Molecular cloning, characterization, and expression of wheat cystatins

We cloned four kinds of cDNAs of wheat cystatins (WCs), WC1, WC2, WC3, and WC4, from the seed. They had 47-68% amino acid sequence similarities to other plant cystatins. WC1, WC2, and WC4 had 63-67% similalities to one another while 93% of amino acids were identical between WC1 and WC3. This suggested that WCI, WC2, and WC4 should be regarded as the isoforms of wheat cystatins. The mRNAs for WC1, WC2, and WC4 were all expressed in seed at an early stage of maturation and, after that, their quantities decreased gradually. However, each of the mRNAs was again expressed one day after the start of germination and the expression continued for the following five days. WC1 seemed to be expressed at a higher level than WC2 and WC4. Immunostaining for looking at site-specific expression of each WC demonstrated that both WC1 and WC4 existed in the aleuron layer and embryo, but in the endosperm the only existing species was WC1. Differences in mRNA level and tissue localization found for the WCs may suggest their differential physiological roles.     

Asparaginyl endopeptidase during maturation and germination of durum wheat

Asparaginyl-endopeptidase activity was detected in endosperms of maturing and germinating wheat seeds. The highest activity was found during maturation before the maximal accumulation of storage proteins. The enzyme activity then decreased in the dry seeds and increased again during germination. The increase of activity during germination required the presence of the embryo. In fact, the activity found in detached endosperms was lower than that found in attached ones. The localization at tissue level of the enzyme reveals differences between maturation and germination: the enzyme was about equally located in the aleurone layer and starchy endosperm during maturation, but solely in the aleurone layer during germination. The asparaginyl enzymes from maturing and germinating seeds had many similar properties, such as pH optimum, pH stability, thermal stability and sensitivity to thiol reagents and to thiol compounds. The results suggest that asparaginyl endopeptidases may be involved in the modification of proproteins of storage proteins during seed maturation and in the degradation of storage proteins deposited in the aleurone layer during germination.     

Purification and partial characteristic of a major gliadin-degrading cysteine endopeptidase from germinating triticale seeds

The endopeptidase of the highest electrophoretic mobility was the main endopeptidase hydrolyzing gliadin in the endosperm of germinated triticale (X Triticosecale Wittm.) grains after three days of imbibition. Activity of this endopeptidase, named EP8 starts to be detectable after two days of imbibition. The appearance of its activity in the endosperm on a second day of imbibition may suggest that EP8 is synthesized in aleurone during germination and/or secreted into the starchy endosperm as an inactive polypeptide during grains development and then activated. EP8 was isolated from the endosperm of germinating triticale seeds and purified 257-fold using ammonium sulphate, ion exchange chromatography on DEAE Sepharose CL-6B and gel filtration on Sephadex G-100. The enzyme was totally inhibited by E-64—class-specific cysteine proteinases inhibitor and activated by thiol compounds. Molecular weight estimated by SDS-PAGE was 39.5 kDa. The optimum pH for the hydrolysis of gliadin was 4.2 and for hemoglobin 5.2. High activity of EP8 against wheat gliadin in vitro suggests that this cysteine endopeptidase plays a major role in the mobilization of storage proteins in the endosperm of germinating triticale grains.     

Differential expression of wheat aspartic proteinases, WAP1 and WAP2, in germinating and maturing seeds

Two aspartic proteinase (AP) cDNA clones, WAP1 and WAP2, were obtained from wheat seeds. Proteins encoded by these clones shared 61% amino acid sequence identity. RNA blotting analysis showed that WAP1 and WAP2 were expressed in both germinating and maturing seeds. The level of WAP2 mRNA expression was clearly weaker than that of WAP1 in all tissues of seeds during germination and maturation. APs purified from germinating seeds were enzymatically active and digested the wheat storage protein, gluten. To elucidate the physiological functions of WAP1 and WAP2 in seeds, we investigated the localisation of WAP1 and WAP2 by in situ hybridisation. In germinating seeds investigated 24h after imbibition, both WAP1 and WAP2 were expressed in embryos, especially in radicles and shoots, scutellum, and the aleurone layer. In maturing seeds, WAP1 was expressed in the whole embryo, with slightly stronger expression in radicles and shoots. WAP1 was also expressed in the aleurone layer 3 weeks after flowering. Strong signals of WAP1 mRNA were detected in the whole embryo and aleurone layer 6 weeks after flowering. On the other hand, WAP2 was scarcely detected in seeds 3 weeks after flowering, and thereafter weak signals began to appear in the whole embryo. WAP1 and WAP2 were expressed widely in germinating and maturing seeds. Such diversity in site- and stage-specific expression of the two enzymes suggests their differential functions in wheat seeds.     

Differentiation of a Functional Aleurone Layer Within the Seed Coat of Sinapis alba L.

The hitherto unresolved ontogenetic origin of the aleurone layerin mustard (Sinapis alba L.) seeds was investigated with lightand electron microscopy. Contrary to previous views, this layerof storage cells is neither derived from the endosperm nor fromthe nucellus, but from a particular cell layer within the innerintegument of the seed coat. These cells differentiate and becomefilled with storage protein and fat concurrently with the maturationof the embryo. They survive seed desiccation and become depletedof storage materials during seed germination. Temporally correlatedwith the germinating embryo, the aleurone cells produce microbodyenzymes, which are controlled by light in a similar fashionin both tissues. Sinapis alba L., mustard, aleurone layer, seed coat, seed formation, germination     

Effects of Ageing on Amylase Activity and Scutellar Cell Structure during Imbibition in Wheat Seed

In wheat seed the scutellum plays an important role in the hydrolysisof stored substrate during germination. This layer is activatedfirst, whilst the aleurone becomes activated later. A good correlationexists between the initiation of visible germination and theappearance of enzyme activity in the scutellum. Enzyme activityin the aleurone becomes apparent only when the germinating seedlingreaches the rapid growth phase. Electron microscopic observationsshow that during the later stages of germination the scutellarcells develop finger like projections. These may serve to absorbendospermic reserves hydrolysed by aleurone amylase. The scutellumof aged non-germinating seeds showed no amylase activity andno finger like projections were produced even after prolongedimbibition.Copyright 1993, 1999 Academic Press Wheat (Triticum aestivum L.), deteriorated, germination, scutellum, scanning electron microscopy, aleurone     

Sugars as repressors of gibberellin-induced synthesis of α-amylase in wheat

In germinating cereal caryopses, α-amylase is synthesized in the aleurone layer and scutellum epithelium. Produced enzyme is released into the endosperm, where starch is hydrolyzed. We investigated the effect of sugars on gibberellic acid (GA)-induced synthesis of this enzyme in both tissues of wheat (Triticum aestivum L.) seeds. α-Amylase synthesis in the embryo was much more sensitive to sugars, and their inhibitory effect was observed at the lower concentrations (10–20 mM), whereas in the aleurone layer the enzyme was only inhibited at a relatively high (above 100 mM) concentration of sugars in the medium. These results point to a specific (repressive) influence of sugars on embryonic α-amylase and probably to its nonspecific (osmotic) effect on the cells of the aleurone layer. It was found that phosphorylated sugars were more effective repressors of α-amylase than nonphosphorylated sugars.     

Expression and Localization of Phosphoenolpyruvate Carboxylase in Developing and Germinating Wheat Grains

Phosphoenolpyruvate carboxylase (PEPC) activity and corresponding mRNA levels were investigated in developing and germinating wheat (Triticum aestivum) grains. During grain development PEPC activity increased to reach a maximum 15 d postanthesis. Western-blot experiments detected two main PEPC polypeptides with apparent molecular masses of 108 and 103 kD. The most abundant 103-kD PEPC subunit remained almost constant throughout the process of grain development and in the scutellum and aleurone layer of germinating grains. The less-abundant 108-kD polypeptide progressively disappeared during the second half of grain development and was newly synthesized in the scutellum and aleurone layer of germinating grains. PEPC mRNA was detected throughout the process of grain development; however, in germinating grains PEPC mRNA accumulated transiently in the scutellum and aleurone layer, showing a sharp maximum 24 h after imbibition. Immunolocalization studies revealed the presence of the enzyme in tissues with a high metabolic activity, as well as in the vascular tissue of the crease area of developing grains. A clear increase in PEPC was observed in the scutellar epithelium of grains 24 h after imbibition. The data suggest that the transiently formed PEPC mRNA in the scutellar epithelium encodes the 108-kD PEPC subunit.     

Characterization of the expression of a wheat cystatin gene during caryopsis development

A cDNA coding for phytocystatin, a protease inhibitor, was isolated from wheat embryos by differential display RT-PCR and the corresponding full-length cDNA (named WC5 for wheat cystatin gene 5) subsequently obtained by RACE. The deduced primary sequence of the protein suggests the presence of a 28 amino acid N-terminal signal sequence and a 100 amino acid mature protein containing the three consensus motifs known to interact with the active site of cysteine peptidases. Northern and western analysis revealed a spatio-temporal pattern of the cystatin gene expression during caryopse development. In the embryo, WC5 was only expressed during early embryogenesis whereas, in seed covering layers, WC5 expression was restricted to the maturation stage of grain development. In addition, immunolocalization experiments showed that cystatin accumulated in the aleurone layer of the maturating seed and in the parenchymal tissues of the embryo scutellum. A recombinant form of the wheat cystatin was shown to be able to inhibit peptidase activities present in whole seed protein extracts. In addition, immunological techniques allowed us to identify two putative target peptidases. The possible roles of the cystatin protein are discussed in relation with tissular localization and putative peptidase targets during seed maturation.     

Protein composition of cotyledons and aleurone grains inLupinus luteus L. Seeds during Germination

The change in protein composition of whole cotyledons and cotyledon aleurone grains ofLupinus luteus L. during seed germination was studied. SDS-polyacrylamide gel electro-phoresis showed a clear change in composition of cotyledon proteins as well as in composition of the aleurone grains during 5 days of seed germination. At this time, both in whole cotyledons as well as in aleurone grains, two subunits of β-conglutin with mol. m. 53 000 and 39 000 were rapidly hydrolyzed. After 5 days of germination traces of α-conglutin subunits could be detected in the cotyledons, whereas in aleurone grains this globulin fraction disappeared. In whole cotyledons and in cotyledon aleurone grains the γ-conglutin subunits with mol. m. 28 000 and 17 000 were not mobilized during the study period. These results indicate that the protein components with lower mol. m. were degraded later than those withhigher mol. m. during seed germination.     

Peptide mixtures can self-assemble into large amyloid fibers of varying size and morphology

Peptide mixtures spontaneously formed micrometer-sized fibers and ribbons from aqueous solution. Hydrolyzed gliadin produced short, slightly elliptical fibers while hydrolyzed wheat gluten, a mixture of gliadin and glutenin, formed round fibers of similar size. Mixing hydrolyzed gliadin with increasing molar amounts of myoglobin or amylase resulted in longer, wider fibers that transitioned from round to rectangular cross section. Fiber size, morphology, and modulus were controlled by peptide mixture composition. Fourier transform infrared (FT-IR) spectroscopy results showed that peptides experienced α to β transitions forming an elementary cross-β peptide secondary structure, indicative of amyloids. Large fiber formation was observed to be dependent on hydrophobic packing between constituent peptides. A model was developed to show how the fiber morphology was influenced by the peptides in the mixture.     

X-ray Analysis Studies of Elements Stored in Protein Body Globoid Crystals of Triticum Grains

Energy-dispersive x-ray analysis was used to investigate the elemental storage within protein bodies, specifically the globoid crystals, in grains of wheat. Areas of the grain investigated included various parts of the embryo, the aleurone layer plus starchy endosperm near the embryo and the aleurone layer plus starchy endosperm farthest from the embryo. Variations did occur grain-to-grain, cell-to-cell and, in certain regions, intracellularly. No protein bodies with electron-dense globoid crystals were found in the starchy endosperm. Generally globoid crystals contained P, K, and Mg in all areas investigated. Globoid crystals from the aleurone layer farthest from the embryo on occasion contained Ca, whereas aleurone globoid crystals near the embryo sometimes contained Fe. In most of the embryo regions examined, a few globoid crystals contained Ca along with P, K, and Mg. No specific pattern to the Ca distribution could be found. Welldefined elemental distribution occurred with Mn. Manganese was found only in globoid crystals located in the base and midregions of the stele in the radicle. Thus, in wheat there is some specific distribution of minerals dependent upon cell type and/or position in the grain.     

Enzymic mechanisms of starch breakdown in germinating rice seeds: 7. Amylase formation in the epithelium

The time sequence analysis of the starch digestion pattern of the thin sectioned germinating rice (Oryza sativa L.) seed specimens using the starch film method showed that at the initial stage amylase activity was almost exclusively localized in the epithelium septum between the scutellum and endosperm. Starch breakdown in the endosperm tissues began afterward; amylase activity in the aleurone layers was detectable only after 2 days. Polyacrylamide gel electrofocusing (pH 4 to 6) revealed nearly the same zymogram patterns between endosperm and scutellum extracts, although additional amylase bands appeared in the endosperm extracts at later germination stages (4 to 6 days). These are presumably attributable to the newly synthesized enzyme molecules in the aleurone cells.     

Changes in the cell wall accompanying drying and maturation determine the ease of isolation of protoplasts from wheat aleurone layers

Cell walls of aleurone tissue of developing wheat grains (25 days post anthesis) are degraded by cellulase to give a good yield of protoplasts. As grains become older the aleurone cell walls become increasingly resistant to cellulase, which is completely ineffective on 45-day old material. Resistance to cellulase is provoked even in aleurone cells of young grains by enforced drying of the grains to 12% water content. This suggests that the decreasing effectiveness of cellulase that occurs as grains develop and mature may be caused partly by the natural loss of water.Resistance to degradation by the aleurone cell walls of older or dried grains is to a large extent overcome by the addition of hemicellulase and xylanase to the digestion mixture but completely if acetyl esterase is also included. Changes involving hemicelluloses and xylans are therefore implicated in the effects of drying or maturation, including acetylation processes especially in grains which are almost mature. Arising from these findings a method has been developed for obtaining a high yield of protoplasts from aleurone layers of mature wheat grains, having a high viability and response to gibberellic acid.Abbreviations dpa days post anthesis - MES 2-(N-morpholino) ethanesulfonic acid - WC water content - SE standard error of the mean     

The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression.

Gene expression in the aleurone and endosperm is highly regulated during both seed development and germination. Studies of alpha-amylase expression in the aleurone of barley (Hordeum vulgare) have generated the current paradigm for hormonal control of gene expression in germinating cereal grain. Gene expression studies in both the aleurone and endosperm tissues of maize (Zea mays) seed have been hampered because of a lack of an efficient transformation system. We report here the rapid isolation of protoplasts from maize aleurone and endosperm tissue, their transformation using polyethylene glycol or electroporation, and the regulation of gene expression in these cells. Adh1 promoter activity was reduced relative to the 35S promoter in aleurone and endosperm protoplasts compared to Black Mexican Sweet suspension cells in which it was nearly as strong as the 35S promoter. Intron-mediated stimulation of expression was substantially higher in transformed aleurone or endosperm protoplasts than in cell-suspension culture protoplasts, and the data suggest that the effect of an intron may be affected by cell type. To examine cytoplasmic regulation, the 5' and 3' untranslated regions from a barley alpha-amylase were fused to the firefly luciferase-coding region, and their effect on translation and mRNA stability was examined following the delivery of in vitro synthesized mRNA to aleurone and endosperm protoplasts. The alpha-amylase untranslated regions regulated translational efficiency in a tissue-specific manner, increasing translation in aleurone or endosperm protoplasts but not in maize or carrot cell-suspension protoplasts, in animal cells, or in in vitro translation lysates.     

Thioredoxin and germinating barley: targets and protein redox changes

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.     

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.