The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley

The bifunctional alpha-amylase/subtilisin inhibitor (BASI) is an abundant protein in barley seeds, proposed to play multiple and apparently diverse roles in regulation of starch hydrolysis and in seed defence against pathogens. In the Triticeae, the protein has evolved the ability to specifically inhibit the main group of alpha-amylases expressed during germination of barley and encoded by the amyl gene family found only in the Triticeae. The expression of the asi gene that encodes BASI has been reported to be controlled by the hormones abscisic acid (ABA) and gibberellic acid (GA). Despite many studies at the gene and protein level, the function of this gene in the plant remains unclear. In this study, the 5'-flanking region (1033 bp, 1033-asi promoter) and the 3'-flanking region (655 bp) of the asi gene were isolated and characterised. The 1033-asi promoter sequence showed homology to a number of ciselements that play a role in ABA and GA regulated expression of other genes. With a green fluorescent protein gene (gfp) as reporter, the 1033-asi promoter was studied for spatial, temporal and hormonal control of gene expression. The 1033-asi promoter and its deletions direct transient gfp expression in the pericarp and at low levels in mature aleurone cells, and this expression is not regulated by ABA or GA. In transgenic barley plants, the 1033-asi promoter directed tissue-specific expression of the gfp gene in developing grain and germinating grain but not in roots or leaves. In developing grain, expression of gfp was observed specifically in the pericarp, the vascular tissue, the nucellar projection cells and the endosperm transfer cells and the hormones ABA or GA did not regulate this expression. In mature germinating grain gfp expression was observed in the embryo but not in aleurone or starchy endosperm. However, GA induced gfp expression in the aleurone of mature imbibed seeds from which the embryo had been removed. Expression in maternal rather than endosperm tissues of the grain suggests that earlier widespread assumptions that the protein is expressed largely in the endosperm may have been largely based on analysis of mixed grain tissues. This novel pattern of expression suggests that both activities of the protein may be primarily involved in seed defence in the peripheral tissues of the seed.     

Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible <Emphasis Type="Italic">WRKY</Emphasis> gene

It is well known that abscisic acid (ABA) antagonizes gibberellin (GA)-promoted seed germination. Recent circumstantial evidence suggests that salicylic acid (SA) also inhibits seed germination in maize and Arabidopsis. Our study shows that SA blocks barley seed germination in a dosage dependent manner. As an initial effort to addressing the mechanism controlling the crosstalk of SA, GA and ABA signaling in barley, we studied the regulation of α-amylases by SA and a WRKY gene whose expression is modulated by these hormones. Assays of α-amylase activity reveal that GA-induced α-amylase production in aleurone cells is inhibited by bioactive SA, but not its analogs, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid. This inhibitory effect is unlikely due to repressing α-amylase secretion or inhibiting α-amylase enzyme activities. Northern blot analyses indicate that SA suppresses GA-induced expression of a barley low pI α-amylase gene (Amy32b). Because our previous data indicate that ABA-inducible and GA-suppressible WRKY genes inhibit the expression of α-amylase genes in rice, we studied the steady state mRNA levels of a barley WRKY gene, HvWRKY38. The expression of HvWRKY38 in barley aleurone cells is down-regulated by GA, but up-regulated by SA and ABA. However, the regulation of HvWRKY38 by SA appears to be different from that of ABA in term of the kinetics and levels of induction. Over-expression of HvWRKY38 in aleurone cells by particle bombardment blocks GA induction of the Amy32b promoter reporter construct (Amy32b-GUS). Therefore, HvWRKY38 might serve as a converging node of SA and ABA signal pathways involved in suppressing GA-induced seed germination. Zhen Xie and Zhong-Lin Zhang contributed equally to this work.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Genome‐wide identification and characterization of microRNAs responding to ABA and GA in maize embryos during seed germination

• MicroRNAs (miRNAs) are an important class of non‐coding small RNAs that regulate the expression of target genes through mRNA cleavage or translational inhibition. Previous studies have revealed their roles in regulating seed dormancy and germination in model plants such as Arabidopsis thaliana, rice (Oryza sativa) and maize (Zea mays). However, the miRNA response to exogenous gibberellic acid (GA) and abscisic acid (ABA) during seed germination in maize has yet to be explored.
• In this study, small RNA libraries were generated and sequenced from maize embryos treated with GA, ABA or double‐distilled water as control.
• A total of 247 miRNAs (104 known and 143 novel) were identified, of which 45 known and 53 novel miRNAs were differentially expressed in embryos in the different treatment groups. In total, 74 (37 up‐regulated and 37 down‐regulated) and 55 (23 up‐regulated and 32 down‐regulated) miRNAs were expressed in response to GA and to ABA, respectively, and a total of 18 known and 38 novel miRNAs displayed differential expression between the GA‐ and ABA‐treated groups. Using bioinformatics tools, we predicted the target genes of the differentially expressed miRNAs. Using GO enrichment and KEGG pathway analysis of these targets, we showed that miRNAs differentially expressed in our samples affect genes encoding proteins involved in the peroxisome, ribosome and plant hormonal signalling pathways.
• Our results indicate that miRNA‐mediated gene expression influences the GA and ABA signalling pathways during seed germination.

     

Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling.     

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes

Lettuce (Lactuca sativa ‘Salinas’) seeds fail to germinate when imbibed at temperatures above 25°C to 30°C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.     

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.     

Biochemical and molecular characterization of three barley seed proteins with antifungal properties

We have purified three proteins from barley (Hordeum vulgare L.) seeds which synergistically inhibit the growth of fungi measured in a microtiter well assay. The proteins are a 26-kDa chitinase, a 30-kDa ribosome-inactivating protein, and a 32-kDa (1-3)-beta-glucanase. Full-length cDNAs encoding them were isolated and sequenced to determine the complete primary structures of the proteins. Northern hybridizations with the cDNAs as probes showed that the corresponding mRNAs accumulate differentially during seed development and germination. Chitinase mRNA accumulates to high levels in aleurone cells during late seed development and early germination, while high levels of mRNA encoding the ribosome-inactivating protein accumulate only in the starchy endosperm during late seed development. The glucanase mRNA accumulates to low levels during seed development and to higher levels in aleurone and seedling tissues during germination. Southern hybridizations showed that the three proteins are encoded by small families of three to eight genes. Their biological roles and potential use in genetic engineering studies are discussed.     

Abscisic acid and gibberellic acid-regulated responses of embryos and aleurone layers isolated from dormant and nondormant barley grains

Dormant and nondormant isogenic barley grains were obtained by maturing grains under short day (SD) or long day (LD) growth conditions, respectively. Hormonal responses of isolated embryos and aleurone layers from these grains were studied. Addition of abscisic acid (ABA) reduced germination rate and percentage of embryos, and induced Rab (ABA-responsive) mRNA in aleurone layers from both types of grain. Embryos and aleurone layers from dormant grains responded stronger to ABA than those from nondormant grains. Gibberellic acid (GA₃) increased the germination rate and percentage of embryos from dormant grains and counteracted the ABA-induced inhibition of embryo germination. GA₃ did not affect the amount of Rab mRNA in aleurone layers, suggesting that expression of the Rab gene has no direct correlation with germination. The stronger response of embryos and aleurone layers from dormant grains to ABA may not be explained by higher endogenous ABA levels, but might be due to differences in hormone signal transduction. Aleurone protoplasts from dormant grains had a higher cytosolic pH than those from nondormant grains. To inhibit the ABA-induced Rab mRNA, a much higher concentration of weak acid was required for aleurone layers from dormant grains than for those from nondormant grains. A possible difference in ABA signal transduction between dormant and nondormant grains is discussed.     

Seed dormancy and ABA signaling: The breakthrough goes on

The seed is an important organ in higher plants, it is an important organ for plant survival and species dispersion. The transition between seed dormancy and germination represents a critical stage in the plant life cycle and it is an important ecological and commercial trait. A dynamic balance of synthesis and catabolism of two antagonistic hormones, abscisic acid (ABA) and giberellins (GAs), controls the equilibrium between seed dormancy and germination. Embryonic ABA plays a central role in induction and maintenance of seed dormancy and also inhibits the transition from embryonic to germination growth. Therefore, the ABA metabolism must be highly regulated at both temporal and spatial levels during phase of dessication tolerance. On the other hand, the ABA levels do not depend exclusively on the seeds because sometimes it becomes a strong sink and imports it from the roots and rhizosphere through the xylem and/or phloem. These events are discussed in depth here. Likewise, the role of some recently characterized genes belonging to seeds of woody species and related to ABA signaling are also included. Finally, although four possible ABA receptors have been reported, not much is known about how they mediate ABA signaling transduction. However, new publications seem to show that almost all these receptors lack several properties to consider them as such.Key words: ABA/GA balance, ABA in woody plants, ABA-receptors, biosynthetic ABA mutants, rhizosphere ABA, seed dormancy