Aleurone Cells Research Articles

A method for obtaining high integrity RNA from developing aleurone cells and starchy endosperm in rice ( Oryza sativa L.) by laser microdissection

Laser microdissection (LMD) is now becoming a powerful tool to elucidate the spatial profiling of gene expression. However, a remaining difficulty is obtaining high integrity RNA from fixed and embedded tissues in order to obtain accurate and reliable expression analyses. This study aimed to develop methods for the preparation of high integrity RNA from aleurone cells and starchy endosperm of developing rice endosperm by LMD. Acetone or 3:1 ethanol:acetic acid (AA), was used for the fixative and 2% carboxymethyl cellulose (CMC) or paraffin, was used for the embedding reagent. AA and CMC were better for identification of cell types and for recovery of intact RNA, although CMC-embedding did not preserve morphology as well as paraffin-embedding. Quantitative RT-PCR revealed that OsSUT1 mRNA encoding a sucrose transporter was localized in aleurone cells and that SDBE mRNA encoding a starch debranching enzyme (pullulanase) was localized in starchy endosperm. Taken together, these results show that our LMD method is suitable for preparing high integrity RNA from aleurone cells and starchy endosperm in rice. mRNA obtained with this method should help to understand the molecular basis for endosperm development.

Salicylic acid inhibits gibberellin-induced alpha-amylase expression and seed germination via a pathway involving an abscisic-acid-inducible WRKY 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 alpha-amylases by SA and a WRKY gene whose expression is modulated by these hormones. Assays of alpha-amylase activity reveal that GA-induced alpha-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 alpha-amylase secretion or inhibiting alpha-amylase enzyme activities. Northern blot analyses indicate that SA suppresses GA-induced expression of a barley low pI alpha-amylase gene (Amy32b). Because our previous data indicate that ABA-inducible and GA-suppressible WRKY genes inhibit the expression of alpha-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.

Gliadain, a gibberellin‐inducible cysteine proteinase occurring in germinating seeds of wheat,Triticum aestivumL., specifically digests gliadin and is regulated by intrinsic cystatins

We cloned a new cysteine proteinase of wheat seed origin, which hydrolyzed the storage protein gliadin almost specifically, and was named gliadain. Gliadain mRNA was expressed 1 day after the start of seed imbibition, and showed a gradual increase thereafter. Gliadain expression was suppressed when uniconazol, a gibberellin synthesis inhibitor, was added to germinating seeds. Histochemical detection with anti-gliadain serum indicated that gliadain was present in the aleurone layer and also that its expression intensity increased in sites nearer the embryo. The enzymological characteristics of gliadain were investigated using recombinant glutathione S-transferase (GST)-progliadain fusion protein produced in Escherichia coli. The GST-progliadain almost specifically digested gliadin into low molecular mass peptides. These results indicate that gliadain is produced via gibberellin-mediated gene activation in aleurone cells and secreted into the endosperm to digest its storage proteins. Enzymologically, the GST-progliadain hydrolyzed benzyloxycarbonyl-Phe-Arg-7-amino-4-methylcoumarin (Z-Phe-Arg-NH(2)-Mec) at K(m) = 9.5 microm, which is equivalent to the K(m) value for hydrolysis of this substrate by cathepsin L. Hydrolysis was inhibited by two wheat cystatins, WC1 and WC4, with IC(50) values of 1.7 x 10(-8) and 5.0 x 10(-8) m, respectively. These values are comparable with those found for GST-progliadain inhibition by E-64 and egg-white cystatin, and are consistent with the possibility that, in germinating wheat seeds, gliadain is under the control of intrinsic cystatins.

The Arabidopsis Aleurone Layer Responds to Nitric Oxide, Gibberellin, and Abscisic Acid and Is Sufficient and Necessary for Seed Dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.

Abscisic Acid and Stress Signals InduceViviparous1Expression in Seed and Vegetative Tissues of Maize

Viviparous1 (Vp1) encodes a B3 domain-containing transcription factor that is a key regulator of seed maturation in maize (Zea mays). However, the mechanisms of Vp1 regulation are not well understood. To examine physiological factors that may regulate Vp1 expression, transcript levels were monitored in maturing embryos placed in culture under different conditions. Expression of Vp1 decreased after culture in hormone-free medium, but was induced by salinity or osmotic stress. Application of exogenous abscisic acid (ABA) also induced transcript levels within 1 h in a dose-dependent manner. The Vp1 promoter fused to beta-glucuronidase or green fluorescent protein reproduced the endogenous Vp1 expression patterns in transgenic maize plants and also revealed previously unknown expression domains of Vp1. The Vp1 promoter is active in the embryo and aleurone cells of developing seeds and, upon drought stress, was also found in phloem cells of vegetative tissues, including cobs, leaves, and stems. Sequence analysis of the Vp1 promoter identified a potential ABA-responsive complex, consisting of an ACGT-containing ABA response element (ABRE) and a coupling element 1-like motif. Electrophoretic mobility shift assay confirmed that the ABRE and putative coupling element 1 components specifically bound proteins in embryo nuclear protein extracts. Treatment of embryos in hormone-free Murashige and Skoog medium blocked the ABRE-protein interaction, whereas exogenous ABA or mannitol treatment restored this interaction. Our data support a model for a VP1-dependent positive feedback mechanism regulating Vp1 expression during seed maturation.

Heat Shock-Induced Changes in Lipid and Protein Metabolism in the Endoplasmic Reticulum of Barley Aleurone Layers

Heat shock in barley aleurone layers induces heat shock protein synthesis and suppresses secretory protein synthesis by selectively destabilizing their mRNAs. In addition, the endoplasmic reticulum (ER) membranes upon which secretory protein mRNAs are translated become vesiculated during heat shock, leading to the hypothesis that ER dissociation and targeted mRNA destabilization are linked mechanistically. Supporting this, ER can be heat adapted, and heat-adapted ER has higher levels of fatty acid saturation in membrane phospholipids which do not vesiculate upon heat shock. Secretory protein mRNAs are also more stable in heat-adapted cells. To understand better heat shock-induced changes in ER membranes, we examined ER membrane proteins and enzymes involved in phosphatidylcholine biosynthesis and phospholipid turnover in heat-shocked aleurone cells. Heat shock significantly increased the activity of phospholipases A2 and D, and shortly thereafter significant but gradual increases in choline kinase and phosphocholine glyceride transferase activities and a sharp increase in phosphorylcholine citidyl transferase activity were observed. Only minor changes were observed in SDS-PAGE analyses of proteins from sonicated ER membranes fractionated on continuous sucrose gradients. Overall, heat shock reduced total lipid in ER membranes relative to protein, and in intact, ultracentrifuged aleurone cells examined by light and electron microscopy the ER band appeared to increase in density. The changes in phospholipid metabolism coupled with the suppression of secretory protein synthesis indicate that in addition to inducing a classic heat shock response, high temperature also induces a classic unfolded protein response in the ER of this secretory cell.

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.

Investigation of ferulate deposition in endosperm cell walls of mature and developing wheat grains by using a polyclonal antibody

A polyclonal antibody has been raised against ferulic acid ester linked to arabinoxylans (AX). 5-O-feruloyl-alpha-L-arabinofuranosyl(1-->4)-beta-D-xylopyranosyl was obtained by chemical synthesis, and was coupled to bovine serum albumin for the immunization of rabbit. The polyclonal antibody designated 5-O-Fer-Ara was highly specific for 5-O-(trans-feruloyl)-L-arabinose (5-O-Fer-Ara) structure that is a structural feature of cell wall AX of plants belonging to the family of Gramineae. The antibody has been used to study the location and deposition of feruloylated AX in walls of aleurone and starchy endosperm of wheat grain. 5-O-Fer-Ara began to accumulate early in aleurone cell wall development (beginning of grain filling, 13 days after anthesis, DAA) and continued to accumulate until the aleurone cells were firmly fixed between the starchy endosperm and the nucelus epidermis (19 DAA). From 26 DAA to maturity, the aleurone cell walls changed little in appearance. The concentration of 5-O-Fer-Ara is high in both peri- and anticlinal aleurone cell walls with the highest accumulation of 5-O-Fer-Ara at the cell junctions at the seed coat interface. The situation is quite different in the starchy endosperm: whatever the stage of development, a low amount of 5-O-Fer-Ara epitope was detected. Contrary to what was observed for aleurone cell walls, no peak of accumulation of feruloylated AX was noticed between 13 and 19 DAA. Visualization of labelled Golgi vesicles suggested that the feruloylation of AX is intracellular. The distribution of (5-O-Fer-Ara) epitope is further discussed in relation to the role of ferulic acid and its dehydrodimers in cell wall structure and tissue organization of wheat grain.

Increased expression of HvGAMYB in transgenic barley increases hydrolytic enzyme production by aleurone cells in response to gibberellin

HvGAMYB is a transcription factor protein, which is part of the gibberellin (GA) signal transduction pathway in barley aleurone cells. It activates GA-regulated genes to cause synthesis of alpha-amylase and other hydrolytic enzymes. In aleurone cells, HvGAMYB is up-regulated by GA prior to hydrolytic enzyme gene expression. In this study we tested the hypothesis that over-expression of HvGAMYB by inserting additional gene copies might increase production of the GA-induced aleurone enzymes. Transgenic lines of barley cv.Golden Promise with extra copies of the HvGAMYB gene were previously created and shown to produce increased amounts of HvGAMYB. In this study grains which were null and homozygous for the transgene were compared, and it was found that increased expression of HvGAMYB resulted in increased amounts of two GA-induced hydrolytic enzymes in both germinating grains and in GA-treated de-embryonated grains. Enzyme production by malting barley is an important quality parameter and these results indicate that it might be possible to increase the production of all GA-induced enzymes in aleurone by increasing the production of HvGAMYB. Such an outcome would be a valuable adjunct to conventional breeding programs.

Common mechanisms regulating expression of rice aleurone genes that contribute to the primary response for gibberellin

During germination of cereal seeds, aleurone cells respond to gibberellins (GA) by synthesizing and secreting hydrolytic enzymes that mobilize the reserved nutrients. It has been shown that products of early GA response genes, like a transcription factor GAMyb, act as key molecules leading to this regulation. Pivotal roles of GAMyb on expression of hydrolase genes have been well documented, whereas regulation of GAMyb expression itself remains obscure. In order to understand virtual mechanisms of the GA-mediated expression of genes, it is important to know how GA control expression of early GA response genes. Using an aleurone transient expression system of rice ( Oryza sativa L.), we examined GA responsive domains of early GA response genes in the aleurone, such as GAMyb and OsDof3. The upstream promoter could not confer GA response. Extensive analyses revealed the presence of enhancer-like activities in a large first intron. In Arabidopsis, intron enhancers have been identified in MADS-box homeotic genes, AGAMOUS ( AG) and FLOWERING LOCUS C ( FLC), in which large introns should not only confer proper gene expressions, but also associate with gene silencing by covalent modifications of both DNA and histone. These evidences prompt us to assign that chromatin-based control might be important for initial GA action. Based on this assumption, we have identified DNA methylation of the GAMyb locus in germinated rice seeds.

The B2 domain of VIVIPAROUS1 is bi-functional and regulates nuclear localization and transactivation

The transcriptional regulator VIVIPA-ROUS1 (VP1) is composed of four functional domains that control different aspects of gene expression during seed development. The B2 domain is required for its role as a transcriptional activator, functioning at the site of transcription and/or for its transport into the nucleus. Previous work showed that the B2 domain was required for transactivation of the Em promoter. We demonstrate that VP1::GFP localizes to the nucleus of barley (Hordeum vulgare) aleurone cells, but when B2 is deleted, nuclear accumulation is lost. However, the B2 domain itself is not sufficient for nuclear localization of GFP::GUS. Using point mutagenesis on the putative NLS within B2, we show that the VP1::GFP still accumulates in the nucleus. Utilizing a comparative approach, through the alignment of B2 domains from various VP1/ABI3 proteins, oincluding the ABI3 orthologs from Physcomitrella patens, revealed the involvement of other conserved amino acids. Mutating VP1 at the conserved threonine on the N-terminal side of the putative NLS and at a conserved arginine-glutamine-arginine sequence on the C-terminal side prevented nuclear localization of VP1. A single amino acid change, from alanine to threonine, within this NLS found in the Arabidopsis abi3-7 mutant prevents transcription of AtEm1 and AtEm6 in vivo. We show that this same mutation in VP1 prevents transactivation of the Em-GUS reporter in barley aleurone but does not interfere with nuclear localization. Our data demonstrate that the B2 domain of VP1 is bifunctional in nature regulating both nuclear localization and transactivation.

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.

Characterization Using Raman Microspectroscopy of Arabinoxylans in the Walls of Different Cell Types during the Development of Wheat Endosperm

The time course and pattern of arabinoxylan deposition in the wheat (Triticum aestivum) endosperm during grain development were studied using Raman spectroscopy. The presence of arabinoxylans (AX) is detected at the beginning of grain filling. At this stage, AX appear more substituted than at the later stages. Feruloylation of AX increases during the grain-filling stage, especially in the case of the aleurone layer. Whatever the stage of grain development, four populations of cells could be defined according to Raman arabinoxylan signatures. In the walls of the aleurone cells, AX appeared to be little substituted and highly esterified with phenolic acids. In the walls of prismatic cells, AX were found to be highly substituted and poorly esterified. Apart from aleurone and prismatic cells, the substitution degree of AX in endosperm was in the same range. Cells in the crease region were distinguished from cells in the starchy endosperm by their lower amount of esterified phenolic compounds.

Ternary complex formation between HvMYBS3 and other factors involved in transcriptional control in barley seeds

The SHAQKYF R1MYB transcription factor (TF) HvMYBS3 from barley is an activator of gene expression both during endosperm development and in aleurone cells upon seed germination. Its mRNA was detected as early as 10 days after flowering in developing barley endosperm, with a peak at 18 days, and in aleurone cells at 8 h after water imbibition, as shown by Northern blot and in situ hybridization analyses. The HvMYBS3 protein expressed in bacteria binds to oligonucleotides containing a GATA core derived from the promoters of: (i) the developing endosperm gene Itr1 (5'-GATAAGATA-3') encoding trypsin inhibitor BTI-CMe, and (ii) the post-germinating aleurone gene Amy6.4 (5'-TATCCAC-3'/5'-GTGGATA-3') encoding a high-pI alpha-amylase. Transient expression experiments in co-bombarded developing endosperms and in barley aleurone layers demonstrated that HvMYBS3 trans-activated transcription both from Itr1 and Amy6.4 promoters, in contrast with a previously reported seed-expressed R1MYB, HvMCB1, which was an activator of Itr1 and a transcriptional repressor of the Amy6.4 gene. In the yeast three-hybrid system, the HvMYBS3 protein formed a ternary complex with BPBF and BLZ2, two important seed TFs. However, no binary interactions could be detected between HvMYBS3 and BLZ2 or between HvMYBS3 and BPBF.

Surface Position, Not Signaling from Surrounding Maternal Tissues, Specifies Aleurone Epidermal Cell Fate in Maize

Maize (Zea mays) endosperm consists of an epidermal-like surface layer of aleurone cells, an underlying body of starchy endosperm cells, and a basal layer of transfer cells. To determine whether surrounding maternal tissues perform a role in specifying endosperm cell fates, a maize endosperm organ culture technique was established whereby the developing endosperm is completely removed from surrounding maternal tissues. Using cell type-specific fluorescence markers, we show that aleurone cell fate specification occurs exclusively in response to surface position and does not require specific, continued maternal signal input. The starchy endosperm and aleurone cell fates are freely interchangeable throughout the lifespan of the endosperm, with internalized aleurone cells converting to starchy endosperm cells and with starchy endosperm cells that become positioned at the surface converting to aleurone cells. In contrast to aleurone and starchy endosperm cells, transfer cells fail to develop in in vitro-grown endosperm, supporting earlier indications that maternal tissue interaction is required to fully differentiate this cell type. Several parameters confirm that the maize endosperm organ cultures described herein retain the main developmental features of in planta endosperm, including fidelity of aleurone mutant phenotypes, temporal and spatial control of cell type-specific fluorescent markers, specificity of cell type transcripts, and control of mitotic cell divisions.

Interactions of two abscisic‐acid induced WRKY genes in repressing gibberellin signaling in aleurone cells

Gibberellins (GA) promote while abscisic acid (ABA) inhibits seed germination and post-germination growth. To address the cross-talk of GA and ABA signaling, we studied two rice WRKY genes (OsWRKY51 and OsWRKY71) that are ABA-inducible and GA-repressible in embryos and aleurone cells. Over-expression of these two genes in aleurone cells specifically and synergistically represses induction of the ABA-repressible and GA-inducible Amy32b alpha-amylase promoter reporter construct (Amy32b-GUS) by GA or the GA-inducible transcriptional activator, GAMYB. The physical interactions of OsWRKY71 proteins themselves and that of OsWRKY71 and OsWRKY51 are revealed in the nuclei of aleurone cells using bimolecular fluorescence complementation (BiFC) assays. Although OsWRKY51 itself does not bind to the Amy32b promoter in vitro, it interacts with OsWRKY71 and enhances the binding affinity of OsWRKY71 to W boxes in the Amy32b promoter. The binding activity of OsWRKY71 is abolished by deleting the C-terminus containing the WRKY domain or substituting the key amino acids in the WRKY motif and the zinc finger region. However, two of these non-DNA-binding mutants are still able to repress GA induction by enhancing the binding affinity of the wild-type DNA-binding OsWRKY71 repressors. In contrast, the third non-DNA-binding mutant enhances GA induction of Amy32b-GUS, by interfering with the binding of the wild-type OsWRKY71 or the OsWRKY71/OsWRKY51 repressing complex. These data demonstrate the synergistic interaction of ABA-inducible WRKY genes in regulating GAMYB-mediated GA signaling in aleurone cells, thereby establishing a novel mechanism for ABA and GA signaling cross-talk.

α-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.

Hordoindolines are Predominantly Expressed in the Aleurone Layer in Late Kernel Development in Barley

In order to isolate grain-specific genes during kernel development, two differential screening methods—suppression subtractive hybridization (SSH) and differential hybridization (DH)—were applied. Two cDNAs encoding hordoindolines known to be related to grain hardness were isolated and designated as Hordeum vulgare indoline a and b (HvIDa and HvIDb). The cDNAs encoding HvIDa and HvIDb contained a 450 bp and a 444 bp open reading frame (ORF) that encoded the putative hordoindoline-a and -b precursors consisting of 150 and 148 amino acids, respectively. The deduced amino acid sequences of both HvIDa and HvIDb contained one tryptophan-rich domain and ten highly conserved cysteine residues. The expression of the HvIDa gene was high at 5 DAF, reached a peak at 8 DAF and decreased slightly until 20 DAF, while the expression of HvIDb began to be detectable at 8 DAF when it was higher than that of other developmental stages and decreased slightly until 20 DAF. The HvIDa and HvIDb genes were predominantly detected in the aleurone cell layers in the late part of kernel development, e.g. at 20 DAF. These findings may provide clues to the molecular mechanisms of kernel development and contribute to the determination of the grain texture in barley.

HvMCB1, a R1MYB transcription factor from barley with antagonistic regulatory functions during seed development and germination

The functional analysis of hydrolase gene promoters induced by gibberellin (GA) in barley aleurone cells upon germination has identified a tripartite GA-response complex (GARC) containing a 5'-TATCCAC-3' box as well as the GA-responsive element (GARE) recognized by GAMYB and the pyrimidine box interacting with the DOF transcription factors BPBF and SAD. We show here that the MCB1 gene encoding a R1MYB protein binds to the 5'-TATCCAC-3' (GATA core) box in vitro and is a transcriptional repressor of a GA-induced amylase (Amy6.4) promoter in bombarded aleurone layers. Northern blot and mRNA in situ hybridization analyses showed that the MCB1 transcripts accumulate in the aleurone cells upon germination, as well as in endosperm tissues during seed development. The HvMCB1 protein expressed in bacteria binds in a specific manner to a 27-mer oligonucleotide containing the 5'-TATCCAC-3' sequence, derived from the promoter region of the Amy6.4 gene. Accumulation of the MCB1 transcript diminished in response to external GA incubation in aleurone cells, and in transient expression experiments HvMCB1 repressed transcription of the Amy6.4 promoter in GA-treated aleurone layers and reversed the GAMYB-mediated activation of this amylase promoter. In contrast, during endosperm maturation HvMCB1 acted as a transcription activator of the seed-specific Itr1 gene promoter through binding to a 5'-GATAAGATA-3' box.

Aleurone Cell Identity Is Suppressed following Connation in Maize Kernels

Expression of the cytokinin-synthesizing isopentenyl transferase enzyme under the control of the Arabidopsis (Arabidopsis thaliana) SAG12 senescence-inducible promoter reverses the normal abortion of the lower floret from a maize (Zea mays) spikelet. Following pollination, the upper and lower floret pistils fuse, producing a connated kernel with two genetically distinct embryos and the endosperms fused along their abgerminal face. Therefore, ectopic synthesis of cytokinin was used to position two independent endosperms within a connated kernel to determine how the fused endosperm would affect the development of the two aleurone layers along the fusion plane. Examination of the connated kernel revealed that aleurone cells were present for only a short distance along the fusion plane whereas starchy endosperm cells were present along most of the remainder of the fusion plane, suggesting that aleurone development is suppressed when positioned between independent starchy endosperms. Sporadic aleurone cells along the fusion plane were observed and may have arisen from late or imperfect fusion of the endosperms of the connated kernel, supporting the observation that a peripheral position at the surface of the endosperm and not proximity to maternal tissues such as the testa and pericarp are important for aleurone development. Aleurone mosaicism was observed in the crown region of nonconnated SAG12-isopentenyl transferase kernels, suggesting that cytokinin can also affect aleurone development.