Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of alpha-amylase-encoding gene expression in germinating seeds and cultured cells of rice.

Four alpha-amylase-encoding cDNA (alpha Amy-C) clones were isolated from a cDNA library derived from poly(A)+RNA of gibberellic acid (GA3)-treated rice aleurone layers. Nucleotide sequence analysis indicates that the four cDNAs were derived from different alpha Amy genes. Expression of the individual alpha Amy gene in germinating seeds and cultured suspension cells of rice was studied using gene-specific probes. In germinating seeds, expression of the alpha Amy genes is positively regulated by GA3 in a temporally coordinated but quantitatively distinct manner. In cultured suspension cells, in contrast, expression of the alpha Amy genes is negatively and differentially regulated by sugars present in the medium. In addition, one strong and one weak carbohydrate-starvation-responsive alpha Amy genes have been identified. Interactions between the promoter region (HS501) of a rice alpha Amy gene and GA3-inducible DNA binding proteins in rice aleurone cells were also studied. A DNA mobility-shift assay showed that the aleurone proteins interact with two specific DNA fragments within HS501. One fragment is located between nt -131 to -170 and contains two imperfect directly repeated pyrimidine elements and a putative GA3-response element. The other fragment is located between nt -92 to -130 that contains a putative enhancer sequence. The interactions between aleurone proteins and these two fragments are sequence-specific and GA-responsive.     

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.     

Relationship of ribonucleic Acid metabolism in embryo and aleurone to alpha-amylase synthesis in barley

RNA metabolism of embryo and aleurone of barley grains (Hordeum vulgare L. cv. Himalaya) was studied to elucidate the role of these tissues in the control of alpha-amylase synthesis and germination. The extent of (3)H-uridine incorporated into various RNA classes of the embryo during the first 12 hours of germination was low but constant. Subsequently, there was a rapid increase in RNA synthesis of all fractions. In the aleurones, after 16 hours, a gradual decrease in (3)H-uridine incorporation was observed, and by the time the synthesis of RNA in the aleurones had stopped, alpha-amylase level was at its highest in the grain.On transfer to accelerated aging conditions (43 C; 85% relative humidity), the grains lost their viability within 4 weeks. That this was due to a rapid deterioration of the embryo and not of the aleurone was apparent in studies on alpha-amylase formation, RNA metabolism, and ATP content in grains in various physiological states reported here. Results presented here also reveal a marked influence of the embryo and GA(3) on the quality of the newly synthesized RNAs. Aleurones which lacked the impulse of embryo or GA(3) were capable of synthesizing RNA but these RNAs were less heterodisperse than RNAs from aleurones which were under the influence of an embryo or GA(3).     

GAMYB controls different sets of genes and is differentially regulated by microRNA in aleurone cells and anthers

GAMYB is a component of gibberellin (GA) signaling in cereal aleurone cells, and has an important role in flower development. However, it is unclear how GAMYB function is regulated. We examined the involvement of a microRNA, miR159, in the regulation of GAMYB expression in cereal aleurone cells and flower development. In aleurone cells, no miR159 expression was observed with or without GA treatment, suggesting that miR159 is not involved in the regulation of GAMYB and GAMYB-like genes in this tissue. miR159 was expressed in tissues other than aleurone, and miR159 over-expressors showed similar but more severe phenotypes than the gamyb mutant. GAMYB and GAMYB-like genes are co-expressed with miR159 in anthers, and the mRNA levels for GAMYB and GAMYB-like genes are negatively correlated with miR159 levels during anther development. Thus, OsGAMYB and OsGAMYB-like genes are regulated by miR159 in flowers. A microarray analysis revealed that OsGAMYB and its upstream regulator SLR1 are involved in the regulation of almost all GA-mediated gene expression in rice aleurone cells. Moreover, different sets of genes are regulated by GAMYB in aleurone cells and anthers. GAMYB binds directly to promoter regions of its target genes in anthers as well as aleurone cells. Based on these observations, we suggest that the regulation of GAMYB expression and GAMYB function are different in aleurone cells and flowers in rice.     

Isolation and identification of glycosylphosphatidylinositol-anchored arabinogalactan proteins and novel beta-glucosyl Yariv-reactive proteins from seeds of rice (Oryza sativa)

Arabinogalactan proteins (AGPs) are highly glycosylated extracellular glycoproteins playing important roles in plant growth and development. We have previously reported the possibility that AGPs are involved in the induction of alpha-amylase by gibberellin (GA) in barley aleurone layers by using the beta-glucosyl Yariv reagent (beta-GlcY), which has been presumed to specifically bind AGPs. In this present study, we isolated beta-GlcY-reactive proteins from rice bran rich in aleurone cells. The N-terminal sequences of classical AGP and AG peptides were determined from hydrophilic fractions obtained by reversed phase HPLC. Interestingly, a novel non-specific lipid transfer protein-like protein (OsLTPL1) and a novel early nodulin-like protein (OsENODL1) were also identified in the more hydrophobic fractions from HPLC as beta-GlcY-reactive proteins. Expression analysis of the genes coding for these proteins was performed. While classical AGP, AG peptides and OsLTPL1 were expressed in various parts of rice, OsENODL1 showed temporally and spatially specific expression in the aleurone layers. This new beta-GlcY-reactive protein is a promising candidate for the extracellular signaling factors of GA action in cereal seeds. Furthermore, the possibility that proteins with the AG glycomodule might react with beta-GlcY may broaden the definition of AGPs.