Barley Aleurone Layers Research Articles

Barley metallothionein isoforms, MT2b2 and MT4, differentially respond to photohormones in barley aleurone layer and their recombinant forms show different affinity for binding to zinc and cadmium.

Metallothioneins (MTs) are metal-binding proteins that have important roles in the homeostasis of heavy metals. In this study, the two MT genes was studied in response to phytohormones using the barley aleurone layer as a kind of model system. The aleurone layer was isolated from barley embryo-less half grains and was incubated for 24h with different phytohormones. Based on the results the genes encoding HvMT2b2 and HvMT4 were down-regulated through gibberellic acid (GA), while they were and up-regulated through salicylic acid (SA). Despite this, these two genes were differentially expressed to other hormones. Furthermore, the proteins HvMT2b2 and HvMT4 were heterologous expressed as GST-fusion proteins in E. coli. The HvMT4 and HvMT2b2 heterologous expression in E. coli gives rise to 10- and 3-fold improvements in the accumulation capacity for Zn2+, respectively. Whereas the transgenic E. coli strain that expresses HvMT2b2 could accumulate Cd2+ three-fold higher than control. The expression of HvMT4 did not affect the accumulation of Cd2+. HvMT4 which is known as seed-specific isoform seems to be able to bind to Zn2+ with good affinity and cannot bind Cd2+. In comparison, HvMT2b2 was able to bind both Zn2+ and Cd2+. Therefore HvMT4 could serve a noteworthy role in zinc storage in barley seeds. The expression of HvMT4 is induced by SA 30-fold, concerning the untreated aleurone layer. Such results could provide good insights for the assessment of the effects of phytohormones in the molecular mechanism involved in essential metal storage in cereal seeds.

HVA22 from citrus: A small gene family whose some members are involved in plant response to abiotic stress

The HVA22 gene has been isolated for the first time from the aleurone layer of barley (Hordeum vulgare). Here, we characterized the HVA22 family from citrus (C. clementina and C. sinensis). Twelve genes, 6 in each species, were identified as well as duplication events for some of them. The ORF size ranged from 235 to 804 bp and the protein molecular weight from 94 to 267 kDa. All the citrus HVA22 protein presented transmembrane location and conserved TB2/DP1/HVA22 region. Phylogenetic and gene expression analyses suggested that some citrus HVA22 play a role in flower and fruit development, and that gene expression may be regulated by hormone or environmental conditions. Other regulation levels were also predicted, such as alternative splicing and post-translational modifications. The overall data indicated that citrus HVA22 may be involved in vesicular traffic in stressed cells, and that CcHVA22d could be involved in dehydration tolerance.

Structural and functional divergence of the Mpc1 genes in wheat and barley

BackgroundThe members of the Triticeae tribe are characterised by the presence of orthologous and homoeologous gene copies regulating flavonoid biosynthesis. Among transcription factors constituting a regulatory MBW complex, the greatest contribution to the regulation of flavonoid biosynthetic pathway is invested by R2R3-Myb-type TFs. Differently expressed R2R3-Myb copies activate the synthesis of various classes of flavonoid compounds in different plant tissues. The aim of this research was the identification, comparison and analysis of full-length sequences of the duplicated R2R3-Myb Mpc1 (Myb protein c1) gene copies in barley and wheat genomes.ResultsThe Mpc1 genes were identified in homoeologous group 4 and 7 chromosomes: a total of 3 copies in barley (Hordeum vulgare L.) and 8 copies in bread wheat (Triticum aestivum L.) genomes. All Mpc1 genes have a similar two-exon structure, and almost all of them are transcriptionally active. The calculation of the divergence time revealed that first duplication between 4 and 7 chromosomes of the common ancestor of the Triticeae tribe occurred about 35–46 million years ago (MYA); the last duplication arised about 16–19 MYA before the divergence Triticum and Hordeum genera The connection between gene expression and the appearance of anthocyanin pigmentation was found for three genes from homoeologous group 4 chromosomes: TaMpc1-A2 (5AL) in wheat coleoptile, HvMpc1-H2 (4HL) in barley lemma and aleurone layer, and HvMpc1-H3 (4HL) in barley aleurone layer. TaMpc1-D4 (4DL) from the wheat genome showed a strong level of expression regardless of the colour of coleoptile or pericarp. It is assumed, that this gene regulates the biosynthesis of uncoloured flavonoids in analysed tissues.ConclusionsThe regulatory R2R3-Myb genes involved in anthocyanin synthesis were identified and characterised in Triticeae tribe species. Genes designated HvMpc1-H2 and HvMpc1-H3 appeared to be the main factors underlying intraspecific variation of H. vulgare by lemma and aleurone colour. TaMpc1-A2 is the co-regulator of the Mpc1–1 genes in bread wheat genome controlling anthocyanin synthesis in coleoptile.

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput.

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (β-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

Sulfur dioxide alleviates programmed cell death in barley aleurone by acting as an antioxidant.

Sulfur dioxide (SO2), a gaseous signaling molecule in animal cells, has recently been found to play a physiological role in plants. Here we studied the role of SO2 in gibberellic acid (GA3)-induced programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The application of the SO2 donor (NaHSO3/Na2SO3, 1:3 M/M) effectively alleviated PCD in barley aleurone layers in a dose-dependent manner with an optimal concentration of 50 μM. Further investigations showed that SO2 reduced the accumulation of hydrogen peroxide (H2O2), superoxide anion (⋅O2−) and malondialdehyde (MDA) in aleurone layers. Moreover, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR) and guaiacol peroxidase (POD) were enhanced by SO2 donor treatment. Meanwhile, lipoxygenase (LOX) activity was attenuated by SO2 donor treatment. Furthermore, an induction of endogenous H2S and NO were also observed in SO2-treated aleurone layers, suggesting interactions of SO2 with other well-known signaling molecules. Taken together, we show that SO2 negatively regulated PCD by acting as an antioxidant to scavenge excessive reactive oxygen species (ROS) generated during PCD.

Identification and characterization of regulatory network components for anthocyanin synthesis in barley aleurone

BackgroundAmong natural populations, there are different colours of barley (Hordeum vulgare L.). The colour of barley grains is directly related to the accumulation of different pigments in the aleurone layer, pericarp and lemma. Blue grain colour is due to the accumulation of anthocyanins in the aleurone layer, which is dependent on the presence of five Blx genes that are not sequenced yet (Blx1, Blx3 and Blx4 genes clustering on chromosome 4HL and Blx2 and Blx5 on 7HL). Due to the health benefits of anthocyanins, blue-grained barley can be considered as a source of dietary food. The goal of the current study was to identify and characterize components of the anthocyanin synthesis regulatory network for the aleurone layer in barley.ResultsThe candidate genes for components of the regulatory complex MBW (consisting of transcription factors MYB, bHLH/MYC and WD40) for anthocyanin synthesis in barley aleurone were identified. These genes were designated HvMyc2 (4HL), HvMpc2 (4HL), and HvWD40 (6HL). HvMyc2 was expressed in aleurone cells only. A loss-of-function (frame shift) mutation in HvMyc2 of non-coloured compared to blue-grained barley was revealed. Unlike aleurone-specific HvMyc2, the HvMpc2 gene was expressed in different tissues; however, its activity was not detected in non-coloured aleurone in contrast to a coloured aleurone, and allele-specific mutations in its promoter region were found. The single-copy gene HvWD40, which encodes the required component of the regulatory MBW complex, was expressed constantly in coloured and non-coloured tissues and had no allelic differences. HvMyc2 and HvMpc2 were genetically mapped using allele-specific developed CAPS markers developed. HvMyc2 was mapped in position between SSR loci XGBS0875-4H (3.4 cM distal) and XGBM1048-4H (3.4 cM proximal) matching the region chromosome 4HL where the Blx-cluster was found. In this position, one of the anthocyanin biosynthesis structural genes (HvF3’5’H) was also mapped using an allele-specific CAPS-marker developed in the current study.ConclusionsThe genes involved in anthocyanin synthesis in the barley aleurone layer were identified and characterized, including components of the regulatory complex MBW, from which the MYC-encoding gene (HvMyc2) appeared to be the main factor underlying variation of barley by aleurone colour.

Isolation of tissues and preservation of RNA from intact, germinated barley grain.

Isolated barley (Hordeum vulgare L.) aleurone layers have been widely used as a model system for studying gene expression and hormonal regulation in germinating cereal grains. A serious technological limitation of this approach has been the inability to confidently extrapolate conclusions obtained from isolated tissues back to the whole grain, where the co-location of several living and non-living tissues results in complex tissue-tissue interactions and regulatory pathways coordinated across the multiple tissues. Here we have developed methods for isolating fragments of aleurone, starchy endosperm, embryo, scutellum, pericarp-testa, husk and crushed cell layers from germinated grain. An important step in the procedure involves the rapid fixation of the intact grain to freeze the transcriptional activity of individual tissues while dissection is effected for subsequent transcriptomic analyses. The developmental profiles of 19611 gene transcripts were precisely defined in the purified tissues and in whole grain during the first 24h of germination by RNA sequencing. Spatial and temporal patterns of transcription were validated against well-defined data on enzyme activities in both whole grain and isolated tissues. Transcript profiles of genes involved in mitochondrial assembly and function were used to validate the very early stages of germination, while the profiles of genes involved in starch and cell wall mobilisation matched existing data on activities of corresponding enzymes. The data will be broadly applicable for the interrogation of co-expression and differential expression patterns and for the identification of transcription factors that are important in the early stages of grain and seed germination.

Immobilisation of barley aleurone layers enables parallelisation of assays and analysis of transient gene expression in single cells

The barley aleurone layer is an established model system for studying phytohormone signalling, enzyme secretion and programmed cell death during seed germination. Most analyses performed on the aleurone layer are end-point assays based on cell extracts, meaning each sample is only analysed at a single time point. By immobilising barley aleurone layer tissue on polydimethylsiloxane pillars in the lid of a multiwell plate, continuous monitoring of living tissue is enabled using multiple non-destructive assays in parallel. Cell viability and menadione reducing capacity were monitored in the same aleurone layer samples over time, in the presence or absence of plant hormones and other effectors. The system is also amenable to transient gene expression by particle bombardment, with simultaneous monitoring of cell death. In conclusion, the easy to handle and efficient experimental setup developed here enables continuous monitoring of tissue samples, parallelisation of assays and single cell analysis, with potential for time course studies using any plant tissue that can be immobilised, for example leaves or epidermal peels.

Ethephon advances the release time of limit dextrinase from gibberellic acid-treated aleurone layer

Abstract The aleurone layer plays a key role in germination by responding to hormone signals from embryo and producing hydrolases. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signaling molecules in an isolated system. In this work, the effect of ethephon on the protein release from gibberellic acid (GA)-treated aleurone layer was presented for two important enzymes in starch degradation: α-amylase and limit dextrinase (LD). The results showed that the release of proteins from aleurone layer to culture supernatant was remarkably increased when GA- treated aleurone layer was exposed to 1000–10000 ppm ethephon. Ethephon enhanced the release of α-amylase and advanced the release time of LD from GA-treated aleurone layer to culture supernatant. The results supports that the release of LD is dependent to cell wall degradation which can be induced by the effect of ethephon.

Monitoring intra- and extracellular redox capacity of intact barley aleurone layers responding to phytohormones

Redox regulation is important for numerous processes in plant cells including abiotic stress, pathogen defence, tissue development, seed germination and programmed cell death. However, there are few methods allowing redox homeostasis to be addressed in whole plant cells, providing insight into the intact in vivo environment. An electrochemical redox assay that applies the menadione-ferricyanide double mediator is used to assess changes in the intracellular and extracellular redox environment in living aleurone layers of barley (Hordeum vulgare cv. Himalaya) grains, which respond to the phytohormones gibberellic acid and abscisic acid. Gibberellic acid is shown to elicit a mobilisation of electrons as detected by an increase in the reducing capacity of the aleurone layers. By taking advantage of the membrane-permeable menadione/menadiol redox pair to probe the membrane-impermeable ferricyanide/ferrocyanide redox pair, the mobilisation of electrons was dissected into an intracellular and an extracellular, plasma membrane-associated component. The intracellular and extracellular increases in reducing capacity were both suppressed when the aleurone layers were incubated with abscisic acid. By probing redox levels in intact plant tissue, the method provides a complementary approach to assays of reactive oxygen species and redox-related enzyme activities in tissue extracts.

Application of LC–MS–MS to identify niacin in aleurone layers of yellow corn, barley and wheat kernels

Previous studies identified type II inclusions in-situ of aleurone layers as niacin reserves and quantified niacin using colorimetric methods. Such identification and quantification was based on the König reaction which lacks specificity because cyanogen bromide reacts with all pyridine derivatives. The aim of this investigation was to define the structure of niacin in aleurone layers and aleurone cell contents of yellow corn, wheat and barley using LC–MS/MS. Aleurone layers were manually separated and cell contents released by ultrasonic processing and the residue and pellets were examined microscopically. Niacin was extracted from the samples by autoclaving in alkali. The extracts were analysed using HPLC and the structural identity of niacin was confirmed using quadrupole time of flight mass spectrometry in positive mode. Niacin concentrations were highest in wheat aleurone and lowest in corn aleurone. The MS/MS spectra of pure niacin and the purported niacin peak in sample extracts showed similar fragmentation patterns. The major ion product occurred at m/z 80 representing loss of CO2 and parent ion at m/z 124 (M + H)+. These findings define some of the structural characteristics of niacin in aleurone layer of cereal grains, and demonstrate the possibility of using an ultrasonic processor to release cell contents.

Variability of barley aleurone layer induced by X-rays

A series of Hordeum vulgare cultivars was irradiated by X-rays to induce mutations in endosperm. Many structural defects of endosperm were revealed in plants irradiated 8 DAF. Change of a cell cycle was especially frequent and this was visible in the form of clones of small or large cells in the aleurone layer. X-irradiation appeared as a successful tool in the study of development.

The Hydrogen Sulfide Donor NaHS Delays Programmed Cell Death in Barley Aleurone Layers by Acting as an Antioxidant.

H2S is a signaling molecule in plants and animals. Here we investigated the effects of H2S on programmed cell death (PCD) in barley (Hordeum vulgare L.) aleurone layers. The H2S donor NaHS significantly delayed PCD in aleurone layers isolated from imbibed embryoless barley grain. NaHS at 0.25 mM effectively reduced the accumulation of superoxide anion (·O2 −), hydrogen peroxide (H2O2), and malondialdehyde (MDA), promoted the activity of superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and decreased those of lipoxygenase (LOX) in isolated aleurone layers. Quantitative-PCR showed that NaHS treatment of aleurone tissue led to enhanced transcript levels of the antioxidant genes HvSOD1, HvAPX, HvCAT1, and HvCAT2 and repressed transcript levels of HvLOX (lipoxygenase gene) and of two cysteine protease genes HvEPA and HvCP3-31. NaHS treatment in gibberellic acid- (GA-) treated aleurone layers also delayed the PCD process, reduced the content of ·O2 −, and increased POD activity while decreasing LOX activity. Furthermore, α-amylase secretion in barley aleurone layers was enhanced by NaHS treatment regardless of the presence or absence of GA. These data imply that H2S acted as an antioxidant in delaying PCD and enhances α-amylase secretion regardless of the presence of GA in barley aleurone layers.

Spatio-temporal appearance of α-amylase and limit dextrinase in barley aleurone layer in response to gibberellic acid, abscisic acid and salicylic acid.

Cereal seed germination involves mobilization of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellin produced by the embryo the aleurone layer synthesizes hydrolases that are secreted to the endosperm for degradation of storage products. In this study analysis of intracellular protein accumulation and release from barley aleurone layers is presented for the important enzymes in starch degradation: α-amylase and limit dextrinase (LD). Proteins were visualized by immunoblotting in aleurone layers and culture supernatants from dissected aleurone layers incubated up to 72 h with either gibberellic acid (GA), abscisic acid (ABA) or salicylic acid (SA). The results show that α-amylase is secreted from aleurone layer treated with GA soon after synthesis but the release of LD to culture supernatants was significantly delayed and coincided with a general loss of proteins from aleurone layers. Release of LD was found to differ from that of amylase and was suggested to depend on programmed cell death (PCD). Despite detection of intracellular amylase in untreated aleurone layers or aleurone layers treated with ABA or SA, α-amylase was not released from these samples. Nevertheless, the release of α-amylase was observed from aleurone layers treated with GA+ABA or GA+SA.

Hydroxyproline and proline inhibit α-amylase from isolated barley aleurone layers

Previously, we reported that 1 mM hydroxyproline appeared to inhibit the gibberellic acid-induced release of α-amylase from isolated <em>Hordeum vulgare</em> L. cv. Himalaya aleurone layers into an incubation medium. Here, we report our attempts to determine the mechanism(s) for this inhibition and whether this inhibition can be caused by other proline analogues. Both 1 mM hydroxyproline and proline inhibited extracellular a-amylase activity without affecting its intracellular activity. This suggested that neither hydroxyproline nor proline impaired the release of a-amylase. Lineweaver-Burk plots revealed that both hydroxyproline and proline uncompetitively inhibited α-amy-lase. Thus, the inhibition is probably an assay artifact resulting from the formation of an enzyme-substrate-hydroxyproline or -proline complex. Because azetidine-2-carboxylic acid, glutamic acid and pipecolic acid did not inhibit extracellular α-amylase activity, the uncompetitive inhibition of a-amylase must be unique to imino aicids as well as their precursors and derivatives which possess a five membered ring.

Gibberellic Acid-Induced Aleurone Layers Responding to Heat Shock or Tunicamycin Provide Insight into theN-Glycoproteome, Protein Secretion, and Endoplasmic Reticulum Stress

The growing relevance of plants for the production of recombinant proteins makes understanding the secretory machinery, including the identification of glycosylation sites in secreted proteins, an important goal of plant proteomics. Barley (Hordeum vulgare) aleurone layers maintained in vitro respond to gibberellic acid by secreting an array of proteins and provide a unique system for the analysis of plant protein secretion. Perturbation of protein secretion in gibberellic acid-induced aleurone layers by two independent mechanisms, heat shock and tunicamycin treatment, demonstrated overlapping effects on both the intracellular and secreted proteomes. Proteins in a total of 22 and 178 two-dimensional gel spots changing in intensity in extracellular and intracellular fractions, respectively, were identified by mass spectrometry. Among these are proteins with key roles in protein processing and secretion, such as calreticulin, protein disulfide isomerase, proteasome subunits, and isopentenyl diphosphate isomerase. Sixteen heat shock proteins in 29 spots showed diverse responses to the treatments, with only a minority increasing in response to heat shock. The majority, all of which were small heat shock proteins, decreased in heat-shocked aleurone layers. Additionally, glycopeptide enrichment and N-glycosylation analysis identified 73 glycosylation sites in 65 aleurone layer proteins, with 53 of the glycoproteins found in extracellular fractions and 36 found in intracellular fractions. This represents major progress in characterization of the barley N-glycoproteome, since only four of these sites were previously described. Overall, these findings considerably advance knowledge of the plant protein secretion system in general and emphasize the versatility of the aleurone layer as a model system for studying plant protein secretion.

Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy

Grain ultrastructure is of utmost importance when designing grain processing procedures in the food industry. In this study, wheat and barley grain components were localised using optical and Raman microscopy. The optical microscopic analyses were performed using several selective stains to localise β-glucan, protein and starch or autofluorescence to image the ferulic acid and other fluorescing substances. Alternatively, Raman microscopy was applied to localise the grain components without any need for preceding staining or other sample pretreatment. Both methods provided consistent information on the grain structures, illustrating the distribution of polysaccharides, aromatics and protein in endosperm and aleurone layers. In aleurone layers of both barley and wheat, a distinct difference between the anticlinal and periclinal cell walls was observed. The anticlinal cell walls were enriched with aromatic substances which were present in remarkably lower concentrations in the inner periclinal cell walls but for barley, an even higher concentration in the outer periclinal cell wall was observed. In addition, Raman spectroscopy illustrated the detailed distribution of substances across the aleurone cell walls: β-glucan was adjacent to proteins and it was deficient in the middle lamella whereas arabinoxylan was enriched in the outer cell wall layers and middle lamella.

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.

Models for gibberellic acid transport and enzyme production and transport in the aleurone layer of barley

Gibberellins are growth hormones produced in the embryo of grain released during germination. They promote growth through the production of enzymes in the aleurone layer surrounding the endosperm. These enzymes then diffuse into the endosperm and produce the sugars required by the growing acrospire. Here we model the transport of gibberellins into and along the aleurone layer, the consequent production of enzymes, and their transport into the endosperm. Simple approximate solutions of the governing equations are obtained which suggest that the enzymes are released immediately behind a gibberellin front which travels with almost constant speed along the aleurone layer. The model also suggests that this propagation speed is determined primarily by conditions near the scutellum–aleurone junction, which may enable the embryo to actively control the germination process.