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United States Department of Agriculture

Agricultural Research Service

Research Project: FUNCTIONAL GENOMICS OF AGRONOMIC TRAITS IN DEVELOPING SEED AND POLLEN IN MAIZE AND SORGHUM
2011 Annual Report


1a.Objectives (from AD-416)
1. Identify and analyze critical genes in sugar metabolism and their relationshp to sugar-hormone signaling during maize seed development, particularly in basal endosperm transfer cells. (NP 301, C4, PS 4B). 1a. Phytohormones and sugar profiles in developing seeds of maize. 1b. Identification of genes in sugar – hormone cross-talk in developing endosperm. 1c. Gene discovery in Basal Endosperm Transfer Layer: 1d. Develop physiological, biochemical, molecular and genetic information and resources that can be used to determine the genetic basis of fungal infection in corn. 2. Determine the bases for defective pollen biogensis, including aberrations in sugar-starch metabolism, associated with heat stress and cytoplasmic male steriligy in sorghum. (NP 301, C4, PS 4B).


1b.Approach (from AD-416)
Developmental profiles of various phytohormones in developing seeds of normal (wild type) and several carbohydrate mutants of known genetic bases in maize will be developed using high throughput chemical approaches, including gas chromatography / mass spectrometry (GC-MS). Contemporary genomic approaches will be used to identify genes that are critical to sugar-hormone cross-talk, especially those related to hormone metabolism, transcription factors and proteins that function as receptors and/or response factors. Such genes in developing seeds will be further analyzed in expression studies using both microarray and single gene approaches to dissect gene networks that may control normal seed development and sink strength, the two most critical components of crop yields. Gene discovery studies based on transcriptome and proteome approaches will be initiated to obtain a functional genomic profile of the Basal Endosperm Transfer Layer (BETL), a highly specialized cell layer known be critical for transport and signaling functions in developing seeds. The emphasis in studying pollen biogenesis in sorghum is to understand the base for defective biochemical, molecular and physiological processes (including aberrations in sugar-starch metabolism) associated with heat stress and cytoplasmic male sterility (CMS). Profiles of differentially expressed genes that characterize the expression of CMS, the restoration of male fertility and heat-induced pollen inviability will be obtained and analyzed through contemporary transcriptome and proteome technologies. 1d. Novel maize peptides associated with pathogen attack will be identified through mining the maize genome sequence for homologs of the defense-regulating peptide AtPep1. Peptides will be biochemically isolated and/or synthesized and applied to manipulate and probe mechanisms of maize defense responses. Genes induced by biotic attack or peptide treatment will be identified through microarray experiments and expression patterns will be characterized and quantified through real-time PCR analysis. Chemical defenses and metabolites induced by biotic attack and/or peptide treatment will be characterized and measured by HPLC, LC-MS, GC-MS and NMR. Differences in gene expression or defense metabolite accumulation in different maize varieties will be assessed using resistant versus susceptible cultivars. The effects of peptide-induced defenses on invading organisms will be assessed through in vivo pathogenicity assays, and in vitro antimicrobial assays. Assays of in vivo and in vitro effects of maize defense responses on mycotoxin production will also be examined through LC-MS and GC-MS. Transgenic plants with knocked out or enhanced expression of candidate signaling genes will be used to delineate signaling mechanisms regulating defense responses to biotic stress.


3.Progress Report
Endosperm in maize seed is of immense economic significance. Of the four cell types in an endosperm, basal endosperm transfer layer (BETL) is vital to normal seed development but very little is known on its structure and function. We developed a micro-dissection method to isolate BETL and performed high throughput molecular and bioinformatics studies which cataloged ~2,500 genes that are preferentially expressed in these cells. These data provide an unprecedented profile on developmental and physiological roles of these cells in seed development.

Auxin, indole-3-acetic-acid (IAA), is a major plant hormone that controls nearly all aspects of plant development, including cell division and cell elongation, the major determinants of normal seed development/seed size and, ultimately, the crop yield. However, very little is known regarding genes and enzymes of IAA biosynthesis in seeds. Our genetic and functional genomic studies based on two IAA-deficient seed mutants have identified three new genes, however, expression pattern of only one of them correlated with the IAA profile.

Studies were initiated to determine whether a novel maize protein, ZmPep1, regulates plant defense responses. Results indicated that ZmPep1-treated plants have increased disease resistance as quantified by changes in levels of hormones, gene expression, defense metabolite production and symptom development.

A novel family of defense metabolites was discovered in fungal-infected maize plants and functionally characterized. Results showed that these defense metabolites accumulate to very high levels in diseased tissues and have antimicrobial activity against several fungal pathogens of maize.


4.Accomplishments
1. A metabolic gene Miniature1 (Mn1) has multiple diverse effects in seed development in maize. All seeds are composed of two major components, endosperm and embryo (germ); the former is a major source of food, feed and fuel in maize and the latter is the source of next generation of crop, and the two are believed to be autonomous in development. However, our data show unambiguously that the two tissues were highly inter-dependent; i.e., reduced mass of the mn1 mutant endosperm led to greatly reduced embryo throughout seed development. How such interaction is manifested is unknown. Additionally, gene expression studies in the Mn1 and mn1 genotypes show an underlying inter-connected network of genes that appear to be coordinatedly regulated by the Mn1 gene. The discovery of such a control is significant because the Mn1 gene regulates seed mass, a major yield trait and a unit of crop productivity.

2. Identification of a novel mechanism regulating maize pathogen defense. Maize is susceptible to a number of fungal pathogens including those which produce mycotoxins that can contaminate grain used for food or livestock feed. Quantitative resistance which is effective against a broad spectrum of fungal pathogens is a desirable but elusive trait in commercial crops. The maize protein signal ZmPep1 induces a number of defense responses and protects against diverse fungal pathogens. Results from studies using ZmPep1 indicate that this protein and related proteins in a number of other crop species can be used to activate plant defense and increase plant resistance to a diversity of pathogens.

3. Identification of novel antimicrobial metabolites produced by maize. The mechanisms used by maize plants to protect against fungal attack are not well understood. The discovery of these antimicrobial compounds, a class of terpenoid phytoalexins termed zealexins revealed a critical defensive strategy employed by maize. These research findings provide a novel biochemical mechanism that may be manipulated to help maize plants defend themselves. Future work will focus on optimization of when and where the plant produces these antimicrobial compounds to protect against fungal pathogens and enhance disease resistance.


Review Publications
Huffaker, A., Dafoe, N.J., Schmelz, E.A. 2011. ZmPep1, an ortholog of Arabidopsis elicitor Peptide 1, regulates maize innate immunity and enhances disease resistance. Plant Physiology. 155(30):1325-1338.

Vaughan, M.M., Tholl, D., Tokuhisa, J.G. 2011. An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana. Plant Methods. 7(5):1-10.

Yamaguchi, Y., Huffaker, A. 2011. Endogenous peptide elicitors in higher plants. Current Opinion in Plant Biology. 14:351-357.

Chourey, P.S., Li, Q., Kumar, D. 2010. Sugar - hormone crosstalk in seed development: Two redundant pathways of IAA biosynthesis are regulated differentially in the invertase-deficient miniature1 (mn1) seed mutant in maize. Molecular Plant. 3:1026-1036.

Rijavec, T., Jain, M., Dermastia, M., Chourey, P.S. 2010. Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopsis of maize. Annals Of Botany. 107:1235–1245.

Xiong, Y., Li, Q., Kang, B., Chourey, P.S. 2011. Discovery of Genes Expressed In Basal Endosperm Transfer Cells in Maize Using 454 Transcriptome Sequencing. Plant Molecular Biology Reporter. 29:835-847.

Ni, X., Wilson, J.P., Buntin, G., Guo, B., Krakowsky, M.D., Lee, R., Cottrell, T.E., Scully, B.T., Huffaker, A., Schmelz, E.A. 2011. Spatial patterns of aflatoxin levels in relation to ear-feeding insect damage in pre-harvest corn. Toxins. 3(7):920-931.

Huffaker, A., Kaplan, F., Vaughan, M.M., Dafoe, N.J., Ni, X., Rocca, J.R., Alborn, H.T., Teal, P.E., Schmelz, E.A. 2011. Novel acidic sesquiterpenoids constitute a dominant class of pathogen-induced phytoalexins in maize. Plant Physiology. 156:2082-2097.

Last Modified: 10/21/2014
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