1. Metabolic and Regulatory Aspects of Seed Quality and Germination. Elucidation of biochemical and developmental pathways which have a key role in seed development, germination and nutritional quality form the basis of the research conducted in the Dyer and Raboy laboratories, respectively.
Two approaches to studying starch biosynthesis are being taken. The first is to study the starch biosynthetic pathway at the biochemical and molecular levels. The genes which encode enzymes active in the cytosol and amyloplast and at the beginning, middle, and end of the starch biosynthetic pathway (sucrose synthase, ADPglucose pyrophosphorylase, and granular-bound starch synthase) have been cloned from maize, spinach, wheat, rice, and potato (Nakata et al. 1991; Olive et al. 1989; Preiss 1988; Shure et al. 1983; Werr et al. 1985; Yu et al. 1992). Consequently, using heterologous probes and PCR-based cloning it is now possible to isolate these genes from barley and study the mechanisms of their regulation. The transcript levels of these genes will be correlated to enzyme activity levels during barley kernel development. This should indicate the degree of coordinant regulation of the starch biosynthetic pathway at the gene and enzyme levels at discreet developmental timepoints and throughout seed development. These studies will begin to define the pathway regulating starch synthesis during development. The promoters of these genes will also be analyzed which will lead to the identification of regulatory factors and genes which control starch biosynthesis. The second approach is to isolate mutants, through sodium azide mutagenesis (Nilan et al. 1975), which perturb starch synthesis or genes regulating starch synthesis. Shrunken seed mutations obtained from these populations will be examined biochemically to determine if the lesion is in a specific starch synthesis gene or at some undefined and perhaps regulatory loci. These studies will genetically and molecularly define the starch biosynthetic and regulatory pathways in barley kernels.
Our current objectives include the isolation and characterization of mutations which specifically perturb the accumulation of seed phytic acid, and the identification and purification of enzymes implicated in seed phytic acid synthesis. These are prerequisites to achieving various basic and applied long term goals. Our studies focus on three cereal crops; barley, wheat and maize. The specific objective of this proposal is to isolate and begin the study of barley phytic acid mutants.
We have isolated two maize mutants, termed low phytic acid 1 (lpa1) and lpa2, in which kernel phytic acid is reduced (by about 66% and 33%, respectively), but which display no other obvious change in kernel phenotype or plant growth habit (unpublished data). These mutants were found using a relatively time-consuming assay for phytic acid. In both cases the observed reduction in kernel phytic acid is essentially entirely accounted for by an increase in inorganic P (Pi). It is interesting that this massiveincrease in Pi has little or no effect on kernel phenotype. We have also identified 1L-myo-inositol 1-monophosphate kinase activity, which we hypothesize catalyzes an early step inphytic acid synthesis, in developing wheat kernels (unpublished data). Optimization of the recovery of this activity in crude extracts and the development of a purification scheme for this activity is currently in progress.
Based on the maize work, we propose to screen for phytic acid mutants in a population of chemically-induced mutants in barley (recently produced by Co-PI Anderson) by first screening for mutants which substantially increase kernel Pi. This approach should be relatively rapid. We will initially screen to identify M2 families containing kernels which display substantial increases in Pi above that typically observed in wild-type kernels, using a modification of the method of Chen et al (1956). M2 families thus identified will be further analyzed using a High Voltage Paper Electrophoresis (HVPE) methodwhich provides optimal fractionation of Pi, and the various myo-inositol phosphates including phytic acid (Raboy et al. 1990). Any phytic acid mutants thus identified will then be the subject of numerous studies:
1) to accurately quantify kernel phytic acid, phosphorus and mineral fractions; 2) to study their inheritance and map location; 3) to assay their effect on seed function and physiology including mineral distribution; 4) to study their biochemistry (do they represent lesions in kinase genes, or in other functions such as metabolite transport?).
The Dyer laboratory has recently shown using in vitro translation and two-dimensional gel electrophoresis that certain mRNAs and soluble proteins are more highly expressed in dormant Avena fatua embryos than in nondormant embryos during early imbibition (Dyer 1992). Differential expression is visible within 3 hours of imbibition and remains elevated through 12 hours, but dormancy-associated mRNAs return to equivalent levels in dormant and nondormant embryos after 24 hours. To isolate genes thus temporally regulated during early imbibition, cDNA libraries were constructed using mRNA isolated from dormant and nondormant embryos imbibed for 6 hours. After subtraction hybridization, 300 colonies were isolated based on differential hybridization, from which nine clones have been selected for preliminary studies. Comparison of the 375 bp open reading frame of clone S21G5 with DNA databases shows that the A. fatua clone possesses 54% identity in a 211 bp overlap with the open reading frame of a transmembrane transport protein (Van Hove et al. 1990) and 62% identity in a 71 bp overlap with a nuclear pore glycoprotein (Wozniak et al. 1989), suggesting the presence of membrane spanning domains.
Dormancy-associated cDNA clones will be characterized on three levels. Gene expression will be quantified in northern hybridization experiments to determine:
Future work on this project will emphasize analysis of the regulation and individual functions of the five Arabidopsis phytochromes. Using the polymerase chain reaction (PCR), phytochrome gene families homologous to the Arabidopsis family have been detected in a wide variety of flowering plants including the agriculturally-important monocot grain species. This is significant in that it suggests that much of what is learned from analysis of the phy genes in Arabidopsis should be applicable to higher plants in general. In Arabidopsis, all five phy genes are expressed at the level of mRNA and, using monoclonal antibodies, three of the five have been shown to be expressed at the protein level (Somers et al. 1991). One future goal in this laboratory is to generate additional monoclonal antibodies that are specific to each of the five phy gene protein products. Using transcript-specific nucleic acid hybridization probes and these antibodies, it will be possible to characterize in detail the tissue distribution and developmental regulation of the phy mRNAs and proteins.
In an effort to define functions for the phytochromes, levels of these receptors in various photomorphogenic mutants of Arabidopsis have been analyzed. A mutant showing aberrant red light regulation of seedling stem elongation was found to be specifically deficient for one of the low abundance light-stable phytochromes, phyB (Somers et al. 1991). Current work on this project involves molecular analysis of the mutant and transgenic complementation of the mutant phenotype with a recombinant phyB gene introduced into mutant plants via Agrobacterium-mediated transformation. Assignment of a physiological function in the control of seedling growth to phyB phytochrome is a major step forward in understanding this receptor family. Further analysis of the Arabidopsis phytochrome family will include attempts to specifically block expression of individual phy genes in transgenic plants using antisense RNA and investigation of the patterns of expression of the various phy genes using phy promoter regions fused to a reporter gene.
In a collaboration between the Stout and Sharrock laboratories, the role of calcium-mediated signal transduction in rapid phytochrome responses, in particular red-light induced leaf movement, will be investigated. Such a rapid physiological effect of phytochrome does not involve transcriptional regulation, thus phytochrome is modulating an intracellular system already in place. Phytochrome may act by elevating the cytosolic free Ca++ concentration (Ca++cyt). Secondary effects may include opening of calcium-gated potassium channels in the membrane (affecting turgor), reorientation of cytoskeletal elements (via the Ca++-modulated protein annexin), and the activation of elements of the inositol-phospholipid second messenger system (Leonard and Hepler 1990; Roberts and Harmon 1992). Using currently available techniques (such as injection of Ca++-sensitive fluorescent dyes) to determine Ca++cyt, the earliest effects of red light stimulation of Ca++cyt in the leaf motor cells will be examined in a variety of leguminous plants.