Progress 09/15/02 to 09/14/05
Outputs
Compounds belonging to the 3-deoxyanthocyanidin class have structural similarities with flavan-4-ols and these include luteolinidin, apigeninidin and their derivatives. These compounds have been shown to accumulate as a site-specific response to the ingress of anthracnose fungus (Colletotrichum sublineolum) in sorghum leaves. The role and importance of sorghum 3-deoxyanthocyanidins in plant disease resistance has been well established but their genetics and regulation of biosynthesis was not very well understood. To study genetic and molecular mechanisms that lead to phytoalexins biosynthesis, we used several well defined sorghum genetic stocks that showed differential accumulation of phytoalexins. First, we focused on isolation and expression characterization of the sorghum transcription factor encoded by yellow seed1 (y1). Our results demonstrated that sorghum plants capable of producing 3-deoxyflavonoid pigments called phlobaohenes require a functional MYB type of
transcription factor encoded by a functional y1 (yellow seed1) gene. Sequence characterization of genomic region of sorghum yellow seed1 shows the presence of two genes which are arranged in a head to tail orientation. The two duplicated gene copies, y1 and y2 are separated by a 9.084 kbp intergenic region, which is largely composed of highly repetitive sequences. The y1 is the functional copy, while the y2 may represent a pseudogene. The y1 gene encodes a R2R3 type of Myb domain protein which regulates the expression of chalcone synthase, chalcone isomerase and dihydroflavonol reductase genes required for the biosynthesis of 3-deoxyflavonoids. Phylogenetic analysis indicates that sorghum y1 and y2 sequences may have arisen by gene duplication mechanisms and represent an evolutionarily parallel event to the duplication of maize homologous genes encoded by pericarp color1 (p1) and p2. Recent results indicate that induction of y1 gene may be responsible for the expression of target
genes including structural genes of the flavonoid and phytoalexin biosynthesis. Second, we focused on one of the biosynthetic enzymes and found that the activity of this cytochrome P450 monoxygenase encoded by a (flavonoid 3-prime-hydroxylase) gene is also required for phytoalexin synthesis. Heterologous expression of the F3-prime-H cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. Further, transcription of the flavonoid 3-prime-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase and expression of these genes was induced within the first 24 hours of fungal challenge. Third, further experiment that involve transfer of sorghum genes into transgenic maize are currently in progress in order to study the expression of flavonoids and phytoalexins in maize tissues.
Impacts
In plants, secondary metabolites play a significant role in growth and development as well as survival during abiotic and biotic stresses. This project has attempted to study the genetic basis of synthesis of a set of compounds that kill the anthracnose fungus and thus are known as phytoalexins. Results of this research are helping to clarify the biosynthetic pathway of phytoalexin compounds in sorghum. The long term impact of this project is to induce phytoalexin biosynthesis in maize leaves to provide resistance against anthracnose. This project has thus developed germplasm screening strategies in order to identify new maize and sorghum lines showing enhanced resistance to the anthracnose fungi. The project is also developing transgenic maize germplasm to directly study application of genes isolated from sorghum. The long term impact of this study will be to improve the ability of maize and other cereal crops to withstand environmental stress.
Publications
- Boddu, J., Svabek, C., Ibraheem, F., Jones, A. D., and Chopra, S. 2005. Characterization of a deletion allele of sorghum yellow seed1 showing loss of 3-deoxyflavonoids. Plant Sci. 169: 542-552.
- Chang, R., Chopra, S., and Peterson, P. 2005. Differential excision patterns of the En-transposable element at the A2 locus in maize relate to the insertion site. Mol. Genet. Genomics. 274: 189-195.
- Carvalho, C., Boddu, J., Zehr, U., Axtell, J., Pedersen, J., and Chopra, S. 2005. Functional characterization of excision and insertion events of Candystripe1 transposon using Y1 regulated pigmentation as a marker in sorghum. Genetica 124: 211-212.
- Boddu, J., Svabek, C., Sekhon, R., Gevens, A., Nicholson, R., Jones, D., Pedersen J., Gustine, D., and Chopra, S. 2004. Expression of a putative flavonoid 3-prime-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins, Physiol. Mol. Plant Path. 65: 101-113.
- Chopra, S. 2005. Tissue specific epigenetic regulation of gene expression of maize pericarp color1 alleles. In Am. Soc. Plant Biologists Abstracts. July 16-20, Seattle, WA. http://abstracts.aspb.org/pb2005/public/P70/8173.html.
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Progress 01/01/04 to 12/31/04
Outputs
When challenged by the anthracnose fungus, sorghum seedlings synthesize and series of compounds through biochemical modifications. Our results demonstrated that sorghum seedlings capable of producing 3-deoxy flavonoid pigments require a functional MYB type of transcription factor encoded by the y1 (yellowseed1) gene. Results indicate that induction of y1 gene may be responsible for the expression of target genes including structural genes of the flavonoid and phytoalexin biosynthesis. We have further focused on one of the biosynthetic enzymes and found that the activity of a cytochrome P450 monoxygenase encoded by a F3'H (flavonoid 3-hydroxylase) gene is also required for phytoalexin synthesis. We first isolated a differentially expressed partial cDNA whose characterization and comparison showed that this sequence corresponds to a flavonoid 3'-hydoxylase. Full length sequence characterization allowed us to establish that sorghum f3'h cDNA encodes a peptide of 517
amino acids that has domains conserved among cytochrome P450 proteins functioning in the flavonoid biosynthetic pathway. Heterologous expression of the f3'h cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. We further found that transcription of the flavonoid 3'-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase, and expression of these genes was induced within the first 24 hours of fungal challenge. These recent results demonstrate that synthesis of apigeninidin and luteolinidin followed the induced expression of the f3'h gene implicating its role in fungal induced expression of sorghum phytolaxins.
Impacts
The results of this research are helping to clarify the biosynthetic pathway of phytoalexin compounds in sorghum. The long term impact of this project is to induce phytoalexin biosynthesis in maize leaves to provide resistance against anthracnose.
Publications
- Sangar, V. 2003. Functional characterization of a Myb-transcription factor required for 3-deoxyflavonoid (phlobaphenes) biosynthesis in sorghum (Sorghum bicolor). MS thesis. The Pennsylvania State University, University Park, PA, 83 pp.
- Sangar, V. and Chopra, S. 2003. Functional analysis of a sorghum myb orthologous transcription factor promoter in transgenic maize. In Annual Maize Genetics Conference Abstracts. March 13-16, 2003, Lake Geneva, WI. 45:P112.
- Svabek, C. and Chopra, S. 2003. Identification and Expression of Flavonoid 3'-Hydroxylase in Maize and Sorghum. In Maize Genetics Conference Abstracts. March 13-16, 2003, Lake Geneva, WI. 45:P170.
- Jiang, C., Gu, J., Chopra, S., Gu, X., and Peterson, T. 2004. Ordered Origin of the Typical Two- and Three-Repeat Myb Genes. Gene 326: 13-22.
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Progress 01/01/03 to 12/31/03
Outputs
When challenged by the anthracnose fungus, sorghum seedlings synthesize and series of compounds through biochemical modifications. Our results showed that maize and sorghum differ in their tissue specific synthesis of 3-deoxyanthocyanidin phytoalexins. We are interested in the genetic and molecular mechanisms that are responsible for induction of these compounds. We have further screened sorghum germplasm and genetically well-defined lines to identify presence of flavonoid phytoalexins through HPLC and Mass Spectrometry. Our results demonstrated that the genotypes capable of producing 3-deoxy flavonoid pigments require a functional MYB type of transcription factor encoded by the y1 (yellowseed1) gene. Molecular characterization of the y1 gene showed that the y1 is orthologous to the maize p1 gene. Further genetic studies demonstrated that synthesis of 3-deoxyanthocyanidin phytoalexin compounds require activity of y1 gene (Chopra et al. 2002). We are currently testing
the precise role of the sorghum y1 gene in regulating different flavonoids in sorghum. We will be testing the expression properties of the sorghum y1 gene in maize transgenic plants and cell cultures to induce novel flavonoids in maize. These plant-pathogen interaction studies are facilitated by the generation and utilization of maize plants carrying promoter-GUS marker gene cassettes.
Impacts
The results of this research will help to define and clarify the biosynthetic pathway of flavonoid compounds in cereal grasses. This information will be needed in designing and breeding for novel crops that may require least amounts of fungicides and pesticides to tackle biotic stress.
Publications
- Chopra, S., Gevens, A., Svabek, C., Peterson, T., and Nicholson, R. 2002. Excision of the Candystripe1 transposon from a hyper-mutable Y1-cs allele shows that the sorghum Y1 gene controls the biosynthesis of both 3-deoxyanthocyanidin phytoalexins and phlobaphene pigments. Physiological and Molecular Plant Pathology 60, 321-330.
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Progress 01/01/02 to 12/31/02
Outputs
Preliminary results showed that maize is unable to produce certain flavonoid phytoalexins when challenged by the anthracnose fungus. We identified presence of these flavonoid phytoalexins in sorghum genotypes that are genetically capable of producing 3-deoxy flavonoid compounds under the regulation of the y1 (yellowseed1) gene. Further, we will be testing the expression properties of the sorghum y1 gene in maize transgenic plants and cell cultures to induce novel flavonoids in maize.
Impacts
The results of this research will help to define and clarify the biosynthetic pathway of flavonoid compounds in cereal grasses. This information will be needed in designing and breeding for novel crops that may require the least amounts of fungicides and pesticides to tackle biotic stress.
Publications
- No publications reported this period
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