Progress 07/15/06 to 07/14/07
Outputs
OUTPUTS: We have created transciptome profiles that correlate with the regulatory role of the phytohormones ABA and GA in maize embryogenesis. A database of these gene expression profiles has been created that links genes, genotypes, developmental stages and probable gene functions. As seed development involves both embryogenesis genes and the distinctive metabolic pathways that create a germination-competent, mature seed, our database will provide the research community with information of agricultural as well as general botanical interest. Another aspect of our recent research output was the creation of genetic stocks to analyze the roles of multiple ROP GTPases on maize kernels. Triple, quadruple and quintuple mutants covering the Type II ROPS were produced as segregating stocks. These developments are of interest to other plant geneticists and molecular biologists. Our work was reported at two scientific meetings: NRI PD's meeting: Washington DC March 12-14 2007 Maize Genetics
Meeting: Chicago 22-24, 2007 Abstracts of the presentation were published for both meetings.
PARTICIPANTS: Kirstin A. Carroll, postdoctoral trainee in Rivin lab, OSU, was in charge of conducting microarray experiments and compiling microarray data. Doris Kulhanek, technician in Rivin lab, OSU, was in charge of production of embryo samples, and validation of microarray results using qRT-PCR. Carol Rivin, PD, OSU, was in charge of genetic experiments involving hormone mutants, and oversaw all molecular biology aspects of the project. John Fowler, co-PD,OSU, was in charge of genetic experiments involving Rop mutants, and oversaw all microarray annotation and database production. Matias Kirst, Assist. Professor at University of Florida, has begun to collaborate with us on statistical analysis of microarray experiments. Alice Barkan, Professor at University of Oregon, has collaborated with us on the creation of an annotated database of maize microarray information. Four undergraduate students conducted genetic and molecular analyses for this project.
TARGET AUDIENCES: Our research and output is aimed at other plant geneticists and developmental biologists. Our work will also be of interest to cereal breeders.
Impacts
Our research has been testing the hypothesis that the abscisic acid (ABA) hormone signaling that governs maturation in cereal embryos is antagonized by gibberellic acids (GA), another class of phytohormones. Our data strongly support the view that both ABA and GA act in maintaining maturation of the embryo, but that the interaction is probably not direct. By microarray gene profiling, we found that GA-deficient embryos have a remodeled transcriptome relative to genetically normal embryos, particularly with regard to genes for sugar metabolism enzymes and for genes associated specifically with maturation phase. From these studies, we have generated a new model of how cereal embryo maturation is regulated by the interplay of phytohormone and metabolic signaling. The interest in hormone-sugar interaction in this context is enhanced by the agricultural importance of cereal seeds; the events of maturation are what make grains both nutritious and storable, and failure of
seeds to mature properly results in significant loss of yield and quality. Moreover, a great fraction of the world's cereal crops are produced from from genotypes with significantly reduced GA signaling. These "Green Revolution" dwarf types were selected for their resistance to lodging, but there may be less obvious physiological and developmental effects on seed development. Dissection of the pathways in maize will be broadly applicable, since maize genes and genetic hierarchies often closely parallel those in other cereals.
Publications
- Carroll, K.A. and Rivin, C. 2007. Practical advice on using the maize oligonucleotide microarray. Maize Genetics Newsletter 81 pg 12
- Carroll, K.A., Kulhanek, D., Fowler, J. and Rivin, C. 2007. Microarray evidence for ABA - GA antagonism during embryo maturation. Maize Genetics Newsletter 81 pg 13
- Carroll, K.A., Kulhanek, D., Fowler, J. and Rivin, C. 2007 Regulatory Circuits in Maize Embryo Development: ABA, GA and the ROP GTPases. Abstract Book NRI PD's meeting, Washington DC, March 12-14, 2007.
- CCarroll, K.A., Kulhanek, D., Fowler, J. and Rivin, C. 2007 Interpreting Mixed Signals in Maize Maturation. Regulatory Circuits in Maize Embryo Development: ABA, GA and the ROP GTPases. Abstract Book Maize Genetics Meeting, Chicago March 22-24, 2007
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Progress 07/15/05 to 07/15/06
Outputs
ABA is a highly conserved hormone signal required to induce maturation phase in developing plant embryos. In maize, ABA-deficient kernels are viviparous and desiccation-intolerant at maturity. Our research program concerns the possible anagonistic interaction of ABA signaling in maturation phase with gibberellic acids (GAs) and a specific family of the Rop GTPases, both of which have been shown to antagonize ABA in other biological contexts. Our overall goal is to test two major hypotheses concerning how these signaling systems might interact to change gene expression and developmental capabilities of the maize embryo during maturation phase. Hypothesis 1. Growth and maturation of the maize embryo, including changes in gene expression, are regulated by ABA-GA balance. Maize embryos deficient in both ABA and GA exhibit the wild-type phenotypes of quiescence and desiccation tolerance, suggesting that the growth and maturation of the maize embryo, including changes in
gene expression are regulated by ABA-GA balance. The peak of active GA precedes that of ABA. We envision that GA signaling may either intercept the ABA signaling pathway to modulate ABA sensitivity, or participate in a negative regulatory mechanism to suppress maturation independently of ABA. This hypothesis is being tested using genomics and genetics approaches. First, we are using two-color microarray analysis and qRT-PCR to compare gene expression in wildtype, ABA-deficient (ABA-), GA-, and ABA-/GA- mutant embryos at pre-maturation and early maturation phases. Our initial experiments strongly support the ABA/GA balance hypothesis: Known maturation genes are much more highly expressed in the double mutant than they are in ABA- mutants. The microarray has also revealed a variety of genes not known to be ABA or maturation-related that show this same pattern. Overall, 89% of genes found to be differentially expressed between WT and ABA- sibling embryos were also significantly different
in ABA-/GA- vs ABA- comparisons. To identify components of a ABA-GA signaling and crosstalk circuit, we have also screened for new EMS mutants in which vivipary is restored in ABA-/GA- seeds. We have several candidate mutants that appear to segregate for this trait. Hypothesis 2: Group 2 Rop GTPases are part of the embryo maturation signaling circuit. This hypothesis was based on the observation that several Type II ROPs appeared to peak in expression with the onset of embryo maturation in maize, and that related ROPs have been shown to negatively regulate ABA signaling in Arabidopsis. 2). Our results do not confirm ROP expression previously reported, but three of the four rops show expression increases in GA- mutant and ABA-/GA- mutant samples, an unexpected result. The data do not fit into a simple model in which GA and ROP, as negative regulators of ABA signaling, would increase or decrease in parallel. Our next steps are to test if ROP protein or activity levels are affected by GA
or ABA, using an antibody we have developed. We have also developed multiply mutant rop lines to test whether removing specific ROPs affects embryo maturation and the proposed ABA/GA signaling circuit.
Impacts
This is a basic research project, not directed toward a direct agricultural goal. The project concerns how genes are regulated during seed maturation, the process that produces seeds that are both storable and nutritious. Our findings will be of interest to breeders and to those interested in engineering cereal crops for enhanced nutritional value, and more robust dormancy.
Publications
- No publications reported this period
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