植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同, 种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中, 醇溶蛋白在粗面内质网合成后形成蛋白质聚集体, 直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后, 根据各自的分选信号进入特定的运输囊泡, 经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中, 然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

水稻谷蛋白57H突变体T360的基因克隆与初步功能分析

谷蛋白是稻米贮藏蛋白的主要组分,其含量和组成直接影响稻米的各项品质指标和营养价值.在水稻胚乳中,谷蛋白首先在内质网中以57 kDa前体的形式合成,之后经由复杂的细胞学转运途径到达蛋白贮藏液泡后完成切割和沉积.尽管经过数十年的研究,已经分离克隆了 10余个谷蛋白合成、转运和沉积的关键基因,但其分子调控网络尚有待完善.本研...     

种子贮藏蛋白的运输、积累和基因表达调控

种子中贮藏蛋白的运输和积累途径主要有：(1)蛋白质合成后经内膜系统转移到蛋白质贮藏液泡(PSV)中积累;(2)合成的蛋白质直接在粗糙内质网的膜囊中积累形成蛋白质体;(3)贮藏蛋白不经高尔基体的加工由粗糙内质网上合成后直接运输到PSV中积累。贮藏蛋白基因的表达受该基因的顺式作用元件和反式作用因子的共同调控,此外染色体的结构也影响贮藏蛋白基因的表达。     

甜荞不同品种不同海拔地区种子蛋白组分含量研究

该研究通过分析甜荞10个品种在4个不同海拔栽培的种子蛋白质组分(清蛋白、球蛋白、醇溶蛋白和谷蛋白)的含量变异,以揭示不同荞麦品种之间以及不同栽培地点甜荞种子蛋白组分的变异规律。结果表明:在所有甜荞品种种子蛋白组分含量中清蛋白谷蛋白球蛋白醇溶蛋白。其中,种植于海拔最低的内蒙古通辽的甜荞种子平均球蛋白含量最高(1.081%),而种植于海拔1 450 m的河北甜荞谷蛋白平均含量最高(2.805%);海拔2 620 m的青海甜荞清蛋白平均含量为4.750%,而在海拔最高的西藏日喀则收获的甜荞种子的醇溶蛋白最高(平均为0.393%)。另外,蒙0530在4个地区的平均种子清蛋白和谷蛋白含量都最高,而球蛋白含量最高的品种是赤甜荞1号,定甜荞2号的种子醇溶蛋白含量最高。双因素方差分析表明,种子清蛋白含量品种间变异达极显著水平,不同地点间的种子醇溶蛋白含量达极显著水平,而地点和品种两个因素对种子球蛋白含量和谷蛋白含量的变异都有极显著影响。相关性分析表明,赤甜荞1号的醇溶蛋白含量与海拔呈显著正相关,蒙0530的球蛋白含量与海拔呈显著负相关,其他品种蛋白组分与海拔的相关性不显著。该研究结果对于甜荞优质品种培育和栽培以及推广都有一定的指导意义。     

3种生态型翅果油树种子贮藏蛋白分析

采用考马斯亮蓝G-250染色法和SDS-PAGE电泳技术对翅果油树3种生态类型种子贮藏蛋白含量进行测定和图谱分析.结果显示:大宫灯、长果型、小宫灯3种生态类型种子中总贮藏蛋白含量分别为33.753%、32.075%和26.633%;3种生态类型种子贮藏蛋白均以谷蛋白含量最高(65.971%～68.267%),其次为球蛋白和清蛋白,醇溶蛋白的含量最低;电泳图谱显示,3种生态类型之间清蛋白、球蛋白、醇溶蛋白和谷蛋白的蛋白质条带信息均存在差异.研究表明,翅果油树3种生态类型间种子贮藏蛋白具有多态性.     

鸡冠花种子蛋白质的提纯及分析

鸡冠花种子干燥后用石油醚脱酯,用凯氏定氮法测定蛋白质含量。按Osbern系统分别提取白蛋白、球蛋白、醇溶蛋白、谷蛋白;用Folin一酚试剂法测定各自相对含量,并用单向和双向SDS—PAGE方法分析种子总蛋白和4类不同溶性蛋白质的组成成分及这些组份的热稳定性。实验发现:鸡冠花种子蛋白质含量达26.04％,白蛋白、球蛋白、醇溶蛋白、谷蛋白的相对含量分别为11.5％、72％、4.6％、12％。SDS—PAGE表明;其种子蛋白中20KD球蛋白成份含量最高,其它一些次要组分为63KD、61KD、15KD、13KD白蛋白成份,58KD、37KD、23KD球蛋白成份,39KD、34KD、25KD谷蛋白成份及26KD醇溶蛋白成份,其中58KD由37KD和20KD两亚基经二硫键连接而成,39KD组份由24.5KD和18KD两亚基通过二硫键连接而成,23KD球蛋白组份遇热沉淀。     

小麦籽粒蛋白质组分的生态变异及与植株氮转化的关系研究

小麦籽粒蛋白质和蛋白质组分含量决定了小麦面团流变学特性和加工品质、籽粒蛋白质组分的形成与植株氮素吸收利用密切相关,且蛋白质组分的合成有一定的顺序性。对不同生态区的小麦籽粒蛋白质组分合成进行了研究。结果表明,蛋白质及组分含量存在明显的基因型差异,徐州点蛋白质和谷蛋白含量显著大于南京点;花期氮素积累量和花后氮素转运量徐州点显著大于南京点,而花后氮同化量南京点显著大于徐州点;清蛋白、醇溶蛋白和谷蛋白含量与籽粒蛋白质含量呈显著正相关,与花后植株氮素转运量呈显著正相关。不同生态点小麦花后发育速率模式基本一致,但南京点发育进程迟于徐州点;在籽粒形成期和灌浆后期,南京点的发育速率较快,降低了清蛋白和谷蛋白的合成,导致南京点籽粒蛋白质含量降低。     

trxS基因对大麦发芽籽粒中蛋白质降解的影响

对转trxS基因大麦籽粒发芽过程中蛋白酶活性、不同蛋白组分含量和贮藏蛋白SDS-PAGE图谱的变化进行了研究。结果表明：与对照相比,转基因籽粒中的蛋白酶活性提高;清蛋白、球蛋白、醇溶蛋白和谷蛋白含量低于对照。SDS-PAGE图谱也表明,转基因籽粒中贮藏蛋白降解快于对照。     

西瓜种子发育和萌发过程中子叶细胞超微结构的变化

西瓜种子子叶内贮存物质开始积累时,细胞质内有大量核糖体、质体、线粒体,内质网片段和囊泡,种子脱水期至成熟期,细胞器的数量减少,成熟种子子叶细胞的细胞壁不连续,几乎观察不到细胞器的存在,种子萌发过程中内质网,线粒体,质体的数目逐渐增多,叶肉细胞的质体发育成叶绿体,种子形成过程中,在子叶细胞大液泡分隔的同时,膨胀的内质网囊泡内积累蛋白质（直径0.1-0.4μm),这些小的蛋白质球体最终进入液泡形成大的蛋白体（直径1-3μm);萌发种子贮存蛋白质被水解的同时,一些脂体进入液泡并被分解,同时液泡融合;脂类物质开始积累的时间早于蛋白质,积累的量较蛋白质多,但在萌发种子中被彻底水解的时间晚于蛋白质,淀粉粒的数量在种子形成时减少,种子萌发时在表皮细胞和叶肉细胞内都重新合成。     

水稻57H突变体系列的胚乳储藏蛋白质表型多样性及其分类(英文)

种子储藏蛋白质主要由谷蛋白、醇溶谷蛋白和球蛋白组成。这些蛋白质在种子发育期被合成后,经过区室化过程,被蓄积在胚乳中。本研究系统分析了水稻57H突变体的表型多样性,旨在为胚乳储藏蛋白质的遗传调节机制的全面解明展示一个新的前景。胚乳蛋白质的SDS-PAGE分析和免疫印迹分析显示了高量含有57kD谷蛋白前体的水稻57H突变体系列具有多样的储藏蛋白质表型。基于其表型的多样性,57H突变体系列被分成了三种类型。与野生型水稻品种相比,所有的类型Ⅰ突变体(glup4,glup6,Glup5,esp2)不仅高量蓄积谷蛋白前体、少量蓄积成熟型谷蛋白的40kD酸性亚基和20kD碱性亚基,而且显著减少了13kD醇溶谷蛋白b组分和26kDa球蛋白的蓄积;类型Ⅱ突变体(Glup1,glup2)不仅高量蓄积谷蛋白前体,还减少了26kDa球蛋白的蓄积;类型Ⅲ突变体(glup3)除了高量蓄积谷蛋白前体、少量蓄积成熟型谷蛋白亚基之外,并没有减少其它储藏蛋白质的蓄积。结果指出了57H变异系列对储藏蛋白的蓄积具有多样的影响,实质上是关于储藏蛋白质区室化的遗传体系。并就该遗传体系对储藏蛋白质的翻译后区室化及其营养性状的可能的影响进行了讨论。     

Deposition of storage proteins

Plants store amino acids for longer periods in the form of specific storage proteins. These are deposited in seeds, in root and shoot tubers, in the wood and bark parenchyma of trees and in other vegetative organs. Storage proteins are protected against uncontrolled premature degradation by several mechanisms. The major one is to deposit the storage proteins into specialized membrane-bounded storage organelles, called protein bodies (PB). In the endosperm cells of maize and rice prolamins are sequestered into PBs which are derived from the endoplasmic reticulum (ER). Globulins, the typical storage proteins of dicotyledonous plants, and prolamins of some cereals are transported from the ER through the Golgi apparatus and then into protein storage vacuoles (PSV) which later become transformed into PBs. Sorting and targeting of storage proteins begins during their biosynthesis on membrane-bound polysomes where an N-terminal signal peptide mediates their segregation into the lumen of the ER. After cleavage of the signal peptide, the polypeptides are glycosylated and folded with the aid of chaperones. While still in the ER, disulfide bridges are formed which stabilize the structure and several polypeptides are joined to form an oligomer which has the proper conformation to be either deposited in ER-derived PB or to be further transferred to the PSV. At the trans-Golgi cisternae transport vesicles are sequestered which carry the storage proteins to the PSV. Several storage proteins are also processed after arriving in the PSVs in order to generate a conformation that is capable of final deposition. Some storage protein precursors have short N- or C-terminal targeting sequences which are detached after arrival in the PSV. Others have been shown to have internal sequence regions which could act as targeting information. In some cases positive targeting information is known to mediate sorting into the PSV whereas in other cases aggregation and membrane association seem to be major sorting mechanisms.     

Wheat α-gliadin and high-molecular-weight glutenin subunit accumulate in different storage compartments of transgenic soybean seed

Wheat seed storage proteins (prolamins) are important for the grain quality because they provide a characteristic texture to wheat flour products. In wheat endosperm cells, prolamins are transported from the Endoplasmic reticulum to Protein storage vacuoles through two distinct pathways—a conventional pathway passing through the Golgi apparatus and an unconventional Golgi-bypassing pathway during which prolamins accumulate in the ER lumen, forming Protein bodies. Unfortunately, transport studies conducted previously achieved limited success because of the seed-specificity of the latter pathway and the multigene architecture of prolamins. To overcome this difficulty, we expressed either of the two families of wheat prolamins, namely α-gliadin or High-molecular-weight subunit of glutenin, in soybean seed, which naturally lacks prolamin-like proteins. SDS-PAGE analysis indicated the successful expression of recombinant wheat prolamins in transgenic soybean seeds. Their accumulation states were quite different—α-gliadin accumulated with partial fragmentation whereas the HMW-glutenin subunit formed disulfide-crosslinked polymers without fragmentation. Immunoelectron microscopy of seed sections revealed that α-gliadin was transported to PSVs whereas HMW-glutenin was deposited in novel ER-derived compartments distinct from PSVs. Observation of a developmental stage of seed cells showed the involvement of post-Golgi Prevacuolar compartments in the transport of α-gliadin. In a similar stage of cells, deposits of HMW-glutenin surrounded by membranes studded with ribosomes were observed confirming the accumulation of this prolamin as ER-derived PBs. Subcellular fractionation analysis supported the electron microscopy observations. Our results should help in better understanding of molecular events during the transport of prolamins in wheat.

     

A C-terminal sequence of soybean beta-conglycinin alpha' subunit acts as a vacuolar sorting determinant in seed cells

In maturing seed cells, many newly synthesized proteins are transported to the protein storage vacuoles (PSVs) via vesicles unique to seed cells. Vacuolar sorting determinants (VSDs) in most of these proteins have been determined using leaf, root or suspension-cultured cells apart from seed cells. In this study, we examined the VSD of the alpha' subunit of beta-conglycinin (7S globulin), one of the major seed storage proteins of soybean, using Arabidopsis and soybean seeds. The wild-type alpha' was transported to the matrix of the PSVs in seed cells of transgenic Arabidopsis, and it formed crystalloid-like structures. Some of the wild-type alpha' was also transported to the translucent compartments (TLCs) in the PSV presumed to be the globoid compartments. However, a derivative lacking the C-terminal 10 amino acids was not transported to the PSV matrix, and was secreted out of the cells, although a portion was also transported to the TLCs. The C-terminal region of alpha' was sufficient to transport a green fluorescent protein (GFP) to the PSV matrix. These indicate that alpha' contains two VSDs: one is present in the C-terminal 10 amino acids and is for the PSV matrix; and the other is for the TLC (the globoid compartment). We further verified that the C-terminal 10 amino acids were sufficient to transport GFP to the PSV matrix in soybean seed cells by using a transient expression system.     

Reducing rice seed storage protein accumulation leads to changes in nutrient quality and storage organelle formation

Rice (Oryza sativa) seed storage proteins (SSPs) are synthesized and deposited in storage organelles in the endosperm during seed maturation as a nitrogen source for germinating seedlings. We have generated glutelin, globulin, and prolamin knockdown lines and have examined their effects on seed quality. A reduction of one or a few SSP(s) was compensated for by increases in other SSPs at both the mRNA and protein levels. Especially, reduction of glutelins or sulfur-rich 10-kD prolamin levels was preferentially compensated by sulfur-poor or other sulfur-rich prolamins, respectively, indicating that sulfur-containing amino acids are involved in regulating SSP composition. Furthermore, a reduction in the levels of 13-kD prolamin resulted in enhancement of the total lysine content by 56% when compared with the wild type. This observation can be mainly accounted for by the increase in lysine-rich proteins. Although reducing the level of glutelins slightly decreased protein storage vacuoles (PSVs), the simultaneous reduction of glutelin and globulin levels altered the inner structure of PSVs, implicating globulin in framing PSV formation. Knock down of 13-kD prolamins not only reduced the size of endoplasmic reticulum-derived protein bodies (PBs) but also altered the rugged peripheral structure. In contrast, PBs became slightly smaller or unchanged by severe suppression of 10- or 16-kD prolamins, respectively, indicating that individual prolamins have distinct functions in the formation of PBs. Extreme increases or decreases in sulfur-poor prolamins resulted in the production of small PBs, suggesting that the ratio of individual prolamins is crucial for proper aggregation and folding of prolamins.     

Multivesicular bodies in developing tobacco seed and mung bean are functionally equivalent

Protein storage vacuoles (PSVs) are the primarily storage organelles in cotyledon cells for protein preservation in seeds. Storage proteins are transported from the endoplasmic reticulum (ER) to the Golgi apparatus for subsequent delivery to PSVs via presumably Golgi-derived dense vesicles (DVs). However, recent studies demonstrated that storage proteins in early stage of developing cotyledon of mung beans reached the multivesicular bodies (MVBs) prior to the detection of DVs, indicating the possible involvement of MVBs in mediating transport of storage proteins during the early stage of seed development. Here, we further show that the MVBs in developing tobacco seeds are functionally and biochemically equivalent to those in developing mung beans. Thus, MVBs in developing tobacco seeds are structurally distinct from DVs, contain both vacuolar sorting receptors (VSRs) and storage proteins, and they are insensitive to treatments of wortmannin and brefeldin A (BFA).     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

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.     

Protein mobilization in germinating mung bean seeds involves vacuolar sorting receptors and multivesicular bodies

Plants accumulate and store proteins in protein storage vacuoles (PSVs) during seed development and maturation. Upon seed germination, these storage proteins are mobilized to provide nutrients for seedling growth. However, little is known about the molecular mechanisms of protein degradation during seed germination. Here we test the hypothesis that vacuolar sorting receptor (VSR) proteins play a role in mediating protein degradation in germinating seeds. We demonstrate that both VSR proteins and hydrolytic enzymes are synthesized de novo during mung bean (Vigna radiata) seed germination. Immunogold electron microscopy with VSR antibodies demonstrate that VSRs mainly locate to the peripheral membrane of multivesicular bodies (MVBs), presumably as recycling receptors in day 1 germinating seeds, but become internalized to the MVB lumen, presumably for degradation at day 3 germination. Chemical cross-linking and immunoprecipitation with VSR antibodies have identified the cysteine protease aleurain as a specific VSR-interacting protein in germinating seeds. Further confocal immunofluorescence and immunogold electron microscopy studies demonstrate that VSR and aleurain colocalize to MVBs as well as PSVs in germinating seeds. Thus, MVBs in germinating seeds exercise dual functions: as a storage compartment for proteases that are physically separated from PSVs in the mature seed and as an intermediate compartment for VSR-mediated delivery of proteases from the Golgi apparatus to the PSV for protein degradation during seed germination.     

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.