This chapter discusses some of the events following germination of barley grains which occur in the aleurone tissue attached to the growing seedling. It describes some of the major hormonal controls affecting expression of alpha-amylase genes in aleurone of barley, discussed the cellular mechanisms by which they operate, and attempted to relate these to germination and early seedling development. Whether the accumulation of abscisic acid (ABA)-inducible mRNAs in the aleurone of dehydrating seedlings occurs in response to ABA or to some other compound is not known. The changes in levels of alpha-amylase mRNA seen in the aleurone of developing seedlings are also seen in isolated aleurone layers treated with gametophytic factor (Ga). The observation that ABA can prevent the GA-induced synthesis of alpha-amylase indicates that aleurone is a tissue which is ABA-responsive, and raises the possibility that there may be a physiological role for its sensitivity to ABA.

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.

To increase brewing yield and efficiency, malts with high extract values, high enzymic activities and good modification are essential. To produce malt that meets these requirements, the barley employed must have minimal post-harvest dormancy and be able to germinate vigorously. The aims of this study were to determine the extent to which some Australian barley varieties changed during post-harvest storage, how these changes influenced germination characteristics, enzyme production and malt quality, and, of the germination tests examined, which gave the best indication of a barley's malting potential.Four commercially grown barley samples were obtained, one from Tasmania and three from Victoria. Each sample was stored at room temperature for one year. At monthly intervals, samples were taken and placed at -18 degrees C. The germinative energy (GE) and germinative index (GI) of these samples were measured. Samples were also micro-malted and the quality of the malt was assessed using standard EBC methodology.Storage at room temperature positively influenced the germination characteristics of all samples, with concomitant improvements in hydrolytic enzyme production during malting and in a number of malt quality parameters. It was found that, of the germination tests examined, the GI consistently correlated with enzyme activities during malting and with various malt quality parameters thus indicating that the GI is a good indicator of malting potential.

GAMYB is a GA-responsive activator of hydrolase gene expression in the aleurone layer of germinated cereal grains. We have isolated a putative GAMYB-binding protein, GMPOZ, which contains a BTB/POZ domain found in certain animal transcriptional regulators. Although BTB/POZ domain proteins are numerous in plants, very few are yet characterized. We found that GMPOZ is nuclear localized and that GMPOZ mRNA is expressed highly in anthers as well as aleurone. Transient silencing of the GMPOZ gene suggests that GMPOZ is involved in hormone responsive gene expression in aleurone.

GAMYB is a gibberellin (GA)-regulated activator of hydrolase gene expression in the aleurone layer of germinating cereal grains. Although it is clear that GAMYB expression is regulated by GA, more remains to be understood about how this transcription factor operates within the GA-response pathway. In order to isolate new components from the GA-response pathway, barley aleurone libraries were screened for GAMYB-binding proteins using a recently developed yeast two-hybrid system, which is compatible with the use of transcription factors as baits. We isolated a new member of the emerging Mak-subgroup of cdc2- and MAP kinase-related protein kinases. We have termed this GAMYB-binding protein KGM (for kinase associated with GAMYB). Transient expression of KGM specifically repressed alpha-amylase promoter activity at the level of GAMYB function but a mutation designed to de-stabilise the activation loop of KGM alleviated this repression. We propose that KGM is a negative regulator of GAMYB function in aleurone that may prevent precocious hydrolase gene expression.

Analyses of abscisic acid (ABA), ent-kaurenoids and gibberellins (GAs) showed that there were major changes in the contents of these compounds associated with germination of after-ripened barley (Hordeum vulgare cv. Schooner and cv. Proctor) grain but not in hydrated dormant grain. Embryos from dormant and after-ripened dry grain contained similar amounts of ABA, of ent-kaurenoids and of GAs, determined by gas chromatography-mass spectrometry-selected ion monitoring. In embryos of after-ripened grain, ABA content decreased rapidly after hydration and ABA appeared to be metabolized (inactivated) to phaseic acid (PA) rather than diffusing into the endosperm or the surrounding medium as previously thought. Similar changes in ABA occurred in hydrated dormant grain during germination in darkness. Accumulation of ent-kaurenoids and GAs, including GA1, the first biologically active GA in the early 13-hydroxylation biosynthetic pathway, occurred to a much greater extent in after-ripened than in dormant grain and these changes occurred mainly after 18 h of hydration when ABA had already decreased and germination was occurring. The block in ent-kaurenoid and GA synthesis in dormant grain appeared to occur prior to ent-kaurene in the biosynthetic pathway. These results are consistent with the view that ABA is the primary effector of dormancy and that after-ripening involves the development of the ability to reduce the amount of ABA quickly following hydration. Accumulation of GAs does not appear to be causally related to loss of dormancy but it does appear to be related to germination.

We have previously identified GAMYB, a gibberellin (GA)-regulated transcriptional activator of alpha-amylase gene expression, in aleurone cells of barley (Hordeum vulgare). To examine the regulation of GAMYB expression, we describe the use of nuclear run-on experiments to show that GA causes a 2-fold increase in the rate of GAMYB transcription and that the effect of GA can be blocked by abscisic acid (ABA). To identify GA-signaling components that regulate GAMYB expression, we examined the role of SLN1, a negative regulator of GA signaling in barley. SLN1, which is the product of the Sln1 (Slender1) locus, is necessary for repression of GAMYB in barley aleurone cells. The activity of SLN1 in aleurone cells is regulated posttranslationally. SLN1 protein levels decline rapidly in response to GA before any increase in GAMYB levels. Green fluorescent protein-SLN1 fusion protein was targeted to the nucleus of aleurone protoplasts and disappeared in response to GA. Evidence from a dominant dwarf mutant at Sln1, and from the gse1 mutant (that affects GA "sensitivity"), indicates that GA acts by regulating SLN1 degradation and not translation. Mutation of the DELLA region of SLN1 results in increased protein stability in GA-treated layers, indicating that the DELLA region plays an important role in GA-induced degradation of SLN1. Unlike GA, ABA had no effect on SLN1 stability, confirming that ABA acts downstream of SLN1 to block GA signaling.

A green fluorescent protein (GFP) gene was cloned between the promoter and 3´ regions from a barley high isoelectric point (pI) α-amylase gene, then inserted into barley. GFP fluorescence was used to locate and quantify expression of the transgene in barley grains following hydration. Light and confocal laser microscopy revealed fluorescence in the known regions of α-amylase synthesis in the scutellar epithelium, aleurone layer and embryonic axis. Fluorescence was quantified using a simple fluorescence assay, which showed induction of the transgene to mirror the induction of α-amylase in aleurone exposed to gibberellic acid. Expression from the transgene was also shown to be inhibited by abscisic acid, in the same way as expression of endogenous α-amylase genes. Overall, the transgenic grain revealed patterns of α-amylase expression before and after germination, and showed strong potential for further studies investigating both α-amylase production and transport of gibberellin in malting grain.

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.

GAMYB is an MYB transcription factor which is expressed in cereal aleurone cells in response to gibberellin (GA). HvGAMYB binds to the TAACAAA box of a high-pl alpha-amylase gene promoter and transcriptionally activates its expression. In this study, we examined the role of HvGAMYB in activating expression of other GA-regulated genes encoding hydrolytic enzymes. In transient expression experiments, HvGAMYB transactivated expression of reporter genes fused to a low-pl alpha-amylase gene promoter, an EII (1-3, 1-4)-beta-glucanase gene promoter and a cathepsin B-like protease promoter. HvGAMYB DNA binding specificity was determined using a PCR-based random site selection using HvGAMYB fusion protein isolated from E. coli. The deduced consensus closely resembled gibberellin response elements in alpha-amylase promoters. Functional analysis of HvGAMYB by transient expression of C terminal HvGAMYB deletions in barley aleurone cells identified two transcriptional activation domains (TADs) which function in transcriptional regulation of both high- and low-pl alpha-amylase promoters. The same TADs were identified using a heterologous yeast expression system. Together, these results indicate that HvGAMYB has two TADs. These domains are C-terminal to its DNA-binding domain.

Barley (Hordeum vulgare L.) seed proanthocyanidins play a primary role in beer colloidal haze formation. The barley ant28‐484 mutation eliminates proanthocyanidin synthesis and beer brewed with barley homozygous or ant28‐484 exhibits intrinsic haze stability. The recessive nature of this mutation, coupled with the fact that the proanthocyanidin‐free phenotype it confers is discernable only in maternal tissues of the seed, makes introgression of ant28‐484 into new cultivars arduous. The goal of this study was to identify molecular markers for use in marker‐assisted selection of ant28‐484. An F2 barley population derived from the cross Arapiles ✕ Caminant was scored for ant28‐484 segregation. DNA pools from F2 plants homozygous for ant28‐484 or the contrasting wild‐type allele were used for bulked segregant RAPD analysis. RAPD bands differentiating the two DNA pools were then analyzed for linkage to ant28‐484 in the F2 population. Five linked RAPD markers were identified, with linkage estimates to ant28‐484 ranging from 0 to 11 centimorgans (cM). Three of the markers were linked in cis to ant28‐484 and two were linked in trans. The three most tightly linked RAPD markers were cloned, and southern analysis indicated that they represent low or single copy sequences. The ant28‐484 mutation was localized to barley chromosome arm 3HL, on the basis of results of southern analysis with wheat‐barley ditelosomic addition lines probed with the cloned RAPD markersa nd on the basis of linkage to the previously mapped isozyme locus Est4. These markers will enhance the efficiency with which ant28‐484 can be introgressed into barley germplasm.

A cDNA clone, OsGAmyb, which encodes a homologue to the barley Myb-like transcription factor, HvGAMyb, was isolated from a rice endosperm cDNA library. The clone was used to show that expression of the OsGAmyb gene in aleurone cells was stimulated by gibberellic acid and the gene product was shown to transactivate an alpha-amylase gene promoter in transient expression analyses.

Isozymes of barley α-amylase were matched to cDNAs that encode them using transient expression in oat aleurone layers. Four cDNAs, including two that are previously unpublished, were inserted into oat aleurone cells by microparticle bombardment. The cDNAs were under the control of theAct1 promoter of rice. Expression levels were sufficient for in-gel detection of enzyme activity following isoelectric focusing of aleurone homogenates. The system has also proved useful in characterizing a hybrid β-glucanase gene.

The occurrence and roles of cGMP were investigated in aleurone layers and protoplasts isolated from barley (cv Himalaya) grain. Levels of cGMP in freshly isolated barley aleurone layers ranged from 0.065 to 0.08 pmol/g fresh weight of tissue, and cGMP levels increased transiently after incubation in gibberellic acid (GA). Abscisic acid (ABA) did not increase cGMP levels in aleurone layers. LY 83583 (LY), an inhibitor of guanylyl cyclase, prevented the GA-induced increase in cGMP and inhibited GA-induced [alpha]-amylase synthesis and secretion. The inhibitory effects of LY could be overcome by membrane-permeant analogs of cGMP. LY also prevented GA-induced accumulation of [alpha]-amylase and GAMYB mRNAs. cGMP alone was not sufficient to induce the accumulation of [alpha]-amylase or GAMYB mRNA. LY had a less dramatic effect on the accumulation of mRNAs encoding the ABA-responsive gene Rab21. We conclude that cGMP plays an important role in GA, but not ABA, signaling in the barley aleurone cell.

Functional analysis of a barley high-pI alpha-amylase gene promoter has identified a gibberellin (GA) response complex in the region between -174 and -108. The sequence of the central element, TAACAAA, is very similar to the c-Myb and v-Myb consensus binding site. We investigated the possibility that a GA-regulated Myb transactivates alpha-amylase gene expression in barley aleurone cells. A cDNA clone, GAmyb, which encodes a novel Myb, was isolated from a barley aleurone cDNA library. RNA blot analysis revealed that GAmyb expression in isolated barley aleurone layers is up-regulated by GA. The kinetics of GAmyb expression indicates that it is an early event in GA-regulated gene expression and precedes alpha-amylase gene expression. Cycloheximide blocked alpha-amylase gene expression but failed to block GAmyb gene expression, indicating that protein synthesis is not required for GAmyb gene expression. Gel mobility shift experiments with recombinant GAMyb showed that GAMyb binds specifically to the TAACAAA box in vitro. We demonstrated in transient expression experiments that GAMyb activates transcription of a high-pI alpha-amylase promoter fused to a beta-glucuronidase reporter gene in the absence of GA. Our results indicate that the GAMyb is the sole GA-regulated transcription factor required for transcriptional activation of the high-pI alpha-amylase promoter. We therefore postulate that GAMyb is a part of the GA-response pathway leading to alpha-amylase gene expression in aleurone cells.

In growth-cabinet experiments heat and water stresses were separately applied to populations of barley plants, cv. Schooner, 17 or 27 days post-anthesis (DPA), after which the mature grain was micromalted and a number of malt quality parameters measured. Water stress at 17 DPA (mid grain-fill) led to decreased grain (1 → 3),(1 → 4)-β-glucan and increased malt extract, (1 → 3),(1 → 4)-β-glucanase, α-amylase and β-amylase, while at 27 DPA (late grain-fill) only extract was increased. Heat stress with maintenance of soil moisture level had no effect on these parameters. Both types of stress reduced yield and grain size. Neither stressed nor unstressed grain showed any dormancy. These findings indicate that the malting performance of Schooner barley is more related to water stress than heat stress. They also suggest that a limited episode of water stress during grain-fill may be beneficial for malting quality.

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)

The starchy endosperm (SE) of the developing grain (caryopsis) of barley (Hordeum vulgare L.) cv Himalaya, as well as that of other barley cultivars examined, acidifies during maturation. The major decrease in pH begins with the attainment of maximum grain dry weight, onset of dehydration, and completion of chlorophyll loss. Acidification is correlated with the accumulation of malate and lesser amounts of citrate and lactate, produced and probably secreted by the pericarp/testa/aleurone (PTA). It is accompanied by large concurrent rises in phosphoeno/pyruvate carboxylase and alcohol dehydrogenase (ADH) activity in the PTA. The activity of seven other enzymes of oxaloacetate and pyruvate metabolism was found to fall or rise only slightly during acidification. Sequential changes in relative amount of ADH isozymes were found in both PTA and SE. The PTA maintained a high respiration rate and adenylate energy charge (AEC) throughout acidification, whereas the SE showed a low respiration rate and rising AEC. The data are consistent with the occurrence of hypoxia in the SE. It is suggested that the above enzyme changes are required for the development of a malate/ethanol fermentation (i.e. a mixed metabolism) in the aleurone layer during maturation.