Action of Sorghum Proteinase on the Protein Bodies of Sorghum Starchy Endosperm

Protein bodies isolated from the starchy endosperm of ungerminatedsorghum exhibited some autolytic activity but seemed incapableof significant self-hydrolysis. Enzyme assay, transmission electronmicroscopy, sodium dodecyl sulphate—polyacrylamide gelelectrophoresis and amino acid analysis revealed that a proteinaseextract from germinated sorghum could degrade the protein bodiesin a manner resembling that which takes place in vivo. The proteinbodies were degraded mainly from the periphery. Glutelin (matrixprotein) was first hydrolysed, followed by the prolamin proteinbody protein. Proteinase extracts from both the germ and endospermof germinated sorghum were capable of degrading the proteinbodies. This finding is consistent with the concept that theproteinase is synthesized in the germ and then secreted intothe starchy endosperm during germination. Key words: Sorghum bicolor, protein body degradation, proteinase.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

The dynamics of protein body formation in developing wheat grain

Wheat is a major source of protein in the diets of humans and livestock but we know little about the mechanisms that determine the patterns of protein synthesis in the developing endosperm. We have used a combination of enrichment with ¹⁵N glutamine and NanoSIMS imaging to establish that the substrate required for protein synthesis is transported radially from its point of entrance in the endosperm cavity across the starchy endosperm tissues, before becoming concentrated in the cells immediately below the aleurone layer. This transport occurs continuously during grain development but may be slower in the later stages. Although older starchy endosperm cells tend to contain larger protein deposits formed by the fusion of small protein bodies, small highly enriched protein bodies may also be present in the same cells. This shows a continuous process of protein body initiation, in both older and younger starchy endosperm cells and in all regions of the tissue. Immunolabeling with specific antibodies shows that the patterns of enrichment are not related to the contents of gluten proteins in the protein bodies. In addition to providing new information on the dynamics of protein deposition, the study demonstrates the wider utility of NanoSIMS and isotope labelling for studying complex developmental processes in plant tissues.     

Delivery of prolamins to the protein storage vacuole in maize aleurone cells

Zeins, the prolamin storage proteins found in maize (Zea mays), accumulate in accretions called protein bodies inside the endoplasmic reticulum (ER) of starchy endosperm cells. We found that genes encoding zeins, α-globulin, and legumin-1 are transcribed not only in the starchy endosperm but also in aleurone cells. Unlike the starchy endosperm, aleurone cells accumulate these storage proteins inside protein storage vacuoles (PSVs) instead of the ER. Aleurone PSVs contain zein-rich protein inclusions, a matrix, and a large system of intravacuolar membranes. After being assembled in the ER, zeins are delivered to the aleurone PSVs in atypical prevacuolar compartments that seem to arise at least partially by autophagy and consist of multilayered membranes and engulfed cytoplasmic material. The zein-containing prevacuolar compartments are neither surrounded by a double membrane nor decorated by AUTOPHAGY RELATED8 protein, suggesting that they are not typical autophagosomes. The PSV matrix contains glycoproteins that are trafficked through a Golgi-multivesicular body (MVB) pathway. MVBs likely fuse with the multilayered, autophagic compartments before merging with the PSV. The presence of similar PSVs also containing prolamins and large systems of intravacuolar membranes in wheat (Triticum aestivum) and barley (Hordeum vulgare) starchy endosperm suggests that this trafficking mechanism may be common among cereals.     

Storage-protein hydrolysis and protein-body breakdown in germinatedZea mays L. seeds

Storage proteins of maize (Zea mays L.) were studied in germinated seeds, as were the proteins of protein bodies isolated from endosperms at different germination times. Major endosperm storage proteins were degraded in a sequential way, glutelin 2 being hydrolysed faster than zein 1. Immunocytochemical labelling of the different protein bodies using the antisera anti-glutelin 2 and anti-zein 1 indicates that the protein bodies were degraded by progressive hydrolysis from their surface. The digestion of glutelin 2 correlated with the disappearance of the protein-body membranes.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Formation mechanism of the internal structure of type I protein bodies in rice endosperm: relationship between the localization of prolamin species and the expression of individual genes

Rice prolamins, a group of seed storage proteins, are synthesized on the rough endoplasmic reticulum (ER) and form type I protein bodies (PB-Is) in endosperm cells. Rice prolamins are encoded by a multigene family. In this study, the spatial accumulation patterns of various prolamin species in rice endosperm cells were investigated to determine the mechanism of formation of the internal structure of PB-Is. Immunofluorescence microscopic analysis of mature endosperm cells showed that the 10 kDa prolamin is mainly localized in the core of the PB-Is, the 13b prolamin is localized in the inner layer surrounding the core and the outermost layer, and the 13a and 16 kDa prolamins are localized in the middle layer. Real-time RT-PCR analysis showed that expression of the mRNA for 10 kDa prolamin precedes expression of 13a, 13b-1 and 16 kDa prolamin in the developing stages. mRNA expression for 13b-2 prolamin occurred after that of the other prolamin species. Immunoelectron microscopy of developing seeds showed that the 10 kDa prolamin polypeptide initially accumulates in the ER, and then 13b, 13a, 16 kDa and 13b prolamins are stacked in layers within the ER. Studies with transgenic rice seeds expressing prolamin-GFP fusion proteins under the control of native and constitutive promoters indicated that the temporal expression pattern of prolamin genes influenced the localization of prolamin proteins within the PB-Is. These findings indicate that the control of gene expression of prolamin species contributes to the internal structure of PB-Is.     

Biosynthesis of storage proteins in developing rice seeds

Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the starchy endosperm protein of rice (Oryza sativa L. Japonica cv Koshihikari) during seed development confirmed that storage protein begins to accumulate about 5 days after flowering. Two polypeptide groups, 22 to 23 and 37 to 39 kilodaltons, the components of glutelin, the major storage protein in rice seed, appeared 5 days after flowering. A 26-kilodalton polypeptide, the globulin component, also appeared 5 days after flowering. Smaller polypeptides (10- to 16-kilodaltons) including prolamin components, appeared about 10 days after flowering. In contrast, the levels of the 76- and 57-kilodalton polypeptides were fairly constant throughout seed development. Transmission electron microscopy and fractionation by sucrose density gradient centrifugation of the starchy endosperms at various stages of development showed that protein body type II, the accumulation site of glutelin and globulin, was formed faster than protein body type I, the accumulation site of prolamin.

The 57-kilodalton polypeptide but not the glutelin subunits was labeled in a 2-hour treatment with [¹⁴C]leucine given between 4 and 12 days after flowering to developing ears. In vivo pulse-chase labeling studies showed the 57-kilodalton polypeptide to be a precursor of the 22 to 23 and 37 to 39 kilodalton subunits. The 57-kilodalton polypeptide was salt-soluble, but the mature glutelin subunits were almost salt insoluble.

In vitro protein synthesis also showed that the mRNAs directly coding the 22 to 23 and 37 to 39 kilodalton components were absent in developing seeds and that the 57-kilodalton polypeptide was the major product. Thus, it was concluded that the two subunits of rice glutelin are formed through post-translational cleavage of the 57-kilodalton polypeptide.

     

The Hydrolysis of Endosperm Protein in Zea mays

Degradation of the major storage proteins in maize endosperm, zein and glutelin, begins during the 2nd day of germination. The protein most abundant in the mature endosperm is degraded most rapidly. The patterns of protein loss are essentially similar in germinating seeds and excised endosperms. Cycloheximide, added at the beginning of the incubation period, prevents the development of α-amylase and protease activities and the disappearance of starch and protein reserves. Late additions (70 hours) of cycloheximide still inhibit the increase in hydrolase activity but have no effect on the hydrolysis of storage reserves. The results indicate that the hydrolytic enzymes are synthesized de novo in the maize endosperm.     

不同倍性水稻胚乳蛋白的差异表达研究

以4份同源四倍体水稻和4份相应的二倍体水稻为研究材料, 对其种子内清、球蛋白(清蛋白和球蛋白)、醇溶蛋白和谷蛋白的含量进行了测定, 利用聚丙烯酰胺凝胶电泳(SDS-PAGE)技术分析了其特异性。结果表明: 同源四倍体水稻胚乳蛋白各组分含量与相应的二倍体相比, 大部分呈增加趋势; 同源四倍体水稻胚乳蛋白的亚基类型与相应的二倍体水稻基本一致, 仅在全蛋白电泳和清、球蛋白电泳中各发现1条差异条带。研究结果认为: 二倍体水稻经过染色体组加倍之后, 同源四倍体水稻重复基因组在蛋白质水平上的表达结果趋于“二倍化”, 即与二倍体水稻表现出相似的特点, 但在蛋白质表达量上前者往往高于后者。基因组多倍化对同源四倍体水稻重复基因组进化最主要的影响并不是体现在蛋白质结构上的差异, 而是体现在蛋白质表达量上的差异。     

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.     

Stored cysteine proteinases start globulin mobilization in protein bodies of embryonic axes and cotyledons during vetch (Vicia sativa L.) seed germination

Inhibition of protein synthesis by cycloheximide during vetch seed germination, did not prevent globulin breakdown as indicated by a decrease in vicilin- and legumin-specific immunosignals on Western blots. Protein bodies isolated from embryo axes and cotyledons of dry vetch (Vicia sativa L.) seeds using a non-aqueous method were found to be free of cytoplasmic and organellar contaminations. Lysates of these purified protein bodies were capable of degrading globulins; this process was blocked by the cysteine proteinase (CPR) inhibitor iodoacetic acid. Protein bodies contained the papain-like CPR2 and CPR4, and the legumain-like CPR VsPB2. In vitro assays showed that albumin extracts from protein bodies degraded oligopeptide substrates in the PepTag-Assay and degraded the legumain substrate N-benzoyl-asparaginyl-p-nitroanilide. We conclude that, during germination, globulin mobilization is initiated by stored CPRs in protein bodies of embryonic axes as well as cotyledons, and that de-novo-formed proteolytic enzymes mainly mediate bulk degradation of stored globulin in cotyledons after germination. Received: 14 February 2000 / Accepted: 16 August 2000     

水稻淀粉胚乳细胞编程性死亡中细胞核变化特征

应用透射电子显微镜技术 ,观察了水稻 (OryzasativaL .)淀粉胚乳细胞编程性死亡过程中核的变化特征。伴随胚乳的发育进程 ,淀粉胚乳细胞核表现出衰退特征 :核变形、染色质凝缩、核膜多处被降解破坏、核基质外泄等。DNALadder显示核内大片段DNA呈严重的弥散状拖尾现象 ,而核内和胞质中在 14 0～ 180bp处有明显的条带。在核衰退的同时 ,其胞质中的粗面内质网、淀粉质体和线粒体等细胞器具有正常的代谢功能 ,细胞仍在合成并积累营养物质 ,淀粉胚乳细胞一边衰退一边行使其功能 ,直至死亡。这些结果表明 ,水稻淀粉胚乳在核衰退的同时 ,细胞仍在积极合成与积累贮藏产物 ,表现为一种特殊形式的植物细胞编程性死亡现象。此外 ,对淀粉胚乳细胞特有的核质关系、植物细胞编程性死亡过程中细胞核的变化等问题进行了讨论。     

水稻淀粉胚乳程序性细胞死亡中的去核化

对水稻品种中籼8836淀粉胚乳细胞的去核化发育阶段的细胞超微结构变化和同期籽粒灌浆速率及相关酶活性的动态进行了观察和分析。开花受精后约在第3天胚乳完成细胞化,花后第5天少数淀粉胚乳细胞启动去核发育过程。核消亡是淀粉胚乳细胞程序性细胞死亡(PCD)的第一步。同一籽粒淀粉胚乳细胞的去核进程是不同步的。花后第13天所有淀粉胚乳细胞都已完成去核过程。在去核过程中,胚乳核的形态变化特征既有动植物PCD的共性又有其特殊性。伴随核降解过程,一部分线粒体解体,表明去核化与线粒体解体有一定联系。在去核化发育阶段,与PCD有关的酶类,如超氧化物歧化酶(SOD)过氧化氢酶(CAT)活性非常高;与淀粉合成有关的酶类,如ADPG焦磷酸化酶、可溶性淀粉合成酶(SSS酶)、淀粉分支酶(或Q酶)也表现出很高的活性。去核化发育阶段籽粒灌浆速率最高,籽粒增重亦最快。淀粉胚乳细胞去核之后,细胞并未立即死亡,这些无核的细胞仍维持正常有序的代谢活动,继续进行淀粉和贮藏蛋白的合成与积累,但上述酶类的活性明显降低,灌浆速率也明显趋缓。淀粉胚乳细胞最终被贮藏物质充满时成为死细胞,完成其程序性死亡过程。Evan’s blue染色鉴定表明淀粉胚乳细胞死亡不同步,细胞死亡在淀粉胚乳组织中是随机发生的。     

水稻淀粉胚乳程序性细胞死亡中的去核化

对水稻品种中籼8836淀粉胚乳细胞的去核化发育阶段的细胞超微结构变化和同期籽粒灌浆速率及相关酶活性的动态进行了观察和分析。开花受精后约在第3天胚乳完成细胞化,花后第5天少数淀粉胚乳细胞启动去核发育过程。核消亡是淀粉胚乳细胞程序性细胞死亡(PCD)的第一步。同一籽粒淀粉胚乳细胞的去核进程是不同步的。花后第13天所有淀粉胚乳细胞都已完成去核过程。在去核过程中,胚乳核的形态变化特征既有动植物PCD的共性又有其特殊性。伴随核降解过程,一部分线粒体解体,表明去核化与线粒体解体有一定联系。在去核化发育阶段,与PCD有关的酶类,如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性非常高;与淀粉合成有关的酶类,如ADPG焦磷酸化酶、可溶性淀粉合成酶(SSS酶)、淀粉分支酶(或Q酶)也表现出很高的活性。去核化发育阶段籽粒灌浆速率最高,籽粒增重亦最快。淀粉胚乳细胞去核之后,细胞并未立即死亡,这些无核的细胞仍维持正常有序的代谢活动,继续进行淀粉和贮藏蛋白的合成与积累,但上述酶类的活性明显降低,灌浆速率也明显趋缓。淀粉胚乳细胞最终被贮藏物质充满时成为死细胞,完成其程序性死亡过程。Evan‘s blue染色鉴定表明淀粉胚乳细胞死亡不同步,细胞死亡在淀粉胚乳组织中是随机发生的。     

Proteolytic activities in the developing seeds of ×Haynaldoticum sardoum

Aminopeptidase, carboxypeptidase and proteinase activities were measured in endosperms from unripe and ripe seeds of ×Haynaldoticum sardoum. Aminopeptidase and proteinase activities were high during the early maturation stages and then decreased. In contrast, carboxypeptidase activity increased with maturation. Localization studies demonstrated that aminopeptidase and carboxypeptidase activities were present in the three tissues examined (pericarp, green layer plus aleurone, and starchy endosperm). Proteinase activity against gliadin was located in the pericarp and in the green layer plus aleurone, but was absent in the starchy endosperm. The presence of proteolytic activities in the outer kernel layers might be correlated to the hydrolysis of transitory protein reserves during the senescence of the seed coat?. Aminopeptidase and carboxypeptidase activities located in the starchy endosperm could participate in the breakdown of protein reserves during the early phases of seed germination.     

Amylases in developing barley seeds

The amylases of developing barley seeds (Hordeum vulgare L. cv. Himalaya) were investigated by colorimetric and electrophoretic methods. Maxima of amylolytic activity appeared in the aleurone layers and starchy endosperm at 5 and 20 days after anthesis. Amylase from 5-day-old aleurone layers could be separated into four rapidly moving bands with α-amylase activity. By 20 days the four bands had been replaced by seven bands of medium mobility. These seven bands of amylase were electrophoretically identical to those observed when mature aleurone layers are treated with gibberellic acid. Immature aleurone layers failed to respond to exogenous gibberellic acid. In the starchy endosperm the seven bands of medium mobility were also present. Calcium-dependent alterations in the electrophoretic mobility and activity of particular bands occurred during the maturation of the starchy endosperm. Treatment of the immature starchy endosperm with papain yielded four forms of β-amylase.     

Evidence for osmotic regulation of hydrolytic enzyme production in germinating barley seeds

α-Amylase levels in intact seeds of barley (Hordeum vulgare L. cv. Himalaya) reach a maximum at 3 to 4 days of germination while gibberellin levels continue to increase beyond 6 days of germination. In contrast to its effect on half seeds, gibberellic acid does not increase the total amount of α-amylase produced in germinating seeds. The inability of gibberellic acid to stimulate α-amylase production is not related to its availability; rather, evidence suggests that a factor(s) in whole seeds prevents further enhancement of α-amylase formation and accumulation. Hydrolysis products accumulate in the subaleurone space of the endosperm of germinating seeds up to concentrations of 570 milliosmolar. Chromatography of these hydrolysis products indicate the presence of maltose and glucose. Calculations based on reducing sugar determinations show that glucose accounts for as much as 57% of the solutes present in the endosperm fluid. Both maltose and glucose in the range of 0.2 to 0.4 M effectively inhibit the production of α-amylase by isolated barley aleurone layers. This inhibition is quantitatively similar to that brought about by solutions of polyethylene glycol and mannitol. On the basis of these data we propose that hydrolysis products which accumulate in the starchy endosperm of germinating seeds function to regulate the production of hydrolytic enzymes by the aleurone layer.     

Enzymic Mechanism of Starch Breakdown in Germinating Rice Seeds: 8. Immunohistochemical Localization of beta-Amylase

Rabbit antiserum against β-amylase isolated from germinating seeds of rice was produced, and its specific cross-reactivity with β-amylase was confirmed by means of Ouchterlony double immunodiffusion and immunoelectrophoresis procedures. The cellular localization of β-amylase was studied by indirect fluorescence microscopy of thin sectioned germinating rice seed specimens (1-day stage) which had been fixed and treated with purified rabbit anti-β-amylase immunoglobulin G followed by conjugation with fluorescein isothiocyanate-labeled goat antirabbit immunoglobulin G. It has been demonstrated that β-amylase is uniformly associated with the periphery of starch granules in the starchy endosperm cells. The finding is discussed in relation to the general notion concerning the presence of the latent form of β-amylase bound to protein bodies in cereal seeds.