Wheat Aleurone Cells Research Articles

Gibberellic-acid-dependent expression of α-amylase in wheat aleurone cells is mediated by target of rapamycin (TOR) signaling

Target of rapamycin (TOR) signaling is an essential nutrient-dependent pathway controlling cell growth in all eukaryotes. TOR signaling is well characterized in yeast and animals but remains poorly investigated in plants. The hormonal action of gibberellic acid (GA) is a crucial factor for wheat germination by inducing the synthesis of α-amylase in wheat aleurone cells. Here we showed that GA promotes the activation of Triticum aestivum TOR (TaTOR) signaling as evidenced by increased phosphorylation of T. aestivum S6K1 (TaS6K1) on its conserved hydrophobic motif together with proteasomal degradation of growth-inhibitory factor Rht-1. GA-dependent activation of TaTOR signaling led to α-amylase synthesis and Rht-1 proteasomal degradation because both GA-dependent events were sensitive to TaTOR inhibition. Using antibodies specific to TaTOR, we successfully identified the presence of endogenous TaTOR protein in terminally differentiated wheat aleurone layers. Additionally, by examining the rapamycin-sensitive phosphorylation of S6K1 as a reliable indicator of endogenous TOR kinase activity, we demonstrated that the activity of TaTOR in aleurone layers is enhanced by GA. Importantly, this stimulation is not associated with the regulation of either TaTOR transcription or the accumulation of TaTOR protein. In yeast and pull-down assays, a robust interaction between TaS6K1 and the N terminus of Rht-1 (amino acids 1–234) was observed, a finding further supported by co-immunoprecipitation of endogenous Rht-1 and TaS6K1. Furthermore, the administration of mTOR inhibitors significantly attenuated GA-induced degradation of endogenous Rht-1 and prolonged the persistence of the complex formed by these two proteins. We propose that TaTOR-TaS6K1 signaling contributes to GA-dependent wheat germination by mediating α-amylase synthesis and controlling proteasomal degradation of Rht-1 in wheat aleurone cells.

TaABI5, a wheat homolog of Arabidopsis thaliana ABA insensitive 5, controls seed germination

Abscisic acid (ABA) response element (ABRE)-binding factors (ABFs) are basic region/leucine zipper motif (bZIP) transcription factors that regulate the expression of ABA-induced genes containing ABRE in their promoters. The amino acid sequence of the wheat bZIP protein, TaABI5, showed high homology to that of Arabidopsis ABA insensitive 5 (ABI5). TaABI5 was classified into the clade of ABI5s in Arabidopsis and rice, unlike TRAB1 of rice, Wabi5 of wheat, and HvABI5 of barley in the bZIP Group A family, by a phylogenetic analysis. TaABI5 was strongly expressed in seeds during the late ripening and maturing stages; however, its expression level markedly decreased after germination. An in situ hybridization analysis showed that TaABI5 mRNA accumulated in seed embryos, particularly the scutellum. In a transient assay using wheat aleurone cells, TaABI5 activated the promoter of Em containing ABRE, which is an embryogenesis abundant protein gene, indicating that TaABI5 acts as a transcription factor in wheat seeds. Furthermore, the seeds of transgenic Arabidopsis lines introduced with 35S:TaABI5 exhibited high sensitivity to ABA and the inhibition of germination. The seed dormancy of the transgenic Arabidopsis lines was stronger than that of Col. These results support TaABI5 playing an important role in mature seeds, particularly before seed germination, and acting as a functional ortholog to Arabidopsis ABI5.

Steeping and germination of wheat (Triticum aestivum L.). I. Unlocking the impact of phytate and cell wall hydrolysis on bio-accessibility of iron and zinc elements

In wheat aleurone cells iron and zinc ions occur as phytate structures. Phytate and arabinoxylan cell wall material resist digestion in the human gastro-intestinal tract resulting in very low mineral bio-accessibility. During wheat germination, enzymes make nutrients available for plant development and, when used in foods, for human uptake. We here provide understanding of the impact of the wheat matrix on mineral bio-accessibility. A multifactor experiment in which wheat was subjected to multiple combinations of steeping time (8–36 h), steeping temperature (15–40 °C), germination time (0–120 h) and germination temperature (15–30 °C) showed that steeping for 36 h at 15 °C and germination for 120 h at 26 °C decreased phytate content from 0.96% to 0.65% of initial dry matter (idm) and increased water extractable arabinoxylan content from 0.48% to 1.34% of idm due to phytase and endoxylanase action, respectively. While the iron and zinc bio-accessibility in the wheat grains was 5 and 3%, respectively, that in the germinated grains was 6 and 8% when the cellular matrix was preserved, and 22 and 21% when it was mechanically disrupted. This study thus revealed that controlled steeping and germination of wheat can substantially improve mineral bio-accessibility when the cell walls are broken down.

Hydrogen sulfide delays GA-triggered programmed cell death in wheat aleurone layers by the modulation of glutathione homeostasis and heme oxygenase-1 expression

Hydrogen sulfide (H2S) is considered as a cellular signaling intermediate in higher plants, but corresponding molecular mechanisms and signal transduction pathways in plant biology are still limited. In the present study, a combination of pharmacological and biochemical approaches was used to study the effect of H2S on the alleviation of GA-induced programmed cell death (PCD) in wheat aleurone cells. The results showed that in contrast with the responses of ABA, GA brought about a gradual decrease of l-cysteine desulfhydrase (LCD) activity and H2S production, and thereafter PCD occurred. Exogenous H2S donor sodium hydrosulfide (NaHS) not only effectively blocked the decrease of endogenous H2S release, but also alleviated GA-triggered PCD in wheat aleurone cells. These responses were sensitive to hypotaurine (HT), a H2S scavenger, suggesting that this effect of NaHS was in an H2S-dependent fashion. Further experiment confirmed that H2S, rather than other sodium- or sulphur-containing compounds derived from the decomposing of NaHS, was attributed to the rescuing response. Importantly, the reversing effect was associated with glutathione (GSH) because the NaHS triggered increases of endogenous GSH content and the ratio of GSH/oxidized GSH (GSSG) in GA-treated layers, and the NaHS-mediated alleviation of PCD was markedly eliminated by l-buthionine-sulfoximine (BSO, a selective inhibitor of GSH biosynthesis). The inducible effect of NaHS was also ascribed to the modulation of heme oxygenase-1 (HO-1), because the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPP) significantly suppressed the NaHS-related responses. By contrast, the above inhibitory effects were reversed partially when carbon monoxide (CO) aqueous solution or bilirubin (BR), two of the by-products of HO-1, was added, respectively. NaHS-triggered HO-1 gene expression in GA-treated layers was also confirmed. Together, the above results clearly suggested that the H2S-delayed PCD in GA-treated wheat aleurone cells was associated with the modulation of GSH homeostasis and HO-1 gene expression.

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.

A Gibberellin-induced Nuclease Is Localized in the Nucleus of Wheat Aleurone Cells Undergoing Programmed Cell Death

The aleurone layer of cereal grains undergoes a gibberellin-regulated process of programmed cell death (PCD) following germination. We have applied a combination of ultrastructural and biochemical approaches to analyze aleurone PCD in intact wheat grains. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay revealed that PCD was initiated in aleurone cells proximal to the embryo and then extended to distal cells. DNA fragmentation and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis revealed PCD of aleurone cells in maize grains, although the process was delayed as compared with wheat. Aleurone cells undergoing PCD showed a rapid vacuolation with high lytic activity in the cytoplasm, whereas the nucleus, which adopted an irregular shape, appeared essentially intact and showed symptoms of degradation at the end of the process. A nuclease activity was identified localized in the nucleus of aleurone cells undergoing PCD, just prior to the appearance of DNA laddering. This nuclease was induced by gibberellic acid treatment and was not detected when gibberellin synthesis was inhibited or in gibberellic acid-insensitive mutants. This nuclease was activated by Ca(2+) and Mg(2+), strongly inhibited by Zn(2+), and showed optimum activity at neutral pH, resembling nucleases involved in apoptosis of animal cells.

Proteins in Walls of Wheat Aleurone Cells

The protein content (1% w/w) of purified walls from aleurone layers is twice that of walls from the starchy endosperm but their amino acid compositions are comparable. Aprotic solvent extraction and specific enzymatic and chemical degradation of wall polysaccharides released some proteins, and treatment with specific proteases released peptides. The aleurone wall residue (18% of the original wall) after (1→3,1→4)-β-glucan and xylan hydrolase digestion contains 4·5% (w/w) protein associated with cellulosic glucan, glucomannan and highly-substituted arabinoxylan and remains autofluorescent. Wall residues after treatment with 1 M sodium hydroxide also remain autofluorescent indicating they retain a significant proportion of their original 1·84% (w/w) of hydroxycinnamic acids and that these are not exclusively attached to the wall polymers by ester linkages. Little protein was extracted from the walls using non-degradative solvents. However, significant quantities were recovered from whole walls and xylanase-treated wall residues by SDS/mercaptoethanol extraction and electroelution. The various protein fractions isolated were characterised by their amino acid compositions and, in some cases, by amino acid sequencing. Three classes of proteins were identified in wall fractions or from proteolysis fragments: glycine-rich proteins (37–86%), proline-rich proteins (11–39%) and proteins with up to 23% serine. Protein-polysaccharide cross-linking through tyrosine-hydroxycinnamic acid dimerisation may be responsible for the alkali-resistant autofluorescence and the insolubility of the protein may be due, in part, to cross-linking through tyrosine-tyrosine bridges. These associations may also contribute to the relative resistance of the inner aleurone wall layer to enzymic dissolution during germination.

Hydroxycinnamic Acids in Walls of Wheat Aleurone Cells

Walls were isolated from sheets of aleurone cells from wheat, freed from associated protein and starch by extraction with phenol-acetic acid-water followed by treatment with porcine pancreatic alpha -amylase, and finally extracted with sodium dodecyl sulphate. Hydroxycinnamic acids (HCA) released from the walls by saponification with 1 M NaOH amounted to 1·84% w/w of wall and comprised ferulic acid (90%), p coumaric acid (10%) and minor amounts (0·006%) of diferulic acids. The isolated walls exhibited an intense blue autofluorescence when irradiated with UV light that was reduced in intensity and became more diffuse after saponification. Acetyl residues (4·9% w/w) were present representing a degree of substitution of two in every nine pentose residues in the wall arabinoxylans. Digestion of the walls with a polysaccharide hydrolase mixture that lacked HCA esterases, released a series of oligomers that contained 50% of the total esterified HCA. These were separated by hydrophobic exclusion chromatography, purified by paper chromatography and two of these were identified by ES-MS and NMR as O -[5- O -feruloyl-α-L-arabinofuranosyl]-(1→3)- O -β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (FAXX) andO -β-D-xylopyranosyl-(1→4)- O -[5- O -feruloyl-α-L-arabinofuranosyl]-(1→3)-O -β-D-xylopyranosyl-(1→4)-β-D-xylopyranose (XFAXX). The extremely low level of esterified diferulic acid indicates that cross-linking between polysaccharide chains by diester-FA is not important, however the esterified FA may play a role in cross-linking polysaccharides to wall proteins.

Patterns of starchy endosperm acidification and protease gene expression in wheat grains following germination

Cereal aleurone responses to gibberellic acid (GA3) include activation of synthesis of hydrolytic enzymes and acidification of the external medium. We have studied the effect of the pH of the incubation medium on the response of wheat (Triticum aestivum) aleurone cells to GA3. De-embryonated half grains show the capacity for GA3-activated medium acidification when incubation is carried out at pH 6.0 to 7.0 but not at lower pHs. In addition, the activating effect of GA3 on the expression of carboxypeptidase III and thiol protease genes is more efficient when the hormone treatment is carried out at neutral pH. In situ pH staining showed that starchy endosperm acidification takes place upon imbibition and advances from the embryo to the distal part of the grain. In situ hybridization experiments showed a similar pattern of expression of a carboxypeptidase III gene, which is up-regulated by GA3 in aleurone cells. However, aleurone gene expression precedes starchy endosperm acidification. These findings imply that in vivo GA perception by the aleurone layer takes place at neutral pH and suggest that the acidification of the starchy endosperm is regulated by GA3 in germinated wheat grains.

Effects of gibberellic acid and environmental factors on cytosolic calcium in wheat aleurone cells

Gibberellins (GAs) control a wide range of physiological functions in plants from germination to flowering. The cellular mechanisms by which gibberellic acid (GA3) acts have been most extensively studied in the cereal aleurone. In this tissue, alterations in cellular calcium are known to be important for the primary response to GA, which is the production and secretion of hydrolytic enzymes. The extent to which cytosolic Ca2+ mediates the early events in GA action, however, is not known. In order to address this question, changes in cytosolic Ca2+ in wheat (Triticum aestivum L. cv. Inia) aleurone cells that occur rapidly after treatment with GA were characterized. In addition, GA-induced changes were compared with changes induced by three environmental stimuli that are known to modify the GA response: osmotic stress, salt (NaCl), and hypoxia. The Ca2+-sensitive dye fluo-3 was used to photometrically measure cytosolic Ca2+. It was found that GA3 induced a steady-state increase in cytosolic Ca2+ of 100–500 nM. This increase was initiated within a few minutes of treatment with GA and was fully developed after 30–90 min. The changes in cytosolic Ca2+ that were induced by GA were distinct from those induced by mannitol, NaCl, or hypoxia. Mannitol caused a steady-state decrease whereas NaCl and hypoxia both increased cytosolic Ca2+. In the case of NaCl this increase was transient but for hypoxia the increase was prolonged as long as hypoxic conditions were maintained. Gibberellin-induced changes in cytosolic Ca2+ were not induced by the inactive GA, GA8, nor did the GA-insensitive wheat mutant, D6899, respond to active GA3 with altered cytosolic Ca2+. It is concluded that changes in cytosolic Ca2+ are an early and integral part of the GA response in aleurone cells. The data also indicate, however, that changes in Ca2+ are not sufficient, by themselves, to induce the GA response of aleurone cells.

Okadaic Acid, a Protein Phosphatase Inhibitor, Blocks Calcium Changes, Gene Expression, and Cell Death Induced by Gibberellin in Wheat Aleurone Cells

Okadaic Acid, a Protein Phosphatase Inhibitor, Blocks Calcium Changes, Gene Expression, and Cell Death Induced by Gibberellin in Wheat Aleurone Cells

Diversity of calcium-efflux transporters in wheat aleurone cells

The ability of plant cells to regulate cellular calcium is dependent upon the action of calcium-transport proteins. Although significant progress has been made in identifying calcium-transporter activities (CaTs) in a number of plant tissues, very little is known about the diversity of CaTs in any single differentiated cell type. We have used isolated membrane vesicles from wheat (Triticum aestivum L.) aleurone cells to identify and characterize the principal CaTs that are responsible for efflux of Ca2+ from the cytosol of this highly differentiated cell. Based on the sensitivity of transport to protonophores and on their buoyant density in isopycnic sucrose density gradients, our data show that at least three prominent CaTs can be distinguished in membrane vesicles from aleurone cells. Two of these CaTs, which we have named Type I and Type III, were insensitive to protonophores and were inhibited by vanadate or erythrosin B (EB). Type I was associated with a membrane fraction enriched in endoplasmic reticulum whereas Type III was associated with the plasma membrane. The third prominent CaT, Type II, was inhibited by protonophores and by nitrate and was associated with a membrane fraction enriched in tonoplast. The three CaTs differed significantly in their intrinsic properties. Type I had the highest affinity for Ca2+ (K m, 0.15 μM), was stimulated by oxalate, inhibited by vanadate and erythrosin B, and was unaffected by exogenous calmodulin. Type II exhibited complex kinetics with regard to Ca2+ and was best described as a combination of a low-affinity (K m, 21 μM) and a high-affinity component (K m, 0.2 μM). Type II CaT was stimulated by calmodulin (1 μM) and oxalate, and was inhibited by dicyclohexylcarbodiimide (500 μM). Finally, Type III had an intermediate affinity for Ca2+ (K m, 2.0 μM), was not stimulated by calmodulin or by oxalate, and was inhibited by low concentrations of erythrosin B (0.1 μM). These data provide one of the first comparative investigations of the Ca2+-transport activities in a single highly differentiated cell type. They indicate that at least three CaTs function to regulate cellular Ca2+ and we speculate that cytosolic Ca2+ may be buffered primarily by Type II and Type III CaTs which are associated with an acidic intracellular compartment, probably the vacuole, and with the plasma membrane.

Hormonal and developmental control of gene expression in wheat

It is likely in plants, as in animal and fungal cells, that development involves the coordinated regulation of sets of genes. It is further likely that when this regulation acts on transcription that the coordination is mediated via trans -acting factors that recognize regulatory elements close to the responsive genes. In wheat (and barley) aleurone cells, a set of genes including those for a-amylase and for other hydrolases show increased expression at the RNA and transcriptional level in response to gibberellic acid. Based on the pattern of expression in various experimental conditions it seems likely that they are a co-regulated set, in the sense described above. However, a comparative analysis of 5' flanking regions has been made and, after the influence of relatedness between different members of gene families is accounted for, no sequence motifs can be identified that could be regulatory elements. More direct methods of analysis for such elements are described involving analysis of expression from natural or artifically constructed sequence variants. There is a second aspect to the regulated expression of aleurone genes when they are expressed non-coordinately and not under the control of gibberellic acid in non-aleurone tissues. In some instances this is because the same gene, expressed from the same promoter, is expressed in the different tissues and suggests that there are multiple regulatory elements close to these genes that respond to different stimuli depending on the stage of development. The a- Amy2 and carboxypeptidase genes of wheat use this strategy. In other instances, however, it can be seen that the dual mode of expression is achieved when multigene families have evolved in which different subsets have a different capability of expression. This strategy is exemplified by the a - Amy1 and a - Amy3 subsets of the a-amylase gene families

?-amylase genes of wheat are two multigene families which are differentially expressed

The α-Amy1 and α-Amy2 genes of wheat produce distinct subsets of α-amylase isozymes which show different patterns of expression in wheat aleurone cells and in developing grain. In order to characterise the organisation and expression of these genes, clones of α-Amy1 and α-Amy2 cDNA have been isolated. The two types of cDNA clone were distinguished within a small library of α-amylase cDNA clones (Baulcombe and Buffard, Planta 157 493-501 [1983]) by restriction endonuclease mapping and by cross hybridisation. The identity of α-Amy1 or α-Amy2 type was assigned from the results of hybrid selected translation analysis in which small subfragments of the cDNA clones were used. These subfragments were derived from the 3' ends of the cDNA and did not cross hybridise between the different types of cDNA. Hybridisation of α-Amy1 and α-Amy2 cDNA probes to restriction enzyme digests of wheat nuclear DNA revealed that these are multigene families located on the group 6 (α-Amy1) and group 7 (α-Amy2) chromosomes. Studies on the levels of α-Amy1 and α-Amy2 mRNA in developing grain and in aleurone tissue indicated that the differences in isozyme expression are due to the patterns of mRNA accumulation. In aleurone tissue the α-Amy1 transcripts accumulate in parallel with other genes which are regulated by gibberellic acid, while the accumulation of α-Amy2 genes is sustained for 36 h longer.

Two sites of phosphatidylcholine synthesis in the wheat aleurone cell

Sub-cellular fractions from radiolabelled wheat aleurone cells were isolated using two procedures. Using both procedures, [ methyl- 14C]choline, was actively incorporated into phosphatidylcholine in the aleurone grain and oleosome fractions, but [U- 14C]glycerol was poorly incorporated. This was most apparent in quiescent seeds and seeds incubated for 14 h or less. It is proposed that phospholipid-synthesis in the aleurone grain and oleosome fractions takes place from pre-existing storage glycerolipids. In contrast, the microsome fraction incorporated both choline and glycerol. This was more predominant in seeds incubated for 24 h or more and presumably reflects turnover of phospholipids in the endomembrane system. The relevance of these results is discussed in relation to the biogenesis of endoplasmic reticulum in germinating wheat seeds.

Gibberellic-acid-regulated expression of ?-amylase and six other genes in wheat aleurone layers

The effect of gibberellic acid (GA3) on gene expression in wheat aleurone cells has been characterised. In-vitro translation of polyadenylated RNA indicated that α-amylase and other messenger-RNA (mRNA) species increase in relative concentration in GA3-treated tissue. At least one mRNA species declines in relative level in response to GA3. There is also a GA3-dependent, four-fold increase in the level of polyadenylated RNA. This effect is largely the result of increased levels of many mRNA species which are also present in untreated tissue. Seven GA3-induced polyadenylated RNA species including the Amyl α-amylase gene product have been cloned as complementary DNA in the plasmid pBR322. These cloned DNAs have been used as hybridisation probes to show that the GA3-induced increase in α-amylase mRNA is more prolonged than the accumulation of the other GA3-regulated mRNA species. A polyadenylated-RNA sequence showing reduced concentration in GA3-treated tissue has also been cloned.

Phospholipid metabolism of wheat grains: Enzymes of the CDP-diglyceride phospholipid biosynthetic pathway

Enzymes of the CDP-diglyceride pathway of phospholipid synthesis, CDP-diacylglycerol synthetase, CDP-diacylglycerol: glycerol 3-phosphate phosphatidyl-transferase and enzymes of phosphatidylserine formation were initially of relatively high specific activities in aleurone cells of wheat and declined upon imbibition. Enzyme activity of phosphatidylinositol synthesis was not detected in dry grains but was present upon imbibition. CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase shifted during imbibition from 85% of the activity in the supernatant of aleurone layers from dry seeds to 98% associated with large particle fractions after 36 hours of imbibition. Phosphatidylserine formation shifted from a dominant location in the 1,500 x g fraction in the dry seed to a predominantly “mitochondrial” location after 36 hours of imbibition. The subcellular distribution of CDP-diacylglycerol synthetase did not change appreciably upon imbibition from that of the dry seed, 75 to 80% of the activity was found in the supernatant. Only CDP-diacylglycerol: glycerol 3-phosphate phosphatidyltransferase showed increased specific activity late in the imbibition period. GA3 accelerated the decrease of already declining activities of the CDP-diglyceride enzymes and the changes in their patterns of distribution, augmented the activities of the phosphatidylinositol synthesizing enzyme, and both accelerated and augmented the increase in the activity of the enzyme of phosphatidylglycerol synthesis which occurred late in imbibition.

An Ultrastructural Analysis of Aleurone Cells: Lamellar Bodies in Aleurone Layers of Wheat Grains during Germination

Aleurone cells of wheat undergo changes during development that encompass two phases, a quiescent early phase and a synthetic late phase. During the early phase, 4-8 h of imbibition, cell ultrastructure shows little change. During the synthetic phase (8-14 h), the cells undergo changes that include the appearance and disappearance of lamellar bodies and a twofold increase in endoplasmic reticulum (ER). Lamellar bodies are predominantly associated with protein bodies and ER. A precursor-product relationship between protein-body-associated and ER-associated lamellar bodies and between lamellar bodies and ER is consistent with morphometric data. No such relationship is evident for the globoids in the protein bodies. The differential distribution of lamellar bodies in aleurone cells may be an illustration of their general involvement in a unique arrangement, organization, or state of membrane lipids.

Endoplasmic Reticulum Formation during Germination of Wheat Seeds

This study demonstrates germination-induced ultrastructural changes in wheat (Triticum aestivum L. cv Arthur) aleurone cells. Seeds imbided for 4 hours in water contained endoplasmic reticulum (ER) or ER-like membranes as vesicles or as short segments of membrane associated with the spherosomes on the periphery of aleurone grains. Aleurone cells incubated between 8 and 10 hours contained abundant ER membranes mainly associated with the nuclear envelope and, to a lesser extent, with the spherosomes surrounding the aleurone grain. The membranes located on the periphery of the nucleus occurred as regions of stacked cisternae. When aleurone cells were analyzed by morphometry, the increase in ER during incubation was found to be greater than 2-fold. During the same incubation period, other organelles did not change significantly. The early increase in ER was not affected by gibberellin incubation. Thus, the rapid proliferation of ER observed during the early stages of germination in aleurone cells of wheat is not likely to be controlled directly by gibberellin.

Synthesis of a Possible Precursor of α-Amylase in Wheat Aleurone Cells

alpha-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific lambda-globulin for alpha-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic alpha-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of alpha-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of alpha-amylase in aleurone cells.