Defense-related signaling by interaction of arabinogalactan proteins and beta-glucosyl Yariv reagent inhibits gibberellin signaling in barley aleurone cells

Arabinogalactan proteins (AGPs) are hydroxyproline-rich glycoproteins present at the plasma membrane and in extracellular spaces. A synthetic chemical, beta-glucosyl Yariv reagent (beta-GlcY), binds specifically to AGPs. We previously reported that gibberellin signaling is specifically inhibited by beta-GlcY treatment in barley aleurone protoplasts. In the present study, we found that beta-GlcY also inhibited gibberellin-induced programmed cell death (PCD) in aleurone cells. We examined the universality and specificity of the inhibitory effect of beta-GlcY on gibberellin signaling using microarray analysis and found that beta-GlcY was largely effective in repressing gibberellin-induced gene expression. In addition, >100 genes were up-regulated by beta-GlcY in a gibberellin-independent manner, and many of these were categorized as defense-related genes. Defense signaling triggered by several defense system inducers such as jasmonic acid and a chitin elicitor could inhibit gibberellin-inducible events such as alpha-amylase secretion, PCD and expression of some gibberellin-inducible genes in aleurone cells. Furthermore, beta-GlcY repressed the gibberellin-inducible Ca2+-ATPase gene which is important for gibberellin-dependent gene expression, and induced known repressors of gibberellin signaling, two WRKY genes and a NAK kinase gene. These effects of beta-GlcY were also phenocopied by the chitin elicitor and/or jasmonic acid. These results indicate that gibberellin signaling is under the regulation of defense-related signaling in aleurone cells. It is also probable that AGPs are involved in the perception of stimuli causing defense responses.     

The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants

Using expression analysis, the role of the sucrose transporter OsSUT1 during germination and early growth of rice seedlings has been examined in detail, over a time-course ranging from 1 d to 7 d post-imbibition. Unlike the wheat orthologue, TaSUT1, which is thought to be directly involved in sugar transfer across the scutellar epithelium, OsSUT1 is not expressed in the scutellar epithelial cell layer of germinating rice and is, therefore, not involved in transport of sugars across the symplastic discontinuity between the endosperm and the embryo. OsSUT1 expression was also absent from the aleurone cells, indicating it is not involved in the transport of sucrose in this cell layer during germination. However, by 3 d post-imbibition, OsSUT1 was present in the companion cells and sieve elements of the scutellar vascular bundle, where it may play a role in phloem loading of sucrose for transport to the developing shoot and roots. This sucrose is most likely sourced from hexoses imported from the endosperm. In addition, sucrose may be remobilized from starch granules which are present at a high density in the scutellar ground tissues surrounding the vasculature and at the base of the shoot. OsSUT1 was also present in the coleoptile and the first and second leaf blades, where it was localized to the phloem along the entire length of these tissues, and was also present within the phloem of the primary roots. OsSUT1 may be involved in retrieval of sugars from the apoplasm in these tissues.     

Expression of the 30 kD cysteine endoprotease B in germinating barley seeds

The expression of a 30 kD cysteine endoprotease (EP-B) was studied by in situ hybridization and immunomicroscopy to clarify its role in germinating barley grains. At the beginning of germination, EP-B mRNA was expressed in the scutellar epithelium and aleurone cells next to the embryo. Later, mRNA levels were highest in the aleurone layer proceeding to the distal end of the grain. During the first day of germination, EP-B protein was strongly localized to the germ aleurone and scutellar epithelium from where the secretion into the starchy endosperm began. Secretion was also observed to proceed along the aleurone layer to the distal end. These results show that EP-B is differentially localized during germination, and both scutellum and aleurone layer are able to synthesize and secrete EP-B protein.     

Ascorbic acid and reactive oxygen species are involved in the inhibition of seed germination by abscisic acid in rice seeds

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.     

The slender rice mutant,with constitutively activated gibberellin signal transduction,has enhanced capacity for abscisic acid level

The slender rice (slr1-1) mutant, carrying a lethal and recessive single mutation, has a constitutive gibberellin (GA)-response phenotype and behaves as if it were saturated with GAs [Ikeda et al. (2001) Plant Cell 13, 999]. The SLR1 gene, with sequence homology to members of the plant-specific GRAS gene family, is a mediator of the GA signal transduction process. In the slender rice, GA-inducible alpha-amylase was produced from the aleurone layer without applying GA. GA-independent alpha-amylase production in the mutant was inhibited by applying abscisic acid (ABA). Shoot elongation in the mutant was also suppressed by ABA, indicating that the slender rice responds normally to ABA. Interestingly, shoot ABA content was 10-fold higher in the mutant than in the wild type, while there was no difference in root ABA content. Expression of the Rab16A gene, which is known to be ABA inducible, was about 10-fold higher in shoots of the mutant than in those of the wild type. These results indicate that constitutive activation of the GA signal transduction pathway by the slr1-1 mutation promotes the endogenous ABA level.     

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.     

Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA₃) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA₃ within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA₃, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA₃ counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.     

Localizing a-Amylase Gene Expression in Germinated Rice Grains

In situ hybridization was used to localize the sites of expressionfor two a-amylase genes (RAmy1A and RAmy3D) in rice (Oryza sativaL.) over five days of germination. Messenger RNAs from bothgenes were initially detected in the scutellar epithelium andappeared at later stages of germination in the aleurone layer.RAmy3D mRNA reached its peak of accumulation 2 days(d) earlierin the scutellar epithelium than RAmy1A mRNA. Both mRNAs continuedto accumulate in the aleurone layer up to 5 d of germination,although RAmy1A mRNA reached significantly higher levels thanRAmy3D mRNA. Overall, the aleurone layer was responsible forproducing the majority of the total grain a-amylase mRNA. (Received July 27, 1991; Accepted November 6, 1991)     

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.     

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.     

alpha-Amylase and programmed cell death in aleurone of ripening wheat grains

Late maturity alpha-amylase (LMA) in wheat is a genetic defect that may result in the accumulation of unacceptable levels of high pI alpha-amylase in grain in the absence of germination or weather damage. During germination, gibberellin produced in the embryo triggers expression of alpha-Amy genes, the synthesis of alpha-amylase and, subsequently, cell death in the aleurone. LMA also involves the aleurone and whilst LMA appears to be independent of the embryo there is nevertheless some evidence that gibberellin is involved. The aim of this investigation was to determine whether the increase in alpha-amylase activity in LMA-prone genotypes, like alpha-amylase synthesis by aleurone cells in germinating or GA-challenged grains, is followed by aleurone cell death. Programmed cell death was seen in aleurone layers from developing, ripe and germinated grains using confocal microscopy and fluorescent probes specific for dead or living cells. Small pockets of dying cells were observed distributed at random throughout the aleurone of ripening LMA-affected grains and by harvest-ripeness these cells were clearly dead. The first appearance of dying cells, 35 d post-anthesis, coincided with the later part of the 'window of sensitivity' in grain development in LMA-prone wheat cultivars. No dead or dying cells were present in ripening or fully ripe grains of control cultivars. In germinating grains, dying cells were observed in the aleurone adjacent to the scutellum and, as germination progressed, the number of dead cells increased and the affected area extended further towards the distal end of the grain. Aside from the obvious differences in spatial distribution, dying cells in 20-24 h germinated grains were similar to dying cells in developing LMA-affected grains, consistent with previous measurements of alpha-amylase activity. The increase in high pI alpha-amylase activity in developing grains of LMA-prone cultivars, like alpha-amylase synthesis in germinating grains, is associated with cell death, providing further evidence for the involvement of gibberellin in the LMA response.