locus to facilitate the introgression of this gene into commercial varieties by marker assisted selection. The polymorphism for the Xuhw89 locus was validated in a large collection of tetraploid and hexaploid wheat germplasm.
Identification of WNAC-1 as the gene responsible for high GPC
Sequencing of the Gpc-B1 physical contig The BACs 409D13 (1030 sub-clones both directions, 10.8× coverage) and 916017 (1270 sub-clones both directions, 20× coverage) were sequenced from the RSL#65 BAC library. These two BACs included the Gpc-B1 flanking loci Xuhw89 and Xucw71 and therefore, the Gpc-B1 gene. The ends of these two BACs overlapped on a 32,224-bp region (100% identical) confirming the connection predicted by the physical map. The sequence of the complete contig was approximately 245-kb.
BAC 409D13 proximal end included part of wheat gene UCW96 (FIG. 1). Wheat gene UCW96 was separated by 856-bp from gene UCW71 which was also close (1142-bp) to gene UHW84. These three wheat genes were in the same orientation as the colinear rice genes 2474.t23, 22474.t22 and 2474.t14, which where the most similar genes in the rice genome, confirming that these three pairs of genes were orthologous (FIG. 1). The region between 22474.t22 and 2474.t14 was larger in rice and included an additional gene 22474.t17, which was mapped in a different wheat chromosome (5L). A non-colinear wheat gene, designated UCW97, was predicted by Genscan 7,440-bp distal to UHW84. The predicted UCW97 protein includes a leucine-rich repeat (LRR), ribonuclease inhibitor (R1)-like domain (amino acids 278-448, cd00116, E=5e−6) and also showed similarity to a leucine-rich repeat (LRR) (domain COG4886, E=1e−8) between amino acids 504 to 831. This predicted gene was not supported by ESTs. The best match in the Triticum EST collection CA733412.1, showed a low level of identity (84%) to be considered a real orthologue.
An additional wheat gene that was not colinear with the rice genes was identified 38.6-kb distal to UCW97 in BAC 409D13. This putative wheat gene, designated UCW87 (SEQ ID NOS:15 and 16), includes a Rhamnogalacturonate lyase domain (pfam06045, first 190 amino acids, E=1e−56). This domain is characteristic of proteins that degrade the rhamnogalacturonan I (RG-I) backbone of pectin. This family contains mainly members from plants, but also contains the plant pathogen Erwinia chrysanthemi. The closest homologues in plants are Arabidopsis NP—172459.1 (68% similarity, E=0) and rice BACBX000498 (79% similar, E=0, chromosome 12). The wheat protein differs from both the rice and the Arabidopsis genes by the absence of introns three to seven. The large exon three from the predicted wheat protein corresponds to exons three to eight in the rice and Arabidopsis putative orthologous proteins.
BAC 916017 revealed the presence of one additional wheat gene that was not colinear with the rice genes. This gene designated WNAC-B1 (SEQ ID NOS:1 and 2) includes a NAM domain (pfam 02365, E=2e−57) characteristic of a large family of plant NAC transcription factors. Reported herein are also the orthologous WNAC-1 copies for the A genome (SEQ ID NOS:5 and 6), D genome (SEQ ID NOS:7 and S), and H genome from barley (SEQ ID NOS:11 and 12). A paralogous copy of this gene designated WNAC-B2 (SEQ ID NOS:9 and 10) was mapped in the centromeric region of the short arm of wheat homoeologous group 2B using deletion lines. This paralogous gene is 91% identical at the DNA level to WNAC-B1 suggesting a relatively recent duplication. The paralogous WNAC-2 copy was also found in barley (FIG. 4, SEQ ID NO:13 and 14). The closest rice gene in rice is OsNAC5, which was mapped on rice chromosome 7 (BAC AP003932) in a region colinear to the centromeric bin of the short arm of wheat chromosome 2. This result suggests that the WNAC-2 copy in wheat is the ancestral gene and that the WNAC-1 is the result of a duplication that occurred in the Triticeae lineage after the Triticeae—rice divergence but before the wheat—barley divergence. The closest rice gene in rice in further described in Example 3. The Arabidopsis NAC genes more closely related to the WNAC-1 gene belong to the NAP subgroup (Ooka, H., et al., DNA Research 10:239-247 (2003)).
Screening for additional recombinant lines to reduce the number of candidate genes To further reduce the list of candidate genes a total of 3,000 gametes from the cross LDN×RSL65 were screened. F2-3 lines to identify recombinants between flanking markers Xuhw89 and Xucw71. Only 4 lines showed recombination between flanking markers Xuhw89 and Xucw71: RSL121, RSL152, RSL300, and RSL28 (Table 1).







TABLE 1





Molecular characterization of critical recombinant lines and linkage with the


phenotype (Gpc-B1 locus) for high grain protein content.
















*D = DIC alleles, L = LDN alleles. A change between gray and white cells indicates a recombination event.






The Gpc-B1 genotype can be accurately evaluated using near isogenic lines segregating only for the 30-cM of the GPC QTL region, combined with 10 replications (1 m rows) organized in a split plot experimental designs (Olmos S., et al., Theor. Appl. Genet. 107:1243-1251 (2003)). The same procedures were used to evaluate the critical lines in at least two independent experiments (Table 1). A line was classified as low-GPC if it was simultaneously significantly lower (P<0.01) than the high-GPC group and non-significantly (NS) different from the low-GPC group. A line was classified as high-GPC if it was simultaneously significantly higher (P<0.01) than the low-GPC control group and NS different from the high-GPC control group. To further validate the GPC determinations the critical lines were backcrossed again to Langdon (low GPC) and sister homozygous lines from the progeny with alternative alleles were compared against each other.
For example, 19 independent RSL300-derived homozygous recombinant lines were compared to both high- (P=0.9478) and low-GPC controls (P<0.001) and also to sister RSL300 lines for which the non recombinant chromosome DIC segment was fixed (P=0.6394). These results demonstrated that the recombinant chromosome in RSL300 carries the WNAC-B1 a allele for high grain protein, and therefore eliminated UCW97 and UHW84 as candidate genes for the Gpc-B1 locus.
In summary, the high-density genetic map combined with the sequencing of the physical contig showed that the Gpc-B1 locus is proximal to the Xuhw89 locus and distal to the Xucw97 locus (Table 1). Only the WNAC-1 gene and the UCW87 putative gene were completely linked to the segregation for GPC and therefore, were the only candidate genes for the Gpc-B1 locus.
Allelic variation at the WNAC-1 and UCW87 candidate genes To determine which of these two genes is Gpc-B1, the allelic variation between the DIC (high-protein parent) and LDN (low protein parent) was analyzed for both the UCW87 and WNAC-B1 candidate genes from chromosome 6B.
UCW87: The DNA genomic sequence of wheat UCW87 for both LDN and DIC alleles showed that the large exon 3 from wheat corresponded to exons 3 to 8 in the rice and Arabidopsis closely related genes (SEQ ID NOS:15 and 17). It is not known if this large structural change affected the functionality of this wheat gene.
The predicted UCW87 protein from the high-GPC parent (SEQ ID NO:16) shows only one amino acid difference relative to the predicted protein of the low-GPC parent (LDN, SEQ ID NO:18). This K (DIC) to N (LDN) mutation is outside the functional Rhamnogalacturonate lyase domain and therefore is not likely to affect the function of this domain. In addition, this mutation involves two polar amino acids with similar hydrophobicity and has therefore, a low probability to alter the protein folding. A K to N mutation is regarded as neutral in the BLOSUM 62 matrix for amino acid substitutions. Finally, a BLASTP analysis of the most similar proteins to UCW87 revealed that the polymorphic K/N amino acids are not located in a conserved position in the protein, suggesting that this amino acid is not critical for the protein function.
In summary, the single amino acid substitution observed between the LDN and DIC UCW87 proteins does not seem to be sufficient to explain the large phenotypic differences observed between the parental lines.
WNAC-B1: On the contrary, a significant difference was observed between the LDN and DIC alleles for the 6BS WNAC-B1 gene. The DIC allele for WNAC-B1, designated WNAC-B1a hereafter (SEQ ID NO:1), showed the five conserved domains characteristic of the NAC transcription factors (SEQ ID NO:2). However, the LDN allele, designated WNAC-B1b showed a 1-bp T insertion after the first 10-bp of the gene (FIG. 4, SEQ ID NO:3) that results in a frame-shift mutation that completely alter the WNAC-1 protein after the third amino acid (SEQ ID NO:4). This mutation is sufficient to produce a non-functional protein for the B genome copy of this transcription factor. Even though there is a second functional copy of the WNAC-1 transcription factor in the A genome, the abundance of functional WNAC-1 protein would be reduced in the plants carrying the LDN allele.
The WNAC-B1b allele associated with low protein content is widely distributed among the cultivated durum varieties. A survey of a world collection of 54 durum lines (Maccaferri, et al., Theor Appl Genet 107:783-797 (2003)) with a molecular marker for the frame-shift mutation revealed that all the tested durum cultivated varieties have the non functional WNAC-B1b allele for low protein content (Table 2). This result indicates that the introduction of the WNAC-B1 a allele from DIC into the cultivated durum varieties has the potential to increase GPC in a large set of tetraploid varieties.







TABLE 2







Durum varieties carrying the WNAC-B1b


allele with the frame-shift mutation.










VARIETY
COUNTRY







Dunfati




Edolic



Durfort
FRANCE



Exeldur
FRANCE



Nefer
FRANCE



Neodur
FRANCE



Minos
GREECE



Adamello
ITALY



Appio
ITALY



Capelli
ITALY



Ciccio
ITALY



Cirillo
ITALY



Colosseo
ITALY



Duilio
ITALY



Karel
ITALY



L35
ITALY



Latino
ITALY



Messapia
ITALY



Ofanto
ITALY



Russello sg7
ITALY



San carlo
ITALY



Saragolla
ITALY



Trinakria
ITALY



Valbelice
ITALY



Valforte
ITALY



Valnova
ITALY



Varano
ITALY



Zenit
ITALY



Appulo
ITALY



Vitron
SPAIN



Aconchi 89
MEXICO



Altar 84
MEXICO



Carcomun ‘s’
MEXICO



Mexicali 75
MEXICO



Inrat 69
TUNISIA



Karim
TUNISIA



Khiar
TUNISIA



Aldura
USA



Aruba
USA



Colorado
USA



Duraking
USA



Durex
USA



Imperial
USA



Kofa
USA



Kronos
USA



Langdon
USA



Leeds
USA



Ocotillo
USA



Produra
USA



Reva
USA



Ria
USA



UC1112
USA



UC1113
USA



Wb 881
USA










Two isogenic lines of tetraploid wheat were developed by backcrossing the WNAC-B1a allele into the durum lines Kofa and UC1113 (which have the nonfunctional WNAC-B1b allele) by six backcross generations followed by selection of homologous lines using molecular markers. In a Randomized Complete Block Design (RCBD) experiment with 10 replications (Buenos Aires, Argentina, 2004) it was demonstrated that the lines with the introgressed WNAC-B1 a allele showed significantly higher levels of PC than the isogenic lines with the allele originally present in those varieties (Table 3).
The introduction of the DIC 6BS segment including the WNAC-B1a allele has been also shown to increase GPC when introduced into hexaploid wheat (Mesfin, A., R. C. Frohberg, and J. A. Anderson, Crop Science, 39:508-513 (1999), Khan, I. A., et al., Crop Science 40: 518-524 (2000)). Two additional isogenic lines of hexaploid wheat were developed by backcrossing the WNAC-B1 a allele into the high-protein HRS breeding line UC1041 and the low-protein HRS variety RSI5. The isogenic lines were developed by six backcross generations followed by selection of homozygous lines using molecular markers, and compared using a split plot design experiment with 20 replications in the field (Davis, Calif. 2004). In this experiment, the lines with the introgressed WNAC-B1a allele showed significantly higher levels of GPC than the isogenic lines with the allele originally present in those varieties. The incorporation of the DIC allele resulted in a grain protein content increase of 0.5% in UC1041 (from 14.2 to 14.7%, P=0.001) and of 1.6% in RSI5 (from 11.9% to 13.5%, P<0.0001). These results confirmed the potential of the WNAC-B1a allele from DIC to increase protein content in hexaploid wheat, both in lines of low and high-GPC.
The introduction of the DIC 6BS segment including the WNAC-B1a allele has been also shown to increase GPC when introduced into hexaploid wheat (Mesfin, A., R. C. Frohberg, and J. A. Anderson, Crop Science, 39:508-513 (1999); Khan, I. A., et al., Crop Science 40: 518-524 (2000)). Six additional isogenic lines of hexaploid wheat were developed by backcrossing the WNAC-B1 a allele into the high-protein HRS breeding lines and varieties UC1037, UC1041, Express, and Yecora Rojo and the low-protein HRS varieties Anza and RSI5. The isogenic lines were developed by six backcross generations followed by selection of homozygous lines using molecular markers. UC1041 and RSI5 were compared using a split plot design experiment with 20 replications in the field at Davis, Calif. (2004); and in an RCBD experiment with 10 replications at Buenos Airs, Argentina (2004). The other isogenic lines were tested only in the second location. In both experiments, the lines with the introgressed WNAC-B1 a allele showed significantly higher levels of GPC than the isogenic lines with the allele originally present in those varieties (Table 3). These results confirmed the potential of the WNAC-B1 a allele from DIC to increase protein content in hexaploid wheat, both in lines of low and high-GPC.







TABLE 3







Effect of the WNAC-B1b on GPC in


different genetic backgrounds.













Signif-



% Protein
Relative
icance












VARIETY
Class
Gpc-B1b
Gpc-B1a
Increase
P value










Davis, CA 04












UC1041
HRS
14.2%
14.7%
3.5%
0.0011


RSI5
HRS
11.9%
13.5%
13.4% 
<0.0001







Argentina 04












UC1041
HRS
13.0%
14.2%
9.4%
<0.0001


RSI5
HRS
10.9%
12.0%
10.1% 
0.0014


Express
HRS
12.2%
13.2%
8.0%
0.0002


UC1037
HRS
12.8%
13.7%
7.4%
0.0075


Anza
HRS
10.8%
11.8%
8.5%
0.0003


Yecora Rojo
HRS
12.4%
13.1%
5.1%
0.0300


Kofa
Durum
12.9%
14.2%
10.6% 
0.0007


UC1113
Durum
11.8%
13.5%
13.9% 
<0.0001


Average



9.0%
Increase







GPC





Davis, CA, USA (04): Split Plot design, 20 replications. Buenos Argentina 04: RCBD 10 replications.






Transcription profile of the candidate genes. The process of N remobilization in wheat is regulated at the leaves and ears rather than at the grain level (Bameix, A. J., and M. R. Guitman, J. Exp. Bot. 44:1607-1612 (1993), Martre, P., et al., Plant Physiol. 133:1959-1967 (2003)). The GPC-B1 gene, follows this general principle as demonstrated by the increased availability of soluble amino acids and proteins in the flag leaves of lines with the WNAC-B1 a allele relative to the lines with the WNAC-B1b allele. This increased concentration of proteins and amino acids in a form that can be readily transported to the grain provides a simple explanation to the increased grain protein content and lower straw residual N observed in the lines carrying the WNAC-B1a allele.
Since the differences in soluble proteins and amino acids were detected after anthesis but not in younger leaves, the candidate gene for the Gpc-B1 locus was expected to show some differences in the flag leaves around the time of anthesis.
No transcripts of UCW87 were observed in the flag leaves or other leaves of LDN or DIC at the time of anthesis. Transcripts were detected only with the UCW87 primers in developing seeds. These results indicate that UCW87 is not transcribed in the critical tissues and at the developmental stages expected for the GPC-B1 gene.
On the contrary WNAC-B1 showed the expected transcription profile based on the physiological experiments. Transcription of the WNAC-B1a, WNAC-B1b, WNAC-A1, and WNAC-B2 dramatically increased in the flag leaves after anthesis and during maturity (FIG. 2, lanes 3-5). No transcripts for the WNAC-1 genes were detected in earlier developmental stages in young green leaves. However a low level of transcripts was detected in senescing yellowing leaves (FIG. 2, lane 6).
Based on their natural allelic variation, transcription profiles, and the predicted function of orthologous genes in other plant species, UCW87 was eliminated as a candidate for the Gpc-B1 locus and concluded that WNAC-B1 is Gpc-B1.
EXAMPLE 2
Effect of the Allelic Variation at the Gpc-B1 Locus on Senescence
Allelic variation at the Gpc-B1 locus has other effects in addition to the observed difference in GPC. A field study comparing several RSLs carrying the WNAC-B1a allele with other carrying the WNAC-B1b allele (Davis, Calif., 2004, split plot design, 20 blocks) showed that the lines with the WNAC-B1 a senesce faster than the lines with the WNAC-B1b allele after anthesis. No significant differences were detected in ear emergence or time of anthesis between the two alleles indicating that the differences in maturation were due to differences in senescence/maturation rates. A few days after the first symptoms of senescence, the RSLs with the WNAC-B1 a allele showed a significantly higher percent of yellow peduncles (P<0.001) and a significantly lower water content in the spikes (P<0.001) than the lines with the WNAC-B1b allele. Lines with WNAC-B1a allele had an average humidity of 25.9% versus lines with LDN allele that had average of 29.5%.
The acceleration in the senescence process in the plants with the WNAC-B1a allele is consistent with the higher levels of soluble proteins and amino acids observed in the flag leaves of the plants with this allele relative to the plants with the WNAC-B1b allele. Soluble proteins are the results of degradation of structural or storage proteins into a form that can be transported to the grain. These results suggest that the WNAC-1 gene plays a significant role in the regulation of the senescence process. It is possible, that the observed difference in grain protein content is one of the multiple effects generated by the differences in senescence rates.
The Arabidopsis NAC genes more closely related to the WNAC-B1 gene have been shown to be upregulated during dark induced (Lin J.-F., S.-H. Wu, The Plant Journal 39:612-628 (2004)) and natural senescence (Guo, Y., Plant Cell and Environment 27:521-549 (2004)) in leaves. The initiation of WNAC-1 transcription in the flag leaves after anthesis is also consistent with the proposed role in the regulation of senescence.
The RSLs tested in the senescence experiment included the critical lines RSL28 and RSL121 facilitating a precise mapping of the difference in senescence. The locus regulating the differences in senescence was mapped between flanking loci Xuhw89 and Xucw71 (Table 1) and completely linked to the WNAC-1 gene. It appears that the different amounts of functional WNAC-1 protein generated by the frame shift mutation were responsible for both the differences in maturity and the differences in GPC content. In conclusion, the modulation of the transcript levels of the WNAC-B1 gene can be used to alter both grain protein content and also the rate of leaf senescence in wheat and other cereals.
EXAMPLE 3
WNAC-B1 Orthologues in Rice and Maize
WNAC-B1 Orthologue in Rice.
The complete genomic sequence of rice was searched to identify the closest homologue of WNAC-B1 in rice (SEQ ID NOS:28 and 29). One rice NAC protein was identified (OsNAC) that had 98% similarity to WNAC-B1 at the protein level within the NAC domain (115 of 117 amino acid residues). This high level of similarity dropped to 84% with the second highest score in the blast search (OsNAC-A). FIG. 4 displays a multiple sequence alignment of the 75 NAC proteins previously reported by Ooka, H., et al., DNA Research 10:239-247 (2003) and the wheat WNAC-B1 protein, followed by a phylogenetic analysis (neighbor-joining method). OsNAC and WNAC-B1 clustered together, while OsNAC-A and all the other rice NAC proteins were located on separate branches (complete tree not shown).
In addition, the promoter region of the WNAC-B1 and OsNAC genes was analyzed to find common regulatory elements. A 40-bp segment with perfect conservation between OsNAC, WNAC-B1 and homoeoallele WNAC-A1 was found. Within this conserved segment, an ABRE-like motif (ACGTG) was identified. This motif has previously been shown to be required for senescence responsive gene expression in the erd1 promoter of Arabidopsis (Simpson et al., 2003). This study also identified an ACG motif 40 bp downstream that was required (along with the ABRE-like motif) to promote senescence associated erd1 expression. These two motifs are perfectly conserved and separated by similar distance (38-40 bp) in the rice, wheat and barley promoters for the WNAC-B1 orthologues. The two presented sources of evidence strongly support the hypothesis that OsNAC is the single orthologue of WNAC-B1 in rice.
The rice orthologue OsNAC is located on chromosome 7, which is colinear with wheat chromosome 2. WNAC-B1 maps to wheat chromosome 6, but has a paralogous copy (WNAC-B2) on wheat chromosome 2. This gene, WNAC-B2, is in perfect colinearity with the rice OsNAC gene and therefore, the original gene that was cloned from wheat chromosome 6 is product of a duplication event that occurred in wheat, but was absent in rice. A QTL for rice soluble protein content in senescing leaf blade was mapped near marker R1357 which is located 1.6 cM proximal to OsNAC (Obara et al. 2001), while Tan et al (2001) mapped a QTL for rice GPC 3.6 cM distal to OsNAC. This colinear location between the wheat and rice NAC genes, along with the colocalization of equivalent QTLs in this region, further supports the hypothesis that OsNAC and WNAC-B1 are orthologues that share a similar function.
Additional evidence is provided by the fact that OsNAC shares similar expression patterns as WNAC-B1 (FIG. 5), with no detectable expression in any tissue until the days prior to anthesis. An electronic search for EST sequences of OsNAC before anthesis or in other tissues was negative, with the only EST identified belonging to a library developed from leaf tissue after anthesis. Taking together the conserved promoter motifs, the sequence similarity, their colinear location on the wheat and rice chromosomes and their similar expression profiles, these results strongly suggest that rice OsNAC is the unique orthologue of wheat WNAC-B1.
WNAC-B1 Orthologue in Maize.
The latest version of the maize genome assembly at the Plant Genome Database (http://www.plantgdb.org) was searched. This search identified a GSS contig (SEQ ID NOS:30 and 31) that encodes a protein (ZmNAC) which has 100% similarity to WNAC-B1 at the protein level within the NAC domain (117 of 117 amino acid residues). This high level of conservation within the NAC domain is even higher than that found for the rice orthologue (OsNAC) providing evidence that this protein (ZmNAC) is an orthologue of the wheat WNAC-B1 protein. The possibility for other WNAC-B1 orthologues in maize exists, although ZmNAC is the most likely true orthologue to WNAC-B1 due to its high conservation within the functional NAC domain.
EXAMPLE 4
Effect of the Allelic Variation at the WNAC-B1 Locus on Senescence
To further assess the senescence phenotype, a time-course of chlorophyll content in the flag leaves of plants grown under field conditions was established (FIG. 6b). Plants with the WNAC-B1a allele underwent earlier chlorophyll degradation than plants with the WNAC-B1b allele, starting 19 days after anthesis (DAA). Significant differences in flag leaf chlorophyll content were detected in the measurements performed at 24 DAA (P<0.006), 26 DAA (P<0.002), 28 DAA (P<0.001) and 32 DAA (P<0.001). Measurements for both alleles converged when the flag leaves were completely yellow (35 DAA). No differences were detected between RSLs and controls in the time of anthesis. The average GPC increase in the RSLs with the WNAC-B1a allele relative to the lines with the WNAC-B1b allele was 12 g/kg (P<0.001, FIG. 2a), confirming that the conditions were appropriate for the expression of the differences attributable to the Gpc-B1 locus.
EXAMPLE 5
Effect of WNAC-B1 on Senescence in Tetraploid and Hexaploid Isogenic Lines
The effect of WNAC-B1 in different genetic backgrounds was tested in three hexaploid (Anza, RSI5, and UC1041) and one tetraploid (Kofa) pairs of isogenic lines (BC6F3). The presence of the WNAC-B1a allele significantly increased GPC (P<0.01) in the three hexaploid isogenic lines (Anza, RSI5, UC1041) with respect to the recurrent parent by an average of 12.1 g kg−1. The tetraploid line Kofa carrying the WNAC-B1a allele showed a smaller increase of GPC (3.3 g kg−1, P=0.26) relative to the recurrent parent (a high-protein content variety), and the differences were not significant in this experiment.
In all four genetic backgrounds, the chlorophyll degradation time-course (FIG. 7) showed more rapid chlorophyll degradation in the lines with the WNAC-B1a allele than in the isogenic lines with the recurrent parent allele. These differences were not originated in flowering time variability since the lines for each isogenic pair reached anthesis at the same time. During the first 20-30 DAA no significant differences in chlorophyll content were detected in the flag leaves, but after that, chlorophyll degradation was faster in the isogenic lines carrying the WNAC-B1a allele (FIG. 7, grey lines with open squares) as compared with the original recurrent parent (FIG. 7, black lines with full squares). These differences were similar across genotypes, with the isogenic lines carrying the WNAC-B1a allele being on average 3-4 days more advanced in their senescence process than the corresponding recurrent parent. The significant differences in chlorophyll content (P<0.05, indicated by arrows in FIG. 7) were maintained until the flag leaves of the isogenic pairs approached complete yellowing.
In addition to chlorophyll degradation, the progression of grain moisture content was measured leading to the observation that the isogenic lines with the WNAC-B1a allele presented significantly lower grain moisture content than the isogenic lines with the recurrent parent allele (P<0.05, FIG. 4). Although isogenic lines of UC1041 were not included in FIG. 8, they also showed significant differences in grain moisture content (P=0.02) between the recurrent UC1041 line (20.2% moisture content) and the UC1041 isogenic line carrying the WNAC-B1a allele (8.9% moisture content). These results indicate that the more rapid chlorophyll degradation in the isogenic lines carrying the WNAC-B1a allele was associated with a shorter grain filling period in all four genotypes. This result is consistent with those observed in the tetraploid recombinant substitution lines (FIG. 6).
EXAMPLE 6
Modulation of Senescence by Altering the Expression Levels of WNAC Using a Transgenic Approach
The RNAi construct was made in the binary vector pMCG161 provided by V. Chandler and built using pCAMBIA 1200. The pMCG161 vector is depicted in FIG. 13 and described on The Plant Chromatin Database website accessible at http://www.chromdb.org/. This vector contains a cassette designed for making inverted repeat transcripts of a gene, flanking loop, which should efficiently produce a double stranded RNA. Expression of the transgene is driven by the 35S promoter followed by the Adh1 intron. A 475-bp construct was made from the functional WNAC-A1 (832-bp to TGA stop codon plus 88-bp from the 5′ UTR) in the sense orientation between restriction sites AscI-AvrII and in antisense orientation between restriction sites SgfI-SpeI. The cloned region excluded the NAC domain to avoid silencing of other NAC genes. The engineered plasmid was co-transformed with UBI:BAR into immature embryos of Bobwhite, a hard white spring wheat, by electrophoresis.
To validate WNAC-B1 as the gene responsible for the differences in senescence observed in the RSLs (FIG. 6a), an RNAi construct was designed that was expected to decrease transcript levels for the WNAC copies on all three genomes (A, B and D), as well as on homologous group 2. A delay in senescence was expected to result in transgenic lines with reduced expression of the WNAC genes, similar to the delay of 4-5 days seen in RSLs with the non-functional WNAC-B1b allele (FIG. 6a). Two independent T, transgenic events were generated (L19-54 and L23-119) that showed a delay in senescence with respect to the non-transgenic controls (FIG. 9). Three different phenotypes were measured to determine their senescence profile: flag leaf chlorophyll degradation, DAA when the peduncle was completely yellow and DAA for the main spike to be completely dry. Plants that were positive for the RNAi construct from event L19-54 presented a delay in flag leaf chlorophyll degradation at 24 days with respect to the control lines that were negative for the transgene (FIG. 9a). These same positive plants had a delay of over 30 days to reach complete yellowing of the peduncle and a two week delay in spike yellowing (FIGS. 9b, c). Interestingly, when the flag leaf and peduncle of the positive plants dried, in many cases they still conserved chlorophyll, (FIG. 10b). This suggests a role of WNAC in chlorophyll degradation in both flag leaf and peduncle, among other senescence related processes. Positive plants had similar physical appearance compared to control plants, produced equal number of tillers and presented no differences in time to anthesis. Similar results were obtained from the second positive event, L23-119.
EXAMPLE 7
Effect of the Allelic Variation at the WNAC-B1 Locus on Zn and Fe Concentration in the Grain
Allelic variation at the WNAC-B1 locus has other effects in addition to the observed difference in GPC and senescence. A field study by Distelfeld A. et al., New Phytologist, 169:753-763 (2006) comparing several RSLs carrying the WNAC-B1a allele with other carrying the WNAC-B1b allele (Haifa, Israel, 2004-2005) showed that the lines with the WNAC-B1a accumulate 10-20% higher Zn and Fe concentration in the grain compared to the lines with the WNAC-B1b allele (FIG. 11). The RSLs with the WNAC-B1a allele showed a significantly higher GPC (P<0.01), Zn concentration (P<0.01) and Fe concentration (P<0.01) than the lines with the WNAC-B1b allele. Lines with WNAC-B1a allele had an average Zn concentration of 61 mg/kg versus the lines with the WNAC-B1b allele that had an average of 56 mg/kg. In this same manner, lines carrying the WNAC-B1a allele had an average grain Fe concentration of 39 mg/kg versus an average of 34 mg/kg in the lines with the WNAC-B1b allele. The expected differences in GPC and senescence were also observed in this experiment. Lines with the WNAC-B1a allele increased GPC by 17 g/kg and had an acceleration in senescence of 3 days (peduncle yellowing) with respect to lines with the WNAC-B1b allele.
The increased accumulation of Zn and Fe in the grain of plants with the WNAC-B1a allele is consistent with the proposed role of WNAC-B1 in modulating senescence and therefore nutrient translocation. Physiological studies concluded that the presence of the WNAC-B1a allele resulted in a more efficient N remobilization from the senescing leaves to the ears during grain filling. This same mechanism may be responsible for the increased Zn and Fe accumulation in the grain. These results suggest that WNAC-B1 plays a significant role in the regulation of the senescence and through this, in the nutrient remobilization process of not only protein, but of other micronutrients such as Zn and Fe, as well.
The RSLs tested in the Zn and Fe experiment included the critical lines RSL28 and RSL121 facilitating a precise mapping of the difference in grain mineral concentration. The locus regulating these differences was mapped within a 250-kb region and completely linked to the WNAC-1 gene. Therefore, the different amounts of functional WNAC-B1 protein generated by the frame shift mutation appears responsible for the differences in senescence, GPC, Zn and Fe content in the grain.
EXAMPLE 8
Transcription Profile of Candidate Genes
Transcription of WNAC-A1, WNAC-B1, and WNAC-B2 was first detected in leaf samples from Zadoks 45 stage, 10 days before anthesis and dramatically increased in the flag leaves after anthesis (Zadoks 65 stage) and during maturity (FIG. 12). The highest transcription level was detected at Zadoks 72 stage, 10 days after anthesis. At this point the estimated mRNA copy number in 50 ng of RNA was significantly higher in WNAC-A1 (119,442) and WNAC-B2 (146,679) as compared to WNAC-B1 (47,867). From this point forward, the mRNA levels of WNAC-A1, WNAC-B1, and WNAC-B2 decreased but remained present in the leaves until complete senescence (Zadoks 87). In addition, expression of the WNAC genes was detected in green spikes and peduncles.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

## Claims

1. A transgenic plant comprising a recombinant nucleic acid encoding a plant NAC protein, wherein said nucleic acid encodes a NAC protein which is at least 95% identical to the amino acid sequence depicted in SEQ ID NO: 2.

2. The transgenic plant of claim 1 wherein said NAC protein is SEQ ID NO: 2.

3. The transgenic plant of claim 1 wherein said nucleic acid is SEQ ID NO:1.

4. The transgenic plant of claim 1 wherein said nucleic acid is operably linked to a heterologous promoter.

5. The transgenic plant of claim 1 wherein said plant is selected from the group consisting of wheat, rice, or maize.

6. An isolated nucleic acid molecule comprising a recombinant expression cassette comprising a nucleic acid sequence encoding a NAC protein which is at least 95% identical to the amino acid sequence depicted in SEQ ID NO: 2.

7. A method of increasing grain protein content or of delaying senescence in a cereal plant, the method comprising introducing into the plant a recombinant expression cassette comprising the nucleic acid of claim 6.

8. The method of claim 7, wherein the NAC nucleic acid is SEQ ID NO: 1.

9. The method of claim 7, further comprising selecting plants exhibiting delayed timing of senescence onset compared to the timing of senescence onset in a control plant.

10. The method of claim 7, further comprising selecting plants exhibiting higher grain protein content compared to a control plant.

11. The nucleic acid molecule of claim 6 wherein said NAC protein is SEQ ID NO: 2.

12. The nucleic acid molecule of claim 6 wherein said nucleic acid sequence is SEQ ID NO:1.

13. The nucleic acid molecule of claim 6 wherein said nucleic acid sequence is operably linked to a heterologous promoter.

14. A method of delaying senescence in a wheat plant, the method comprising inhibiting expression of an endogenous WNAC gene in the plant, wherein the endogenous WNAC gene encodes a protein at least 95% identical to SEQ ID NO: 2.

15. The method of claim 14, wherein the method is carried out by introducing into the plant a construct encoding an interfering RNA molecule.