Gibberellins are not required for rice germination under anoxia

Production of -amylase during the germination of rice grains is thought to play an important role for tolerance to anoxia of these cereal grains. Under aerobic conditions -amylases production is enhanced in response to gibberellins produced by the embryos, but the role of these hormones is less clear under anoxia. In this paper we analysed -amylase gene expression in a rice mutant (Tan-ginbozu) severely impaired in gibberellin biosynthesis. Expression of -amylase genes others than the gibberellin-induced Amy1A gene is observed. The expression of the Amy3D gene, which does dot require gibberellins to be induced, is high under anoxia in the Tan-ginbozu mutant suggesting that germination under anoxia can proceed thanks to the activity of the -amylase isoform encoded by the Amy3D gene. Amy3D gene expression is repressed in the presence of high levels of soluble carbohydrates, indicating that the anaerobic expression of this gene can be triggered by a lower carbohydrate content of rice grains kept under anoxia. Germination under anoxia of Tan-ginbozu grains can proceed even in absence of exogenously-added gibberellic acid. Overall, results indicate that gibberellins are not required for the anaerobic germination of rice grains.     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Sugar Repression of a Gibberellin-Dependent Signaling Pathway in Barley Embryos

Increasing evidence shows that sugars can act as signals affecting plant metabolism and development. Some of the effects of sugars on plant growth and development suggest an interaction of sugar signals with hormonal regulation. We investigated the effects of sugars on the induction of [alpha]-amylase by gibberellic acid in barley embryos and aleurone layers. Our results show that sugar and hormonal signaling interact in the regulation of gibberellic acid-induced gene expression in barley grains. The induction of [alpha]-amylase by gibberellic acid in the aleurone layer is unaffected by the presence of sugars, but repression by carbohydrates is effective in the embryo. [alpha]-Amylase expression in the embryo is localized to the scutellar epithelium and is hormone and sugar modulated. The effects of glucose are independent from the effects of sugars on gibberellin biosynthesis. They are not due to an osmotic effect, they are independent of abscisic acid, and only hexokinase-phosphorylatable glucose analogs are able to trigger gene repression. Overall, the results suggest the existence of an interaction between the hormonal and metabolic regulation of [alpha]-amylase genes in barley grains.     

Calcium-dependent induction of novel proteins by abscisic acid in wheat aleurone tissue of different developmental stages

Aleurone tissue of mature wheat (Triticum aestivum L. cv. Sappo) grains make novel polypeptides in response to abscisic acid (ABA), but only in the presence of Ca²⁺. Effects of ABA plus Ca²⁺ include up- and down-modulation of other polypeptides. The ABA-induced polypeptides appear not to be the 21-kilodalton (kDa) amylase inhibitor which has been reported to be ABA-inducible in barley.Aleurone tissue from developing grains of different ages failed to respond to ABA plus Ca²⁺ in any way. Endogenous ABA levels were determined by monoclonal radioimmunoassay in developing, mature, and sensitised developing tissues. The ABA level rose to a maximum at 35 days post anthesis but was not detectable in mature cells. Developing layers sensitised to gibberellic acid (GA) showed decreased levels of ABA, similar to those in mature tissue, concurrent with acquired responsiveness to GA in respect of its induction of -amylase. However, these sensitised cells still remained non-responsive to added ABA in terms of modulation of polypeptide pattern, though they did respond to ABA in the blocking of GA-induced -amylase production. The role of protein phosphorylation in signal transduction was examined. The implications of these findings are discussed with reference to the role of ABA in developing and mature aleurone cells.Abbreviations ABA Abscisic acid - dpa days post anthesis - GA₃ Gibberellic acid - kDa kilodalton - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Inhibition of α-Amylase Acting in Hexaploid Triticale Lines by Exogenous Abscisic Acid

Hexaploid triticale introgressive lines developed after recombination of A-genome with A^m-genome of diploid wheat (Triticum monococcum) were analysed in respect of grains responsiveness to exogenous ABA treatment. This was assessed by in vivo bioassay as grain germination indices, and by α-amylase assay as quantity of synthesised α-amylase measured with the technique of radial diffusion in agarose gel. The results showed an important diminishing of seedling length caused by ABA (variable in different lines) as well as genotype dependant variability of α-amylase synthesis inhibition. The differences of ABA responsiveness were seen both in whole grains and in embryoless half-grains as a direct reaction of the aleurone layer. Variation of grain sensitivity to ABA treatment compared with two sprouting resistance indices showed a significant correlation with Falling Number values in grains, but not with a dormant grains germination in spikes. This is an evidence that in triticale precocious starch decompose in unripened and ungerminated grains is dependent on genotype ABA-responsiveness of the aleurone layer. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

α-Amylase Expression under Anoxia in Rice Seedlings: An Update

Rice grains can germinate under anoxia, while most other cereals fail to behave similarly. The ability of rice grains to degrade starch, thanks to the successful production of -amylase even in the absence of oxygen is likely one of the factors allowing rice to germinate anaerobically. In this paper we review the most recent results concerning the physiology and molecular biology related to the expression of -amylase genes under anoxia. The current view is that expression of sugar-starvation induces isoforms of -amylase playing a major role during germination under anoxia, while gibberellin induces -amylase predominating under aerobic conditions.     

Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid

The patterns of protein synthesis in barley aleurone layers treated with gibberellic acid (GA₃) and abscisic acid (ABA) are compared with the patterns observed in wheat germ in vitro translation assays directed by RNA isolated from similarly treated layers. When used alone, GA₃ and ABA both induce the formation of new translatable mRNAs and cause new proteins to be synthesized. The effects of GA₃ are more dramatic than those of ABA. In GA₃-treated tissues, overall protein synthesis is redirected to produce large quantities of α-amylase and a few other GA₃-induced proteins, while other protein synthesis is reduced or stopped. Large amounts of new translatable mRNA for α-amylase are also induced such that the dominant in vitro translation product is α-amylase. These changes are blocked by the simultaneous addition of ABA to the tissue. In GA₃ plus ABA-treated layers, few changes in protein synthesis in vivo are observed when compared to protein synthesis in untreated tissue, although the induction of mRNA for α-amylase and the other GA₃-induced mRNAs does occur. This indicates that ABA does not interfere with GA₃ induction of translatable mRNAs but prevents the translation of these mRNAs in vivo. Thus ABA and potentially GA₃ regulate the translation of proteins in vivo in barley aleurone layers.     

The effects of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers studies using an α-amylase cDNA clone

Summary Two cDNA clones were characterized which correspond to different RNA species whose level is increased by gibberellic acid (GA₃) in barley (Hordeum vulgare L.) aleurone layers. On the criteria of amino terminal sequencing, amino acid composition and DNA sequencing it is likely that one of these clones (pHV19) corresponds to the mRNA for -amylase (1,4--D-glucan glucanohydrolase, EC 3.2.1.1.), in particular for the B family of -amylase isozymes (Jacobsen JV, Higgins TJV: Plant Physiol 70:1647–1653, 1982). Sequence analysis of PHV19 revealed a probable 23 amino acid signal peptide. Southern hybridization of this clone to barley DNA digested with restriction endonucleases indicated approximately eight gene-equivalents per haploid genome.The identity of the other clone (pHV14) is unknown, but from hybridization studies and sequence analysis it is apparently unrelated to the -amylase clone.Both clones hybridize to RNAs that are similar in size (1500b), but which accumulate to different extents following GA₃ treatment: -amylase mRNA increases approximately 50-fold in abundance over control levels, whereas the RNA hybridizing to pHV14 increases approximately 10-fold. In the presence of abscisic acid (ABA) the response to GA₃ is largely, but not entirely, abolished. These results suggest that GA₃ and ABA regulate synthesis of -amylase in barley aleurone layers primarily through the accumulation of -amylase mRNA.Abbreviations ABA abscisic acid - CHA cyclohepta-amylose - CMC carboxymethyl cellulose - GA₃ gibberellic acid     

Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Abscisic-acid and gibberellin action in developing kernels of triticale (cv. 6A190)

Abscisic-acid (ABA) levels were determined in triticale 6A190 kernels at various stages of development from anthesis to maturity. ABA reached a maximum at ca. 22 d post-anthesis and declined rapidly 12 d later. Associated with drying of the kernel at maturity there was a rapid increase in the endogenous level of -amylase, apparently based upon de-novo synthesis. Simultaneously there were visible signs of degradation of the large starch grains in the starchy endosperm. Regulation of -amylase production in the kernel by exogenous gibberellic acid (GA₃) was only evident in the almost mature kernel (30–40 d after anthesis) and then only if these kernels were first dried artificially. Furthermore, little -amylase mRNA could be detected prior to kernel maturity and water loss. Thus, the high levels of gibberellin (GA) that have been found early in kernel development in cereals do not appear to control the later production of -amylase and onset of kernel germination in the ear of triticale. However, the presence of high levels of ABA until maturity could prevent early germination and premature production of -amylase. Kernels of triticale 6A190 are characteristically shrivelled and non-dormant at maturity. The relevance of changes in the capacity of kernels to respond to and produce GA and ABA is discussed in relation to problems of harvest dormancy in cereals.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Contribution of aleurone layer and scutellum to α-amylase synthesis and secretion in wheat and rice grains

Intact aleurone layers from wheat ( Triticum aestivum L. cv Camp Rémy) and rice ( Oryza sativa L. cv Cigalon) grains both contained and secreted more α-amylase (EC 3.2.1.1.) than did the corresponding scutellar tissues. This discrimination was already evident at the earliest stages of germination at which the tissues could be isolated, and became more pronounced upon subsequent germination and growth. Isoenzyme patterns obtained upon isoelectric focusing showed a considerable polymorphism of the α-amylases of each cereal. The enzyme polymorphism pattern was the same in the aleurone layer and in the scutellum, but some secondary constituents appeared to be more specific for the one or the other of the tissues. Moreover, the isozymes found in the tissues were the same as those found to be secreted. A third α-amylase antigen which differs from the well established α₁ and α₁₁ forms was identified in the germinating wheat grains. The presence of Ca²⁺ in the secretion medium favoured maximum secretion of α-amylases from the wheat scutellum and aleurone layers, whereas it inhibited the secretion of the enzymes from the rice aleurone layer.     

Aleurones from a Barley with Low [alpha]-Amylase Activity Become Highly Responsive to Gibberellin When Detached from the Starchy Endosperm

The physiological and molecular bases for contrasting [alpha]-amylase phenotypes were examined in germinating seeds of two barley (Hordeum vulgare L.) cultivars, Morex and Steptoe. Morex is a high-quality malting barley that develops high [alpha]-amylase activity soon after germination. Steptoe is a feed barley that develops only low [alpha]-amylase activity levels during this period. The expression of all high- and low-isoelectric point (pl) [alpha]-amylase isozymes is reduced in Steptoe. The amount of [alpha]-amylase mRNA per gram of seedling tissue is correspondingly lower in Steptoe. Southern blot analysis revealed that the cultivars have the same copy number and organization for most high- and low-pl genes. Steptoe seedlings or embryoless half-seeds produce little [alpha]-amylase in response to exogenous applications of gibberellic acid (GA3) compared with Morex. However, when isolated aleurones of both cultivars are treated with GA3, they produce similar amounts of high- and low-pl [alpha]-amylase RNAs. This suggests that a factor in the starchy endosperm is responsible for lowered [alpha]-amylase response in Steptoe. The factor is probably not abscisic acid (ABA), since the two cultivars have similar concentrations of ABA during germination.     

Hormonal Status in Wheat and Rice Seedlings under Anoxia

A comparative analysis of the effects of anoxia on growth, fresh weight gain, and phytohormones in wheat (Triticum aestivum L.) and rice (Oryza sativa L.) seedlings was performed. In both plant species, a total cessation of root growth occurred during the initial hours of anoxia. In an anaerobic environment, the fresh weight of wheat seedlings decreased. An increase in the shoot length and weight under the stress conditions was found only in rice seedlings. During the initial hours of anoxia, the level of free ABA in wheat and rice tissues increased manifold, and the accumulation of a free ABA form occurred at the expense of the hydrolysis of its bound forms. The IAA content in plant tissues also increased. In wheat, the accumulation of IAA was short, but in rice, a high hormone level was retained during the entire experiment, and, as a result, its concentration exceeded that of ABA. A level of cytokinins in the tissues of both plant species was affected by anoxia to a lesser extent than that of other phytohormones. This level somewhat decreased under anoxia similarly to the level in darkness under aeration. It is suggested that IAA accumulation in hypoxia-tolerant rice seedlings under anoxia favors maintenance of shoot growth and simultaneous inhibition of root growth. At the same time, in the hypoxia-sensitive wheat, an increase in the ABA level resulted in growth cessation.