Cereal Aleurone Research Articles

Endosperm cell death: roles and regulation in angiosperms.

Double fertilization in angiosperms results in the formation of a second zygote, the fertilized endosperm. Unlike its embryo sibling, the endosperm is a transient structure that eventually undergoes developmentally controlled programmed cell death (PCD) at specific time points of seed development or germination. The nature of endosperm PCD exhibits a considerable diversity, both across different angiosperm taxa and within distinct endosperm tissues. In endosperm-less species, PCD might cause central cell degeneration as a mechanism preventing the formation of a fertilized endosperm. In most other angiosperms, embryo growth necessitates the elimination of surrounding endosperm cells. Nevertheless, complete elimination of the endosperm is rare and, in most cases, specific endosperm tissues persist. In mature seeds, these persisting cells may be dead, such as the starchy endosperm in cereals, or remain alive to die only during germination, like the cereal aleurone or the endosperm of castor beans. In this review, we explore current knowledge surrounding the cellular, molecular, and genetic aspects of endosperm PCD, and the influence environmental stresses have on PCD processes. Overall, this review provides an exhaustive overview of endosperm PCD processes in angiosperms, shedding light on its diverse mechanisms and its significance in seed development and seedling establishment.

Aquaporin activity of barley tonoplast intrinsic proteins is involved in the delay of the coalescence of protein storage vacuoles in aleurone cells

The coalescence of protein storage vacuoles (PSVs) is one of the most prominent cellular changes occurring in cereal aleurone cells during germination. This structural change is highly coupled with the functional transition of this organelle from a storage compartment to a lytic section. Gibberellic acid (GA) promotes this process, whereas abscisic acid (ABA) prevents it. Previously, we demonstrated that ABA-inducible HvTIP3;1 plays a decisive role in ABA-mediated prevention of PSV fusion. In this follow-up study, we examined whether the aquaporin activity of tonoplast intrinsic protein (TIP) is related to its preventive effect on PSV fusion using various functional mutants. The defective forms of aquaporin (HvTIP3;1m and HvTIP3;1ΔNPA-GFPs for HvTIP3;1, and HvTIP1;2m for HvTIP1;2) were found to be less effective than the usual form in delaying the PSV fusion process occurring in GA-treated cells. In contrast, overexpression of HvTIP3;1m reduced the preventive effect of ABA on PSV fusion. Upon inhibition of aquaporin activity using mercury, PSV fusion occurred to a greater extent in ABA-treated barley protoplasts. These data suggest that the aquaporin activity of TIP is involved in the deterrent effect of TIP on PSV coalescence. TIP3-GFP barley transgenic seeds showed prolonged expression of the TIP3;1 transcript. Moreover, PSV fusion progressed at a much slower rate compared to wild type. Additionally, the degradation of storage proteins was not as efficient, suggesting that a metamorphic transition of PSVs to lytic organelles is closely correlated with the disappearance of HvTIPs and the PSV fusion process.

Plant Stature Related receptor-like Kinanse2 (PSRK2) acts as a factor that determines stem elongation toward gibberellins response in rice.

Gibberellins (GAs) are a family of plant hormones that are important to multiple aspects of plant growth and development, especially stem elongation. A PSRK2 was obtained through screening and identifying RLK dominant negative mutants. Phenotype of the loss-of-function mutants, psrk2-DN and psrk2-RNAi, showed that PSRK2 could influence the length of the uppermost and fourth internodes, indicating that PSRK2 might regulate cell division in the intercalary meristems and/or cell elongation in the internodes. Moreover, the expression pattern showed that PSRK2 was strongly expressed in the joined-nodes after the start-up of reproductive growth, but undetectable in leaves. PSRK2 expression was also found to be induced by GA3, and PSRK2 was involved in GA signaling in cereal aleurone cells, and PSRK2 influence the relative length of the second leaf sheaths in seedling stage. These results indicate PSRK2 is a component of GA signaling pathway that controls stem elongation by negatively regulating GA responses. Abbreviations: Os: Oryza sativa; At: Arabidopsis thaliana; RNAi: RNA interfere; DN: Dominate Negative; SMART: Simple Modular Architecture Research Tool; Uni : Uniconazol; PSRK2: Plant Stature Related receptor-like Kinase 2; RLK: Receptor-like Kinase; GA: Gibberellin; IAA: indole-3-acetic acid; BL: Brassinosteroid.

Histone Deacetylase Is Required for GA-Induced Programmed Cell Death in Maize Aleurone Layers.

Recent discoveries have shown that epigenetic regulation is an integral part of phytohormone-mediated processes. The phytohormone gibberellin (GA) triggers a series of events in cereal aleurone cells that lead to programmed cell death (PCD), but the signaling cascade mediating GA-induced PCD in cereal aleurone layers remains largely unknown. Here, we showed that histone deacetylase (HDAC) activity gradually increased relative to histone acetyltransferase (HAT) activity, leading to a global decrease in histone H3 and H4 acetylation levels during PCD of maize (Zea mays) embryoless aleurone layers after 3 d of treatment with GA. HDAC inhibition prevented GA-induced PCD in embryoless aleurone cells, whereas HAT inhibition resulted in PCD even in the absence of GA. Hydrogen peroxide concentrations increased in GA- or HAT inhibitor-treated aleurone cells due to reduced levels of reactive oxygen species scavengers. Hydrogen peroxide-treated aleurone cells showed no changes in the activity or expression of HATs and HDACs. We show that it is possible to predict whether epigenetic modification enzymes serve as a regulator of the GA-triggered PCD signaling pathway in maize aleurone layers. Taken together, these findings reveal that HDAC activity is required for GA-induced PCD in maize aleurone layers and regulates PCD via the reactive oxygen species-mediated signal transduction pathway.

Heme Oxygenase-1 Delays Gibberellin-Induced Programmed Cell Death of Rice Aleurone Layers Subjected to Drought Stress by Interacting with Nitric Oxide.

Cereal aleurone layers undergo a gibberellin (GA)-regulated process of programmed cell death (PCD) following germination. Heme oxygenase-1 (HO-1) is known as a rate-liming enzyme in the degradation of heme to biliverdin IXα, carbon monoxide (CO), and free iron ions (Fe2+). It is a critical component in plant development and adaptation to environment stresses. Our previous studies confirmed that HO-1 inducer hematin (Ht) promotes the germination of rice seeds in drought (20% polyethylene glycol-6000, PEG) conditions, but the corresponding effects of HO-1 on the alleviation of germination-triggered PCD in GA-treated rice aleurone layers remain unknown. The present study has determined that GA co-treated with PEG results in lower HO-1 transcript levels and HO activity, which in turn results in the development of vacuoles in aleurone cells, followed by PCD. The pharmacology approach illustrated that up- or down-regulated HO-1 gene expression and HO activity delayed or accelerated GA-induced PCD. Furthermore, the application of the HO-1 inducer Ht and nitric oxide (NO) donor sodium nitroprusside (SNP) not only activated HO-1 gene expression, HO activity, and endogenous NO content, but also blocked GA-induced rapid vacuolation and accelerated aleurone layers PCD under drought stress. However, both HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) and NO scavenger 2-(4-carboxyphenyl0-4, 4,5,5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO) reserved the effects of Ht and SNP on rice aleurone layer PCD under drought stress by down-regulating endogenous HO-1 and NO, respectively. The inducible effects of Ht and SNP on HO-1 gene expression, HO activity, and NO content were blocked by cPTIO. Together, these results clearly suggest that HO-1 is involved in the alleviation of GA-induced PCD of drought-triggered rice aleurone layers by associating with NO.

Histone Acetylation is Involved in Gibberellin-Regulated sodCp Gene Expression in Maize Aleurone Layers.

The cereal aleurone layer plays an important role in seed germination, and reactive oxygen species (ROS) in aleurone layers act as crucial signal molecules in this progression. Recent studies have revealed that epigenetic modification is involved in plant development and seed germination. However, little is known about a possible relationship between histone modification and the ROS signaling pathway in cereal aleurone layers during seed germination. Here, we found that the expression of both histone acetyltransferases (HATs) and histone deacetylases (HDACs) was increased gradually during seed germination, accompanied by an increase in global acetylation levels of histones H3 and H4 in maize aleurone layers. The acetylation was found to be promoted by GA(3) and suppressed by ABA. However, when the HDAC inhibitor trichostatin A (TSA) was used, the increased H3K9ac and H4K5ac level correlated with an inhibition of the germination. These results indicated that the overall histone acetylation in the aleurone layers is not required for germination. Similarly these two hormones, GA(3) and ABA, exerted opposed effects on the expression of the ROS-related gene sodCp. Furthermore, chromatin immunoprecipitation experiments showed that the promoter region of the sodCp gene was hyperacetylated during germination, and this acetylation was promoted by GA(3) and inhibited by both ABA and TSA. These results suggested that GA(3)-mediated expression of the sodCp gene in aleurone layers is associated with histone hyperacetylation on the promoter and coding region of this gene, consequently leading to an accumulation of H(2)O(2) which regulated production of α-amylase during seed germination.

Comprehensive gene expression analysis of rice aleurone cells: probing the existence of an alternative gibberellin receptor.

Current gibberellin (GA) research indicates that GA must be perceived in plant nuclei by its cognate receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1). Recognition of GA by GID1 relieves the repression mediated by the DELLA protein, a model known as the GID1-DELLA GA perception system. There have been reports of potential GA-binding proteins in the plasma membrane that perceive GA and induce α-amylase expression in cereal aleurone cells, which is mechanistically different from the GID1-DELLA system. Therefore, we examined the expression of the rice (Oryza sativa) α-amylase genes in rice mutants impaired in the GA receptor (gid1) and the DELLA repressor (slender rice1; slr1) and confirmed their lack of response to GA in gid1 mutants and constitutive expression in slr1 mutants. We also examined the expression of GA-regulated genes by genome-wide microarray and quantitative reverse transcription-polymerase chain reaction analyses and confirmed that all GA-regulated genes are modulated by the GID1-DELLA system. Furthermore, we studied the regulatory network involved in GA signaling by using a set of mutants defective in genes involved in GA perception and gene expression, namely gid1, slr1, gid2 (a GA-related F-box protein mutant), and gamyb (a GA-related trans-acting factor mutant). Almost all GA up-regulated genes were regulated by the four named GA-signaling components. On the other hand, GA down-regulated genes showed different expression patterns with respect to GID2 and GAMYB (e.g. a considerable number of genes are not controlled by GAMYB or GID2 and GAMYB). Based on these observations, we present a comprehensive discussion of the intricate network of GA-regulated genes in rice aleurone cells.

Differentiation mechanism and function of the cereal aleurone cells and hormone effects on them.

The cereal aleurone cells differentiate from the endosperm epidermis with the exception of endosperm transfer cells. Aleurone cells contain proteins, lipids, and minerals, and are important for digesting the endosperm storage products to nurse the embryo under effects of several hormones during the seed germination. The differentiation of aleurone cells is related to location effect and special gene expression. Moreover, the differentiation of aleurone cells is probably affected by the cues from maternal tissues. In the paper, differentiation mechanism and function of aleurone cells and hormone effects on them are reviewed. Some speculations about the differentiation mechanism of aleurone cells are given here.

Programmed cell death in barley aleurone cells is not directly stimulated by reactive oxygen species produced in response to gibberellin

The cereal aleurone layer is a secretory tissue that produces enzymes to hydrolyze the starchy endosperm during germination. We recently demonstrated that reactive oxygen species (ROS), produced in response to gibberellins (GA), promoted GAMyb expression, which induces α-amylase expression in barley aleurone cells. On the other hand, ROS levels increase during programmed cell death (PCD) in barley aleurone cells, and GAMyb is involved in PCD of these cells. In this study, we investigated whether the ROS produced in response to GA regulate PCD directly by using mutants of Slender1 (SLN1), a DELLA protein that negatively regulates GA signaling. The wild-type, the sln1c mutant (which exhibits gibberellin-type signaling even in the absence of GA), and the Sln1d mutant (which is gibberellin-insensitive with respect to α-amylase production) all produced ROS in response to GA, suggesting that ROS production in aleurone cells in response to GA is independent of GA signaling through this DELLA protein. Exogenous GA promoted PCD in the wild-type. PCD in sln1c was induced even without exogenous GA (and so without induction of ROS), whereas PCD in Sln1d was not induced in the presence of exogenous GA, even though the ROS content increased significantly in response to GA. These results suggest that PCD in barley aleurone cells is not directly stimulated by ROS produced in response to GA but is regulated by GA signaling through DELLA protein.

TamiR159 Directed Wheat TaGAMYB Cleavage and Its Involvement in Anther Development and Heat Response

In Arabidopsis and rice, miR159-regulated GAMYB-like family transcription factors function in flower development and gibberellin (GA) signaling in cereal aleurone cells. In this study, the involvement of miR159 in the regulation of its putative target TaGAMYB and its relationship to wheat development were investigated. First, we demonstrated that cleavage of TaGAMYB1 and TaGAMYB2 was directed by miR159 using 5′-RACE and a transient expression system. Second, we overexpressed TamiR159, TaGAMYB1 and mTaGAMYB1 (impaired in the miR159 binding site) in transgenic rice, revealing that the accumulation in rice of mature miR159 derived from the precursor of wheat resulted in delayed heading time and male sterility. In addition, the number of tillers and primary branches in rice overexpressing mTaGAMYB1 increased relative to the wild type. Our previous study reported that TamiR159 was downregulated after two hours of heat stress treatment in wheat (Triticum aestivum L.). Most notably, the TamiR159 overexpression rice lines were more sensitive to heat stress relative to the wild type, indicating that the downregulation of TamiR159 in wheat after heat stress might participate in a heat stress-related signaling pathway, in turn contributing to heat stress tolerance.

Presence of base excision repair enzymes in the wheat aleurone and their activation in cells undergoing programmed cell death

Cereal aleurone cells are specialized endosperm cells that produce enzymes to hydrolyze the starchy endosperm during germination. Aleurone cells can undergo programmed cell death (PCD) when incubated in the presence of gibberellic acid (GA) in contrast to abscisic acid (ABA) which inhibits the process. The progression of PCD in aleurone layer cells of wheat grain is accompanied by an increase in deoxyribonuclease (DNase) activities and the internucleosomal degradation of nuclear DNA. Reactive oxygen species (ROS) are increased during PCD in the aleurone cells owing to the β-oxidation of triglycerides and inhibition of the antioxidant enzymes possibly leading to extensive oxidative damage to DNA. ROS generate mainly non-bulky DNA base lesions which are removed in the base excision repair (BER) pathway, initiated by the DNA glycosylases. At present, very little is known about oxidative DNA damage repair in cereals. Here, we study DNA repair in the cell-free extracts of wheat aleurone layer incubated or not with phytohormones. We show, for the first time, the presence of 8-oxoguanine-DNA and ethenoadenine-DNA glycosylase activities in wheat aleurone cells. Interestingly, the DNA glycosylase and AP endonuclease activities are strongly induced in the presence of GA. Based on these data we propose that GA in addition to activation of nuclear DNases also induces the DNA repair activities which remove oxidized DNA bases in the BER pathway. Potential roles of the wheat DNA glycosylases in GA-induced oligonucleosomal fragmentation of DNA and metabolic activation of aleurone layer cells via repair of transcribed regions are discussed.

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.

Loss-of-function of DELLA protein SLN1 activates GA signaling in barley aleurone

Gibberellic acid (GA) is an important signaling molecule that participates in many aspects of plant growth and development. While the importance of this hormone is clear, the transcriptional regulatory networks involved are still being characterized. The cereal aleurone, particularly the barley aleurone, has been used as a classic model to study GA and GA signaling for many years, and these studies have significantly contributed to our understanding of GA in plant biology. The objective of this study was to characterize the transcripts regulated through the DELLA protein SLN1, a negative regulator of the GA signaling pathway. To detect the transcripts, Affymetrix Barley 1 GeneChips were hybridized with RNA extracted from barley aleurone treated with GA or aleurone of the DELLA mutant sln1c without GA treatment. The transcripts detected, in term of both expressed genes and their function, were highly similar between the GA-treatment and the sln1c mutant. These results from a genome-wide transcript analysis provide evidence that SLN1 in the GA signal transduction pathway controls almost all GA-induced genes in the barley aleurone.

Gibberellin Modulates Anther Development in Rice via the Transcriptional Regulation of GAMYB

Gibberellins (GAs) play important roles in regulating reproductive development, especially anther development. Our previous studies revealed that the MYB transcriptional factor GAMYB, an important component of GA signaling in cereal aleurone cells, is also important for anther development. Here, we examined the physiological functions of GA during anther development through phenotypic analyses of rice (Oryza sativa) GA-deficient, GA-insensitive, and gamyb mutants. The mutants exhibited common defects in programmed cell death (PCD) of tapetal cells and formation of exine and Ubisch bodies. Microarray analysis using anther RNAs of these mutants revealed that rice GAMYB is involved in almost all instances of GA-regulated gene expression in anthers. Among the GA-regulated genes, we focused on two lipid metabolic genes, a cytochrome P450 hydroxylase CYP703A3 and beta-ketoacyl reductase, both of which might be involved in providing a substrate for exine and Ubisch body. GAMYB specifically interacted with GAMYB binding motifs in the promoter regions in vitro, and mutation of these motifs in promoter-beta-glucuronidase (GUS) transformants caused reduced GUS expression in anthers. Furthermore, a knockout mutant for CYP703A3 showed gamyb-like defects in exine and Ubisch body formation. Together, these results suggest that GA regulates exine formation and the PCD of tapetal cells and that direct activation of CYP703A3 by GAMYB is key to exine formation.

An Abscisic Acid-Induced Protein, HVA22, Inhibits Gibberellin-Mediated Programmed Cell Death in Cereal Aleurone Cells

Plant HVA22 is a unique abscisic acid (ABA)/stress-induced protein first isolated from barley (Hordeum vulgare) aleurone cells. Its yeast homolog, Yop1p, functions in vesicular trafficking and in the endoplasmic reticulum (ER) network in vivo. To examine the roles of plant HVA22, barley HVA22 was ectopically expressed in barley aleurone cells. Overexpression of HVA22 proteins inhibited gibberellin (GA)-induced formation of large digestive vacuoles, which is an important aspect of GA-induced programmed cell death in aleurone cells. The effect of HVA22 was specific, because overexpression of green fluorescent protein or another ABA-induced protein, HVA1, did not lead to the same effect. HVA22 acts downstream of the transcription factor GAMyb, which activates programmed cell death and other GA-mediated processes. Moreover, expression of HVA22:green fluorescent protein fusion proteins showed network and punctate fluorescence patterns, which were colocalized with an ER marker, BiP:RFP, and a Golgi marker, ST:mRFP, respectively. In particular, the transmembrane domain 2 was critical for protein localization and stability. Ectopic expression of the most phylogenetically similar Arabidopsis (Arabidopsis thaliana) homolog, AtHVA22D, also resulted in the inhibition of vacuolation to a similar level as HVA22, indicating function conservation between barley HVA22 and some Arabidopsis homologs. Taken together, we show that HVA22 is an ER- and Golgi-localized protein capable of negatively regulating GA-mediated vacuolation/programmed cell death in barley aleurone cells. We propose that ABA induces the accumulation of HVA22 proteins to inhibit vesicular trafficking involved in nutrient mobilization to delay coalescence of protein storage vacuoles as part of its role in regulating seed germination and seedling growth.

High level of conservation between genes coding for the GAMYB transcription factor in barley (Hordeum vulgare L.) and bread wheat (Triticum aestivum L.) collections

The transcription factor GAMYB is involved in gibberellin signalling in cereal aleurone cells and in plant developmental processes. Nucleotide diversity of HvGAMYB and TaGAMYB was investigated in 155 barley (Hordeum vulgare) and 42 wheat (Triticum aestivum) accessions, respectively. Polymorphisms defined 18 haplotypes in the barley collection and 1, 7 and 3 haplotypes for the A, B, and D genomes of wheat, respectively. We found that (1) Hv- and TaGAMYB genes have identical structures. (2) Both genes show a high level of nucleotide identity (>95%) in the coding sequences and the distribution of polymorphisms is similar in both collections. At the protein level the functional domain is identical in both species. (3) GAMYB genes map to a syntenic position on chromosome 3. GAMYB genes are different in both collections with respect to the Tajima D statistic and linkage disequilibrium (LD). A moderate level of LD was observed in the barley collection. In wheat, LD is absolute between polymorphic sites, mostly located in the first intron, while it decays within the gene. Differences in Tajima D values might be due to a lower selection pressure on HvGAMYB, compared to its wheat orthologue. Altogether our results provide evidence that there have been only few evolutionary changes in Hv- and TaGAMYB. This confirms the close relationship between these species and also highlights the functional importance of this transcription factor.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-008-0777-4) contains supplementary material, which is available to authorized users.

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.

Enrichment and Identification of Integral Membrane Proteins from Barley Aleurone Layers by Reversed-Phase Chromatography, SDS-PAGE, and LC−MS/MS

The plasma membrane of the cereal aleurone layer is the site of perception of germination signals and release of enzymes to the starchy endosperm. Analysis of membrane proteins is challenging due to their hydrophobicity and low abundance; thus, little is known about the membrane proteins involved in seed germination. A membrane fraction highly enriched for the plasma membrane H+-ATPase was prepared from barley aleurone layers by aqueous two-phase partitioning. Because detergent and salt washes did not efficiently remove soluble proteins from the membrane preparations, an alternative procedure was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral membrane proteins with relatively low levels of soluble contaminating proteins. Forty-six proteins associated with barley aleurone plasma membranes were identified, including proteins with more than 10 transmembrane domains. Among the identified proteins were two new isoforms of the plasma membrane H+-ATPase, two proteins possibly involved in ion-channel regulation, and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination using a proteomics approach.

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.

Transcriptional Responses to Gibberellin and Abscisic Acid in Barley Aleurone

Cereal aleurone has been established as a model system to investigate giberrellin (GA) and abscisic acid (ABA) responses. Using Barley 1 GeneChip, we examined the mRNA accumulation of over 22 000 genes in de-embryonated barley aleurone treated with GA and ABA. We observed that 1 328 genes had more than a threefold change in response to GA treatment, whereas 206 genes had a more than threefold change in response to ABA treatment. Interestingly, approximately 2.5-fold more genes were up-regulated than down-regulated by ABA. Eighty-three genes were differentially regulated by both GA and ABA. Most of the genes were subject to antagonistic regulation by ABA and GA, particularly for genes related to seed maturation and germination, such as genes encoding late embryogenesis abundant proteins and storage mobilization enzymes. This supports the antagonistic roles of GA and ABA in seed maturation and seed germination. Interestingly, we observed that a significant percentage of the genes were coordinately regulated by both GA and ABA. Some GA-responsive genes encoded proteins involved in ethylene, jasmonate, brassinosteroid and auxin metabolic and signaling transduction pathways, suggesting their potential interaction with the GA response. We also identified a group of transcription factor genes, such as MYB and Homeobox genes, that were differentially regulated by GA. In addition, a number of GA- and/or ABA-responsive genes encoded components potentially involved in GA and ABA signal transduction pathway. Overall, the present study provides a comprehensive and global view of transcript expression accompanying the GA and ABA response in barley aleurone and identifies a group of genes with potential regulatory functions in GA- and ABA-signaling pathways for future functional validation. (Managing editor: Li-Hui Zhao)