Cereal Aleurone Layer Research Articles

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.

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.

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.

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.

Global Patterns of Gene Expression in the Aleurone of Wild-Type and dwarf1 Mutant Rice

The cereal aleurone layer is a model system for studying the regulation of transcription by gibberellin (GA) and abscisic acid (ABA). GA stimulates and ABA prevents the transcription of genes for alpha-amylases and other secreted hydrolytic enzymes, but how GA and ABA affect the transcription of other genes is largely unknown. We characterized gene expression in rice (Oryza sativa) aleurone using a half-genome rice microarray. Of the 23,000 probe sets on the chip, approximately 11,000 hybridized with RNA from rice aleurone treated with ABA, GA, or no hormone. As expected, GA regulated the expression of many genes, and 3 times as many genes were up-regulated by GA at 8 h than were down-regulated. Changes in gene expression resulting from ABA treatment were not consistent with the hypothesis that the role of ABA in this tissue is primarily to repress gene expression, and 10 times more genes were up-regulated by ABA at 8 h than were down-regulated by ABA. We also measured transcript abundance in aleurone of dwarf1 (d1) mutant rice. The d1 protein is the sole alpha-subunit of heterotrimeric G-proteins in rice. Genes up-regulated by GA or ABA had higher expression in wild type than in d1 aleurone, and genes down-regulated by GA had lower expression in wild type relative to d1 aleurone. The d1 mutation did not result in a decrease in sensitivity to GA at the level of transcription. Rather, changes in transcript abundance were smaller in the d1 mutant than in wild type.

Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar‐dependent and gibberellin action involves SLENDER1 but not GAMYB

Storage oil is a major constituent in the cereal aleurone layer. The aim of this study was to investigate how gibberellin (GA) and abscisic acid (ABA) regulate conversion of oil to sugar in barley aleurone. The activity of the glyoxylate cycle enzyme isocitrate lyase (ICL) was surveyed in eight barley cultivars. Surprisingly, some cultivars do not require GA for the induction of ICL (e.g. Himalaya), whereas some do (e.g. Golden Promise). Furthermore, in Golden Promise, GA also stimulates triacylglycerol breakdown and enhances the net flux of carbon from acetate to sugar. In contrast, ABA strongly represses ICL activity and the flux of carbon from oil to sugar in both Golden Promise and Himalaya. Biolistics using a promoter reporter showed that GA and ABA regulate ICL at the level of transcription. Studies using barley and rice mutants and pharmacological agents show that GA-dependent induction of ICL activity is mediated by SLENDER1 and requires cGMP, but does not involve the transcription factor GAMYB. Gibberellin and ABA therefore act antagonistically to regulate gluconeogenesis in the aleurone layer as well as controlling the production and secretion of hydrolases into the starchy endosperm. We suggest that the variation between different barley cultivars might be a result of selective breeding to alter seed dormancy.

Functional dissections between GAMYB and Dof transcription factors suggest a role for protein-protein associations in the gibberellin-mediated expression of the RAmy1A gene in the rice aleurone.

In the germinated cereal aleurone layer, gibberellic acids (GA) induce expression of a number of genes encoding hydrolytic enzymes that participate in the mobilization of stored molecules. Previous analyses suggest that the key events controlling the GA-regulated gene expression in the aleurone are formation of active transcription machinery referred to as the GA responsive complex, followed by recruiting GAMYB. In general, bipartite promoter contexts composed of the GA-responsive element and the pyrimidine box are observed within the regulatory regions of cereal GA-responsive genes. Protein factors that recognize each promoter sequence were identified and distinct effects on the GA-mediated activation of gene expression have been also investigated; however, the connection and intercalation between two promoter motifs remain obscure. In this study, I have evaluated cooperative function of GAMYB and a pyrimidine box-binding protein OsDOF3 that influenced the promoter activity of the most predominant GA-responsive gene (RAmy1A) of rice (Oryza sativa). Transient expression of OsDOF3 in the germinated aleurone prolonged GAMYB function on the reporter expression in the absence of GA. The synergistic effect required a set of DNA bindings of two proteins on the RAmy1A promoter region. The yeast two-hybrid assay showed the physical interaction of GAMYB and OsDOF3 in yeast cells, indicating that the association of GAMYB and OsDOF3 may be a functional unit in transcription regulation. The results showed the accessory function of OsDOF3 responsible for a dosage-dependent mediation of GA signaling that leads to high-level expression of physiological target genes.

Crosstalk Between Gibberellin and Abscisic Acid in Cereal Aleurone

The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In cereal aleurone layers, the expression of genes encoding hydrolytic enzymes needed for seedling growth, such as α-amylases and proteases, is induced by GA but suppressed by ABA. In addition, ABA induces the expression of genes that may play a role in the establishment of stress tolerance. Because of these well-defined biochemical and molecular markers, the cereal aleurone layers have been used as a convenient system for studying GA/ABA actions. Both gain- and loss-of-function approaches have been followed by the constitutive or the RNAi-mediated knockdown expression of specific regulatory molecules. The GA signaling pathway is anchored by the transcription factor GAMyb, which interacts with a specific region in the promoter of GA up-regulated genes, and an upstream regulatory molecule SLN1 (SLR1) that appears to be a functional homolog of the Arabidopsis GAI/RGA regulatory proteins. It is established that the ABA induction and suppression of gene expression follow two distinct signaling pathways, with the former requiring a transcription factor ABI5, but inhibited by a protein phosphatase 2C, and the latter mediated by a protein kinase PKABA1 and an unknown factor. The ABA suppression action has been pinpointed to be upstream from the formation of functional GAMyb but downstream from the site of action of SLN1 (SLR1).

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.

Hormonal regulation of a cysteine proteinase gene, EPB-1, in barley aleurone layers: cis- and trans-acting elements involved in the co-ordinated gene expression regulated by gibberellins and abscisic acid.

The synthesis of EPB, a cysteine proteinase responsible for the degradation of seed endosperm storage proteins in barley (Hordeum vulgare), is induced by gibberellins (GA) and repressed by abscisic acid (ABA). The EPB gene family consists of two very similar members, EPB-1 and EPB-2, with the former being more highly induced by GA. We have functionally characterized the cis-acting elements in the EPB-1 promoter and determined that a gibberellin response element (GARE), a pyrimidine box and an upstream element are necessary for GA induction. By comparison with the promoters of alpha-amylase genes, which are also induced by GA, we suggest that GARE is coupled with the upstream element and the pyrimidine box to form a GA response complex. In addition, we have shown that the 3'-untranslated/untranscribed region of the EPB-1 gene is required for a low background expression in the absence of GA. Constitutive expression of a transcription factor, GAMyb, in the absence of GA leads to the transactivation of EPB-1 expression in a dosage dependent manner with the highest level comparable to that in fully GA-induced tissue. Co-expression of a truncated version of GAMyb containing only the DNA binding domain blocks the GA-induction of EPB-1, further supporting the role of GAMyb in the regulation of gene expression. Although ABA is very effective in blocking the GA induction of EPB-1, it has no effect on the GAMyb-mediated expression of EPB-1. We suggest that ABA acts upstream of the formation of functional GAMyb which co-ordinates the hormonal regulation of a diverse group of genes in cereal aleurone layers, including those encoding EPB and alpha-amylases.

An abscisic acid-induced protein kinase, PKABA1, mediates abscisic acid-suppressed gene expression in barley aleurone layers.

The phytohormone abscisic acid (ABA) induces genes-encoding proteins involved in desiccation tolerance and dormancy in seeds, but ABA also suppresses gibberellin (GA)-responsive genes encoding hydrolytic enzymes essential for postgermination growth. A unique serine/threonine protein kinase, PKABA1 mRNA, up-regulated by ABA in seeds, has been identified. In this report, the effect of PKABA1 on the signal transduction pathway mediating ABA induction and suppression of genes has been determined in aleurone layers of barley seeds. Two groups of gene constructs were introduced to barley aleurone layers by using particle bombardment: the reporter constructs containing the coding sequence of beta-glucuronidase gene linked to hormone-responsive promoters and the effector constructs containing the coding region of protein kinases linked to a constitutive promoter. Constitutive expression of PKABA1 drastically suppressed expression of low- and high-pI alpha-amylase and protease genes induced by GA. However, the presence of PKABA1 had only a small effect on the ABA induction of a gene encoding a late embryogenesis abundant protein, HVA1. Our results indicate that PKABA1 acts as a key intermediate in the signal transduction pathway leading to the suppression of GA-inducible gene expression in cereal aleurone layers.

Hormonal signalling in cereal aleurone

The cereal aleurone is an excellent model system for hormonal signalling in plants. When treated with gibberellins (GAs), cereal aleurone layers and aleurone protoplasts initiate signalling cascades that result in the synthesis and secretion of hydrolytic enzymes, most notably α-amylases. Abscisic acid (ABA) antagonizes the effects of GA and stimulates the production of ABA-up-regulated proteins. Receptors for GA and ABA have been localized to the aleurone cell PM, and evidence suggests that another ABA receptor functions within the cytoplasm. Cytosolic and membrane-bound second messengers have been identified and signal transduction pathways are beginning to be understood. Transacting factors that regulate the transcription of hormonally induced genes have been cloned and bring us closer to linking cytosolic signals to changes in gene expression. In this review, recent data pertaining to hormonal signalling in cereal aleurone are summarized. Relationships between signalling and effector molecules are discussed, and models for hormone-induced signalling pathways are proposed.

Plant Growth Hormones: Internal Control Points

Examples of control points involved in regulation of the availability or action of certain plant hormones are presented. The effects of divalent ions, environmental factors, and other hormones on hormone synthesis, release, or action are illustrated The role of gibberellins (GAs) in promoting cell enlargement and division in intact plants is analyzed in view of the paradoxical inhibition of cell division in tissue and suspension culture by GA3. The importance of the intact subapical region as a target site is underlined, but the possibility is raised that GAs play only an indirect role in regulation of cell multiplication at this site. The cereal aleurone layer as a target site for GA action is discussed with the objective of identifying the primary control points. Analysis of existing information most favors a role for GAs in (1) stimulating synthesis of mRNA, and (2) intervening in transcription or translation. There is a paucity of evidence for specific binding sites for plant hormones. With the except...

Early effects of gibberellic acid on barley aleurone layers

The most obvious effects resulting from the application of the hormone gibberellic acid (GA) to cereal aleurone layers are the secretion of metabolites and the de novo synthesis and secretion into the medium of enzymes, mostly hydrolases ( Varner, 1964; Varner et al., 1965; Chrispeels and Varner, 1967 a,b; Jacobsen and Varner, 1967). For that reason research on the mode of action of GA has focused on the elucidation of the roles of protein synthesis and RNA synthesis in the responses. The present report follows observations that the effects of GA occur sequentially ( Pollard, 1968). Furthermore, we have found that GA enhances the secretion of more than 20 metabolites and enzymes. It is desirable to identify the earliest of these reponses since they are most likely to be near the primary action of the hormone. We wish to report (1) the effect of GA on the secretion of soluble carbohydrates and several phosphatases, the earliest effects it has been possible to detect and (2) data relating the effects of the hormone to other aspects of metabolism, especially protein synthesis, RNA synthesis and respiration.