Location: Chemistry Research2012 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 developing maize seed contributes to more than 80% of the seed weight. We have identified basal endosperm transfer layer (BETL) as vital to normal seed development and seed mass accumulation. A global profile of proteins in normal and a mutant seed with aberrant BETL is now obtained using high throughput proteome technologies. The results show a total of approx. 2,500 proteins; of these 131 were differentially expressed in the mutant relative to the normal, including genes and proteins related to sugar metabolism, hormone biosynthesis and pathogen response during seed development. A better understanding of BETL will identify genes critical to seed mass determination, a unit of crop yields in maize. Developing maize seeds synthesize the highest levels of plant hormone auxin, indole-3-acetic-acid (IAA), of all plant organs; yet no IAA-deficient seed mutants are available to understand its role in various processes of developing seeds. We have now identified a single gene mutant, defective endosperm18 (de18) that is IAA-deficient, is greatly reduced in the expression of an IAA biosynthetic gene, and is also associated with the reduced seed mass. This is the first such functional demonstration of IAA in seed weight accumulation. Further studies on additional IAA mutants/genes in seeds will lead to identification of new genes of agronomic significance. A microarray experiment was performed to examine downstream responses regulated by ZmPep peptides. Results uncovered an array of defense genes. Subsequent validation of microarray data by quantitative polymerase chain reaction (qPCR), metabolite analysis and bioassay revealed the ZmPeps are important regulators of maize defense against herbivores. Two candidate receptors for ZmPep, ZmPEPR1 and ZmPEPR2, were identified in silico via homology to known AtPEPR receptors. Both were cloned and heterologous expression assays with ZmPEPR1 demonstrated a functional interaction with all five ZmPep peptides.
1. The Miniature1 (Mn1) gene has multiple diverse effects in seed development in maize. All seeds are composed of two major components, endosperm – a storage tissue, and embryo – a source of next generation of the crop. These two divergent tissues are believed to be autonomous in development. However, we observed that the two are highly inter-dependent throughout seed development. How such interaction is manifested is unknown. Gene expression studies on the Mn1 and mn1 seeds show an underlying inter-connected network of genes that are coordinately regulated by the Mn1 gene. The discovery of such a control by a single metabolic gene is significant because the Mn1 gene regulates seed mass, a major yield trait and a unit of crop productivity.
2. Cytokinin (CK) in seed development. CK is a major plant hormone with a critical role in cell division, and numerous other functions in a plant. In developing seeds, it may be causal to the increased sink size through greater number of cells; thus it is critical to understand how and where it is synthesized. Collective results obtained by the ARS Researchers at Gainesville, FL from biochemical, cellular and molecular studies show that a large proportion of CK is transported from maternal tissues, in addition to a local synthesis in developing seeds. Our previous studies have shown developmentally controlled programmed cell death (PCD) of unknown mechanism that removes certain cells in a developing seed. Because of the cellular co-localization of high CK levels and the PCD, it is hypothesized that these two phenomena are inter-dependent and are essential to facilitate greater transport of water and nutrition to a developing seed. The role of such antagonistic functions by CK, cell division and cell destruction, in normal seed development is of fundamental significance in understanding seed development.
3. Identification of a novel mechanism regulating maize anti-herbivore defenses. Damage caused by insect herbivores results in yield losses and renders plants more susceptible to infection by fungal pathogens; including those which produce mycotoxins that can contaminate grain used for food or livestock feed. Knowledge of molecular mechanisms regulating defenses against herbivores in crop plants is desirable but little is known. The maize protein signal ZmPep3 induces a number of anti-herbivory defense responses including proteinase inhibitors, benzoxazinoids and volatile terpenes. Topical ZmPep3 application conferred enhanced resistance to caterpillar damage. Results from studies using ZmPep3 indicate that this protein and related proteins in a number of other crop species can be used to activate plant anti-herbivore defenses and increase plant resistance to herbivore damage and subsequent pathogen infection.
4. Identification of receptors interacting with ZmPep peptide signals. Signal peptides and their receptors initiate plant responses by working together like a key in a lock. Knowledge of how a signal and its receptor interact is highly desirable and permits manipulation of plant responses such as enhancing resistance to pathogens and herbivores. Despite the desirability of this knowledge, only a very few signal-receptor interactions in plants are known. Our discovery of the maize receptors that interact with the ZmPep peptides elucidates a critical mechanism employed by maize to activate defense responses. These research findings provide a novel biochemical mechanism that may be manipulated to help maize plants defend themselves against both herbivores and pathogens.
Chourey, P.S., Li, Q., Cevallos-Cevallos, J. 2011. Pleiotropy and its limited dissection through a metabolic gene Miniature1 (Mn1) that encodes a cell wall invertase in developing seeds of maize. Plant Science. 184:45-53.