Barley Aleurone Layers Research Articles

Identification of thioredoxin target disulfides in proteins released from barley aleurone layers

Thioredoxins are ubiquitous disulfide reductases involved in a wide range of cellular processes including DNA synthesis, oxidative stress response and apoptosis. In cereal seeds thioredoxins are proposed to facilitate the germination process by reducing disulfide bonds in storage proteins and other targets in the starchy endosperm. Here we have applied a thiol-specific labeling approach to identify specific disulfide targets of barley thioredoxin in proteins released from barley aleurone layers incubated in buffer containing gibberellic acid.

Tetrazolium Staining, Mitochondria, and Barley Quality

Triphenyl tetrazolium chloride (TTC) staining of individual isolated barley aleurone layers was carried out under aseptic conditions and the amount of formazan generated under a standardized incubation period was quantified. The nature of the tests minimized or excluded the influences of grain damage, ageing or contaminating microorganisms. Embryos from selected Triumph barley grains, having intensely or poorly staining aleurone layers, were grown on to maturity. Collected grain was analysed and embryos from the highest-staining progeny from the high-staining aleurone line and the lowest-staining progeny of the low-staining aleurone line were grown on to yield grain of the second selected generations. Aleurone layers of the high-staining line continued to stain intensely, respired strongly, and generated large amounts of α-amylase in response to gibberellic acid and embryos from these grains respired and grew vigorously, as did whole grains. In contrast, the aleurone layers of grains from the low-staining line continued to stain poorly and respired weakly, generated little α-amylase in response to gibberellic acid, the embryos respired slowly and lacked vigour and, like the whole grains, grew poorly. It is proposed that these results are the consequence of the selection procedure separating lines with superior and inferior mitochondria. If this apparently novel approach using TTC staining is confirmed as being valid, it opens a new way for plant breeders to select for more vigorous cereal lines.

Hydrogen peroxide affects abscisic acid binding to ABAP1 in barley aleurones

Dramatic increases in H2O2 levels have been observed following abscisic acid (ABA) treatment of plant tissues. Following ABA treatment in aleurone cells, H2O2 reached transient levels of approximately 115 micromol/L H2O2. To determine whether ABA perception was modified by such changes, the effect of H2O2 on a recently characterized ABA-binding protein (ABAP1), cloned from barley aleurone layers, was examined. ABA binding to the protein was weakened by H2O2 in a concentration-dependent manner. A concentration of 75 micromol/L H2O2 gave a 50% decline in ABA binding in a reaction following first-order kinetics, indicative of binding-site susceptibility to its microenvironment. We monitored the unfolding of ABAP1 using steady-state and time-resolved tryptophan fluorescence, while following the capacity of ABAP1 to bind ABA. ABA binding decreased by 50% following ABAP1 denaturation with 1 mol/L guanidine hydrochloride or 2 mol/L urea, while the maximum emission spectra (lambda emi) red shifted from 338 to 347 nm at 3.5 mol/L guanidine hydrochloride and 5 mol/L urea. However, only a slight blue shift of lambda emi was observed following either ABAP1 incubation with H2O2 or binding to (+)-ABA (physiologically active ABA). The equilibrium ABA dissociation rate accelerated in the presence of 250 micromol/L H2O2, with the half-time dissociation reduced to 8 min. A comparison of inactivation kinetics and conformational changes shows that inactivation of ABAP1 occurs before any noticeable conformational change. This suggests that the ABA binding site is highly responsive to its microenvironment and is situated in a region that is more flexible than the protein molecule as a whole. The results demonstrate that H2O2, generated by ABA treatment of aleurone layers, is sufficient to affect the ABA-binding capacity of ABAP1, suggesting that this may be another level of control of ABA signal transduction.

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.

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.

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.

Redox-sensitive target detection in gibberellic acid-induced barley aleurone layer

Reactive oxygen species (ROS) are involved in redox regulation by their capacity to reversibly oxidize cysteine residues. This regulation is used by cells to modulate and integrate different responses to extracellular stimuli. In the barley ( Hordeum vulgare L.) aleurone layer, gibberellic acid (GA 3) is perceived at the plasma membrane and induces the synthesis and secretion of α-amylase. All aleurone membrane systems participate in the elaboration of this response. During these events, ROS are generated as a by-product from intense lipid metabolism. Therefore, we hypothesized that redox regulation may be operating in the GA 3-induced response. To test this hypothesis, we measured if GA 3 treatment induced changes in the redox state of aleurone membrane-associated proteins. Membrane proteins with sulfhydryl and disulfide groups were isolated from reduced and in situ NEM-alkylated microsomal fractions, respectively. Each fraction was enriched by thiol-affinity chromatography and separated by two-dimensional electrophoresis. The in vivo redox state of each membrane protein present in GA 3-treated and -untreated tissue was determined. Results showed that GA 3 induced the reduced state in 17 constitutive proteins and the oxidized state in another 5. These data indicate that redox changes occur in membrane proteins after GA 3 signaling in the aleurone layer.

Cytosolic calcium is involved in the regulation of barley hemoglobin gene expression

Hemoglobin gene expression is upregulated during hypoxia. To determine whether the induction occurs via similar mechanisms that have been proposed for other hypoxically induced proteins, barley (Hordeum vulgare L.) aleurone layers were treated with various agents that interfere with known components of signal transduction. Ruthenium red, an organelle calcium channel blocker, inhibited anoxia-induced hemoglobin (Hb) and alcohol dehydrogenase (EC 1.1.1.1) (Adh) gene expression in a dose-dependent manner. The divalent ionophore, A23187, combined with EGTA also dramatically reduced anoxia-induced Hb and Adh expression. Normal induction of Hb by anoxia in EGTA-treated cells was restored by adding exogenous Ca2+ but not Mg2+, suggesting that cytosolic calcium is involved in Hb and Adh regulation. W-7, a calmodulin antagonist, did not affect anaerobically induced Hb and Adh expression even though it induced Hb under aerobiosis. A3, a protein kinase inhibitor, did not significantly affect anaerobically induced Hb, but did significantly upregulate the gene under aerobic conditions. The results indicate that calmodulin-independent anaerobic alteration in cytosolic Ca2+ and protein dephosphorylation are factors in Hb induction.

The barley cystatin gene (Icy) is regulated by DOF transcription factors in aleurone cells upon germination

A genomic clone (Icy gene) encoding a barley cystatin has been characterized. The gene contains one intron interrupting its ORF that encodes a protein of 107 amino acid residues. A DNA fragment of -1058 bp upstream of the ATG translation initiation codon has been sequenced and several promoter deletions fused to the beta-glucuronidase (GUS; uidA gene) reporter gene obtained. Transient expression assays in different barley tissues have indicated that a -631 bp promoter fragment was sufficient for full activity. In bombarded barley aleurone layers the GUS-driven expression by this promoter is repressed by GA(3) incubation, as is the accumulation of the Icy transcripts detected. A spatial and temporal location of cystatin transcripts by in situ hybridization techniques indicates that this gene is ubiquitously expressed and its transcripts are particularly abundant in leaves and roots, and in seeds, both during development and upon germination. Two DOF transcription factors, SAD and BPBF, previously described as involved in gene expression regulation in seeds, interact specifically in vitro with oligonucleotides containing DOF binding-sites derived from the Icy gene promoter. Transient expression experiments in co-bombarded aleurone layers demonstrate that BPBF strongly represses transcription of the native Icy promoter, even when co-transfected with SAD that behaves as an activator in this in vivo system.

Isolation and Identification of Glycosylphosphatidylinositol-Anchored Arabinogalactan Proteins and Novel β-Glucosyl Yariv-Reactive Proteins from Seeds of Rice (Oryza sativa)

Arabinogalactan proteins (AGPs) are highly glycosylated extracellular glycoproteins playing important roles in plant growth and development. We have previously reported the possibility that AGPs are involved in the induction of alpha-amylase by gibberellin (GA) in barley aleurone layers by using the beta-glucosyl Yariv reagent (beta-GlcY), which has been presumed to specifically bind AGPs. In this present study, we isolated beta-GlcY-reactive proteins from rice bran rich in aleurone cells. The N-terminal sequences of classical AGP and AG peptides were determined from hydrophilic fractions obtained by reversed phase HPLC. Interestingly, a novel non-specific lipid transfer protein-like protein (OsLTPL1) and a novel early nodulin-like protein (OsENODL1) were also identified in the more hydrophobic fractions from HPLC as beta-GlcY-reactive proteins. Expression analysis of the genes coding for these proteins was performed. While classical AGP, AG peptides and OsLTPL1 were expressed in various parts of rice, OsENODL1 showed temporally and spatially specific expression in the aleurone layers. This new beta-GlcY-reactive protein is a promising candidate for the extracellular signaling factors of GA action in cereal seeds. Furthermore, the possibility that proteins with the AG glycomodule might react with beta-GlcY may broaden the definition of AGPs.

Effect of Ecdysterone on Morphological and Physiological Processes in Plants

In order to elucidate the physiological role of phytoecdysteroids in plants, we investigated the effects of exogenous ecdysterone (ECD) and phytohormones (IAA, GA3, and 24-epibrassinolide (EBL)) on the growth of wheat coleoptiles and Arabidopsis thaliana seedlings (wild-type ecotype Columbia (Col) and its det2 mutant), on α-amylase activity in the barley aleurone layer, and on the pigment content in the kidney bean senescent leaves. The range of effective ECD concentrations depended on the type of a reaction to be regulated. The regulation of growth processes was affected by a wide range of ECD concentrations (10–13–10–5 M), whereas some metabolic processes, such as the activation of α-amylase and the retardation of leaf yellowing, by a narrow range, that is, 10–9–10–7 M and 10–9–10–8 M, respectively. We noted the synergetic effect of ECD and IAA on coleoptile elongation, the antagonistic effect of ECD and EBL on coleoptile elongation, as well as the antagonistic action of ECD and GA3 on coleoptile elongation and α-amylase activity. The data obtained demonstrate that ECD is a physiologically active compound. ECD might be supposed to act as a source of sterols or a regulator of IAA and protein synthesis. The effects of this regulator seems to be brought about by its interaction with the EBL and GA3 receptors.

Purification and Characterization of a Barley Aleurone Abscisic Acid-binding Protein

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.

Metabolism of hydrogen peroxide during reserve mobilization and programmed cell death of barley (Hordeum vulgare L.) aleurone layer cells.

During germination, aleurone layer cells of barley (Hordeum vulgare) grains synthesize and secrete hydrolytic enzymes (principally alpha-amylase) in response to gibberellic acid (GA); shortly thereafter, the aleurone layer cells undergo programmed death. Gluconeogenesis of lipid reserves within aleurone cells, which supports this hydrolytic enzyme synthesis, results in the generation of H(2)O(2), which is catabolized by glyoxysomal catalase. Lowered amounts of catalase may contribute to aleurone cell death because of a compromised capacity to cope with reactive oxygen species generated by glyoxysomes and mitochondria. In the presence of GA, cells of intact aleurone layers underwent programmed death between 18 and 48 h; in the presence of ABA, no cell death was evident over 60 h. The capacity of GA-treated layers to metabolize exogenous H(2)O(2) increased steadily over the first 24 h, during the stage of lipid mobilization and the major synthesis and secretion of alpha-amylase; thereafter, this capacity declined markedly. In contrast, cells of ABA-treated aleurone layers exhibited little change in their capacity for H(2)O(2)-metabolism. Glyoxysomal catalase increased in activity over the first 12-24 h of GA treatment, which was accompanied by an increase in catalase-1 transcripts between 12 and 18 h. Catalase protein and activity declined after 24 h in GA-treated layers, prior to the onset of rapid programmed death at 30 h. These data suggest that a decline in glyoxysomal catalase precedes death of aleurone cells and may indeed contribute to an increase in cellular oxidative stress.

The heat-shock response of developing barley aleurone layers

AbstractAleurone layers of mature germinating barley (Hordeum vulgare, cv. Himalaya) grains respond to heat shock by synthesizing heat-shock proteins (HSPs) and by selectively suppressing the synthesis of proteins normally translated by endoplasmic reticulum (ER)-bound ribosomes. To determine if this also was the case during seed development, we investigated the synthesis of proteins translated by ER-bound ribosomes in heat-shocked aleurone layers isolated from developing barley grains. The optimal induction temperature for the heat shock response in developing aleurone layers was 37.5°C, and temperatures above 42°C inhibited translation. HSPs with apparent molecular masses of 71.1, 66.2, 57.8, 19.1 and 18.8 kDa were induced. Other studies have shown that, in gibberellic acid (GA)-induced aleurone layers from mature barley grains, these temperatures were 40°C and 45°C, respectively. Furthermore, in developing aleurone layers, mRNAs encoding proteins translated by ER-bound ribosomes (mRNAs for a lipid transfer protein and a putative amylase/protease inhibitor) remained stable during heat shock. The ER membranes themselves remained in stacks, but the lumen became distended with electron-dense material. Heat shock prevented the movement of proteins from the ER into the rest of the endomembrane pathway. In contrast, other studies show that in mature, GA-induced aleurone layers, heat shock dissociates ER stacks and blocks translation, but the processing of secretory proteins in the endomembrane pathway is not inhibited. The observation that the same tissue at different developmental stages may respond differently to heat shock indicates that components of the heat-shock response are developmentally regulated. This system provides an opportunity to better understand the nuances of the heat-shock response, especially the post-transcriptional gene regulatory mechanisms that occur.

Heat shock response of warm‐incubated barley aleurone layers

Heat shock suppresses secretory protein synthesis in GA(3)-stimulated barley (Hordeum vulgare cv. Himalaya) aleurone layers by selectively destabilizing their mRNAs and dissociating the stacked rough endoplasmic reticulum (ER) lamellae upon which they are translated. Heat shock also increases phosphatidylcholine (PC) synthesis, and these PC molecules have increased levels of fatty acid saturation. This appears to be adaptive, for aleurone layers maintained at heat shock temperatures for 18 h resynthesize secretory protein mRNAs, rebuild stacked ER lamellae, and resume secretory protein synthesis. In the present study aleurone layers were incubated at warmer than normal pre-heat shock temperatures to determine whether this would favor the formation of heat-resistant ER lamellae that could continue secretory protein synthesis during heat shock. Western blot and SDS-PAGE analyses showed that such treatment did not induce heat shock protein (HSP) synthesis, but it preserved significant secretory protein synthesis during heat shock. Northern hybridizations revealed that levels of mRNAs encoding secretory proteins were several-fold elevated as compared to 25°C preincubated controls, and transmission electron microscopic observations revealed stacked ER lamellae. Thin layer and gas chromatography showed that PC molecules in warm-incubated barley aleurone layers had more fatty acid saturation than did controls. These observations indicate that previous incubation temperature influences both the induction of HSP synthesis and the suppression of normal protein synthesis in the heat shock response. However, we found that it does not affect the temperature at which heat shock becomes lethal.

A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone.

Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5'-CCTTTT-3'. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point alpha-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.

Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell Death in Barley Aleurone Layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

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.

The diffusive transport of gibberellins and abscisic acid through the aleurone layer of germinating barley grain: a mathematical model.

A mathematical model of the diffusive transport of abscisic acid (ABA) and gibberellins (GAs) through the aleurone layer of barley (Hordeum vulgare L.) grain is presented. The model consists of two partial differential equations describing the accumulation of phytohormone in the apoplastic and symplasmic compartments of the aleurone layer, both spatially and temporally. The mathematical model contains the morphology of the barley grain and the physicochemical properties of the two phytohormones. A mathematical derivation of the accumulation ratios for the two phytohormones between the symplast and apoplast under equilibrium conditions resulted in different distribution mechanisms for GAs and ABA. A sensitivity analysis of the accumulation ratio for GAs indicated high sensitivity to the apoplastic pH and the membrane potential, whereas the accumulation ratio for ABA proved to be most sensitive to the pH difference between the apoplast and symplast. The diffusive transport time for GAs to the basal site of the aleurone layer as calculated with the mathematical model is within a physiologically plausible timescale according to experimental data from the literature. Abscisic acid cannot be transported by diffusion to the end of the aleurone layer as quickly as GAs, according to model simulations. Therefore, the functional role of ABA in germination is likely to be in the vicinity of the embryo.

Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone.

Gibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, and Cat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.