Progress 07/15/02 to 07/15/06
Outputs
This project had three major goals. The first was to conduct genome-scale comparative analyses of noncoding sequences in cereal crop species, with an emphasis on maize and rice. The second goal was to evaluate the utility of such sequence comparisons to identify sequences important for regulation of gene expression in cereals, using the starch biosynthetic genes as a model system. The final objective was to functionally test predicted regulatory sequences in transgenic promoter-reporter gene assays. This project was one of the first to assess the utility of methods developed for microbial and mammalian genomes for the comparison of noncoding sequences in plant species. These analyses established which methods were most successful at identifying known regulatory sequence elements and baseline criteria for identifying conserved noncoding sequences in cereal crop genes. This study also demonstrated that noncoding sequences in cereal genomes are evolving at a greatly
accelerated rate relative to mammalian genomes. In addition to publication, the results of these CNS analyses can be accessed at the following website: http://cropsci.uiuc.edu/faculty/moose/PromoterComparisons.htm. Though successful in describing the general properties of CNS in cereal genes, the results from the first phase of the project were not as informative in predicting important regulatory sequence elements in the cereal starch biosynthetic genes. Attempts to confirm the functional properties of CNS through biochemical tests of DNA-protein interaction did not produce definitive results. Because noncoding sequences for many of the maize starch biosynthetic genes were available from 32 diverse maize genotypes, we shifted our emphasis to comparisons of noncoding sequence variation within maize. The number of observed haplotypes ranged from only three for the brittle2 gene to nearly 30 for the waxy1 gene, suggesting which genes harbor potential regulatory differences that might be
exploited by breeding. The extreme variability within 900-bp upstream of the waxy1 transcription start site suggests these sequences may not contribute significantly to waxy1 expression. Transgenic maize lines where the waxy1 flanking noncoding sequences drive the expression of the GUS reporter protein have been characterized. The introduced waxy1 noncoding sequences program patterns of gene expression that are similar to those observed for the endogenous waxy1 gene. The impact of introducing the same noncoding sequences into genetic backgrounds with different levels of endosperm starch accumulation is in progress. Preliminary analysis indicates background-dependent differences in both the intensity and developmental onset of reporter gene expression, which reflect the relative starch concentrations in the mature grain. The basis for these differences is under investigation. Continued analyses of the type described here may reveal much about patterns of regulatory sequence variation
within maize and other cereal crop species.
Impacts
Starch synthesis in cereals is one of the most important metabolic pathways to world agriculture. This project employs a comparative genomics approach to identify candidate regulatory elements in cereal starch biosynthesis genes. An improved understanding of the molecular mechanisms that regulate starch biosynthesis gene expression may offer novel strategies for the modification of starch quantity and quality in cereal grains.
Publications
- Guo, H. and Moose, S.P. 2003. Conserved noncoding sequences among cultivated cereal genomes identify candidate regulatory sequence elements and patterns of promoter evolution. Plant Cell 15: 1143-1158.
- Moose, S.P. 2004. Comparisons of regulatory sequences in the cereals-implications for breeding. Proceedings of the 40th Annual Illinois Corn Breeder's School.
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Progress 01/01/05 to 12/31/05
Outputs
The project has now moved into the final stage of functionally testing promoter sequences in transgenic maize. Transgenic maize lines where the waxy1 (Wx1) flanking sequences drive the expression of the GUS reporter protein have been characterized. The introduced Wx1 noncoding sequences program patterns of gene expression that are similar to those observed for the endogenous Wx1 gene. The transgenes are specifically active in the endosperm and pollen, and reporter gene expression is highest during the period of maximal starch deposition in the endosperm. The impact of introducing the same noncoding sequences into genetic backgrounds with different levels of endosperm starch accumulation is in progress. Preliminary analysis indicates background-dependent differences in both the intensity and developmental onset of reporter gene expression, which reflect the relative starch concentrations in the mature grain. The basis for these differences is under investigation.
Continued analyses of the type described here may reveal much about patterns of regulatory sequence variation within maize and other cereal crop species.
Impacts
Starch synthesis in cereals is one of the most important metabolic pathways to world agriculture. This project employs a comparative genomics approach to identify candidate regulatory elements in cereal starch biosynthesis genes. An improved understanding of the molecular mechanisms that regulate starch biosynthesis gene expression may offer novel strategies for the modification of starch quantity and quality in cereal grains.
Publications
- No publications reported this period
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Progress 01/01/04 to 12/31/04
Outputs
Due to the extremely high variability observed within noncoding sequences between maize, rice, wheat, barley, and sorghum, the comparative approach to identify important regulatory sequences encountered difficulties. Because sequences for the noncoding regions of the starch biosynthetic genes for a number of different maize genotypes were available, we shifted our emphasis to comparisons of noncoding sequence variation within maize. An initial study focused on sequences of promoter regions for the brittle2 (bt2), shrunken2 (sh2), and waxy1 (wx1) genes, which were obtained from 32 maize inbred lines selected to represent the majority of allelic diversity in maize breeding lines. The bt2 promoter sequences exhibited very little variation among the 32 inbred lines, where only three haplotypes were observed due to five single nucleotide polymorphisms (SNPs). Sh2 promoter sequences showed greater variability compared to bt2, where nine haplotypes resulting from 14 SNPs and
four insertion-deletions (indels) were observed in the 32 inbred lines. The allelic relationships estimated from the promoter sequences do not correlate well with the genetic relationships among the inbred lines estimated from pedigree and genotyping data. The allelic variability at sh2 indicates that a number of sh2 promoter haplotypes are functionally interchangeable in agronomically important lines. If particular sh2 promoter haplotypes or their combinations are indeed significantly superior to another for grain yield, then opportunities exists to test this through directed breeding. The wx1 gene represents an extreme case of promoter sequence variability, where essentially all 30 inbred lines have a unique haplotype, caused primarily by different combinations of 16 indels ranging from 5-40bp in length. Each of these wx1 alleles are qualitatively functional (e.g. no visible mutant phenotype), but potential quantitative variability in wx1 expression has not yet been directly
examined. Another interpretation of the extreme variability in the wx1 promoter is that the 900-bp sequence immediately flanking the wx1 transcription start site does not contribute to wx1 expression and hence is neutral to wx1 function. This latter hypothesis is supported by the observations that no significant CNS were found in comparisons of maize and rice wx1 and sequences beyond 900-bp upstream of wx1 were necessary to program reporter gene expression in transgenic maize and rice similar to that observed for the endogenous wx1 genes. Continued analyses of the type described here may reveal much about patterns of regulatory sequence variation within maize and other cereal crop species.
Impacts
The yield, nutritional content, and processing qualities of cereal grains are largely determined by the amount and composition of starch within the seed. Despite the fact that many of the genes that participate in starch biosynthesis within cereal seeds are known and their enzymatic properties are fairly well understood, little is known about how the expression of these genes is coordinately regulated during seed development. This research employs a comparative genomics approach to identify candidate promoter regulatory elements in cereal starch biosynthesis genes. Our results show that promoter sequences are evolving rapidly in the cereals, limiting the utility of the comparative approach. Including sequences from more than two cereal species greatly improves success and has led to the identification of candidate regulatory sequences in the promoters of the corn shrunken2 gene and its orthologs. Further analyses of these sequences will improve our understanding of
the molecular mechanisms that regulate starch biosynthesis gene expression in corn and rice, two of the most important cereal crops. The results from this research may offer novel strategies for the modification of starch quantity and quality in cereal grains.
Publications
- Moose, S.P. 2004. Comparisons of regulatory sequences in the cereals-implications for breeding. Proceedings of the 40th Annual Illinois Corn Breeders School. pp. 65-76. http://imbgl.cropsci.uiuc.edu/index.html.
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Progress 01/01/03 to 12/31/03
Outputs
This research has two objectives. The first is to evaluate the utility of performing cross-species comparative sequence analyses of orthologous cereal genes to identify sequences important for regulation of gene expression using the starch biosynthetic genes as a model system. The second is to characterize the interactions between protein regulatory factors and DNA sequences that regulate the expression of starch biosynthetic genes in cereal grains. Genomic DNA sequences from 13 different sets of orthologous starch biosynthesis genes from corn and rice were obtained from Genbank. Sequences from barley, sorghum and wheat were also obtained. PCR-based strategies were used to clone the promoter sequences from the corn dull1 and brittle1 genes. Using these sequences and a larger dataset with 68 additional corn-rice orthologous gene pairs, we determined that the VISTA algorithm (http://www-gsd.lbl.gov/vista/) had the highest frequency of success in predicting known
regulatory motifs and the lowest background. Further evaluations with > 300 cereal genes showed that the minimum criteria for a conserved promoter sequence is at least 20-bp in length for two-way comparisons or the presence of the sequence in at least three different species. Fourteen such elements were found in comparisons of nine corn and rice starch biosynthesis genes, with four of these also being present in the shrunken2 orthologs. Tests were performed to assess whether similar computational approaches might identify promoter sequence motifs that are conserved among different genes within the starch biosynthetic pathway, which would be candidates for coordinate regulation of these genes during endosperm development. Two such candidate elements were identified, one being common to the sugary1 and starch branching enzyme 2a genes and the other shared between the shrunken1 and shrunken2 genes from both corn and rice. These studies established baseline criteria by which comparisons
of orthologous corn and rice gene promoter sequences can be employed to identify regulatory elements that contribute to conserved patterns of gene expression. This work also demonstrated that promoter sequences are rapidly evolving among grass species, which has important implications for the further application of comparative sequence approaches and for mechanisms of evolution among cereal genes.
Impacts
The yield, nutritional content, and processing qualities of cereal grains are largely determined by the amount and composition of starch within the seed. Despite the fact that many of the genes that participate in starch biosynthesis within cereal seeds are known and their enzymatic properties are fairly well understood, little is known about how the expression of these genes is coordinately regulated during seed development. This research employs a comparative genomics approach to identify candidate promoter regulatory elements in cereal starch biosynthesis genes. Our results show that promoter sequences are evolving rapidly in the cereals, limiting the utility of the comparative approach. Including sequences from more than two cereal species greatly improves success and has led to the identification of candidate regulatory sequences in the promoters of the corn shrunken2 gene and its orthologs. Further analyses of these sequences will improve our understanding of
the molecular mechanisms that regulate starch biosynthesis gene expression in corn and rice, two of the most important cereal crops. The results from this research may offer novel strategies for the modification of starch quantity and quality in cereal grains.
Publications
- Guo, H. and Moose, S.P. 2003. Conserved noncoding sequences among cultivated cereal genomes identify candidate regulatory sequence elements and patterns of promoter evolution. Plant Cell 15: 1143-1158.
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Progress 01/01/02 to 12/31/02
Outputs
This research has two major objectives. The first is to evaluate the utility of performing comparative sequence analyses between corn and rice to identify sequences important for regulation of gene expression in cereals, using the starch biosynthetic genes as a model system. The second objective is to characterize the interactions between protein regulatory factors and DNA sequences that regulate the expression of starch biosynthetic genes in cereal grains. We have made progress towards each of these objectives. Genomic DNA sequences from 13 different sets of orthologous starch biosynthesis genes from corn and rice were obtained from Genbank. Where available, sequences from barley, sorghum and wheat were also obtained. PCR-based strategies resulted in the cloning of promoter sequences from the maize dull1 and brittle1 genes. Using these sequences as well as a larger dataset that included 68 additional maize-rice orthologous gene pairs, it was determined that the VISTA
algorithm (http://www-gsd.lbl.gov/vista/) had the highest frequency of success in predicting known regulatory motifs and the lowest background. Further evaluations showed that the minimum criteria for a conserved promoter sequence is at least 20-base pairs in length for two-way comparisons or the presence of the sequence in at least three different species. Fourteen such elements were found in comparisons of nine corn and rice starch biosynthesis genes, with four of these also being present in the shrunken2 orthologs from sorghum. Tests were also performed to assess whether similar computational approaches might be able to find promoter sequence motifs that are conserved among different genes within the starch biosynthetic pathway, which would be candidates for coordinate regulation of these genes during endosperm development. Two such candidate elements were identified, one being common to the sugary1 and starch branching enzyme 2a genes and the other shared between the shrunken1 and
shrunken2 genes from both corn and rice. Thus, the above studies established baseline criteria by which comparisons of orthologous corn and rice gene promoter sequences can be employed to identify regulatory elements that contribute to conserved patterns of gene expression. It is expected that this approach can be extended to any pair of orthologous corn and rice genes. Those sequence elements conserved between corn and rice gene promoters are being tested for their ability to interact with endosperm nuclear proteins. Preliminary results using gel shift assays have identified DNA-protein interactions with a sequence element from the waxy promoter that is conserved between corn and rice. Similar experiments are in progress for conserved sequence elements identified within the promoters of the shrunken2, sugary1, and starch branching enzyme1 genes.
Impacts
Starch synthesis in cereals is one of the most important metabolic pathways to world agriculture. The yield, nutritional content, and processing qualities of cereal grains are largely determined by the amount and composition of starch within the seed. Despite the fact that many of the genes that participate in starch biosynthesis within cereal seeds are known and their enzymatic properties are fairly well understood, little is known about how the expression of these genes is coordinately regulated during seed development. This research intends to improve our understanding of the molecular mechanisms that regulate starch biosynthesis gene expression in corn and rice, two of the most important cereal crops. The results from this research may offer novel strategies for the modification of starch quantity and quality in cereal grains.
Publications
- No publications reported this period
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