Characterization of an anther-specific glycoprotein in Lilium longiflorum

Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther-specific glycoprotein (LLA-75). LLA-75 protein bound to concanavalin A, suggesting that it was a glycoprotein. Monospecific anti-LLA-75 antibodies were prepared to investigate its distribution during anther development. Immunoblot analyses of total protein from floral and vegetative organs show that LLA-75 glycoproteins accumulated to detectable levels primarily in one discrete stage of anther development, during the microspore and early pollen phase when the tapetum is actively secreting. A cross-reactive protein with molecular mass of 43.0 kD was also observed. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the four isoforms of LLA-75 glycoprotein ranged from isoelectric point 5.6 to 6.1. These affinity purified antibodies to LLA-75 cross-reacted with anther proteins in two dicot species of the Bignoniaceae. In situ localization data using antirabbit immunoglobulin G (IgG) conjugated with gold particles was not definitive but demonstrated that LLA-75 glycoprotein in lily anthers occurred prominently in tapetal tissue and all other tissues to a lesser degree.     

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

Endosomal sorting complex required for transport proteins ISTL1 and LIP5 are required for exocytosis of both plasma membrane and secreted proteins in tapetal cells during microspore formation.     

A Comparative Study of Anther and Pollen Development in Male Fertile and Male. Sterile Green Onion (Allium fistulosum L.)

Anther and pollen development in male-fertile and male-sterile green onions was studied. In the male-fertile line, both meiotic microspore mother ceils and tetrads have a callose wall. Mature pollen grains are 2-celled. The elongated generative cell with two bended ends displays a PAS positive cell wall. The tapetum has the character of both secretory and invasive types. From microspore stage onwards, many oil bodies or masses accumulate in the cytoplasm of the tapetal cells. The tapetum degenerates at middle 2-celled pollen stage. In male-sterile line, meiosis in microspore mother cells proceeds normally to form the tetrads. Pollen abortion occurs at microspore with vacuole stage. Two types of pollen abortion were observed. In type I, the protoplasts of the microspores contract and gradually disintegrate. At the same time the cytoplasm of microspores accumulates oil bodies which remain in the empty pollen. The tapetal cells behave normally up to the microspore stage and early stage of microspore abortion, but contain fewer oil bodies or masses than those in the male-fertilt line. At late stage of microspore abortion, three forms of the tapetal ceils can be observed: (1) the tapetal cells with degenerating protoplasts become flattened, (2) the tapetal cells enlarge but protoplasts retractor, (3) the cells break down and tile middle layer enlarges. In type Ⅱ, the cytoplasm degenerates earlier than the nucleus of the microspores and no protoplast is found in the anther locule. There are fibrous thickenings iii the endothecium of both types. It is difficult to verify whether the tapetum behavior and pollen abortion is the cause or the effect.     

Microspore separation in the quartet 3 mutants of Arabidopsis is impaired by a defect in a developmentally regulated polygalacturonase required for pollen mother cell wall degradation

Mutations in the QUARTET loci in Arabidopsis result in failure of microspore separation during pollen development due to a defect in degradation of the pollen mother cell wall during late stages of pollen development. Mutations in a new locus required for microspore separation, QRT3, were isolated, and the corresponding gene was cloned by T-DNA tagging. QRT3 encodes a protein that is approximately 30% similar to an endopolygalacturonase from peach (Prunus persica). The QRT3 protein was expressed in yeast (Saccharomyces cerevisiae) and found to exhibit polygalacturonase activity. In situ hybridization experiments showed that QRT3 is specifically and transiently expressed in the tapetum during the phase when microspores separate from their meiotic siblings. Immunohistochemical localization of QRT3 indicated that the protein is secreted from tapetal cells during the early microspore stage. Thus, QRT3 plays a direct role in degrading the pollen mother cell wall during microspore development.     

Characterization of two subclasses of PR-10 transcripts in lily anthers and induction of their genes through separate signal transduction pathways

The lily PR-10 belongs to a family of intracellular pathogenesis-related (IPR) proteins. Genomic Southern analysis indicates that the PR-10 is encoded by a family of multiple genes. Seven heterogeneous cDNA clones encoding lily PR-10 from Lilium longiflorum are divided into two subclasses based on sequence comparison and Southern hybridization. A 82% overall sequence similarity was found between the two subclasses (represented by PR-10c and d). The two cDNAs include an open reading frame of 474 bp encoding 157 amino acids. 5'- and 3'-untranslated regions exhibit low similarity, but similarity is high in the coding region. The lily PR-10 genes are induced by abscisic acid (ABA) and methyl jasmonate (MeJA) in the anther and various other organs of lily plants. The induction of PR-10 genes by ABA and MeJA in lily anthers occurs by two separate signal transduction pathways. The protein phosphatase inhibitor okadaic acid inhibits the MeJA-induced expression of PR-10 genes downstream of MeJA. In addition, the protein kinase inhibitor staurosporine inhibits the MeJA-induced expression of PR-10 genes, implying that an activity of staurosporine-sensitive protein kinases exists downstream of MeJA in the anther. However, okadaic acid does not inhibit the ABA-induced expression of PR-10 genes whereas staurosporine does. These observations suggest that, in addition to the known pathway that ABA induces gene expression by activating JA or MeJA, a MeJA-independent pathway of ABA induction exists in the anther. The alternative pathway of ABA induction involves a staurosporine-sensitive protein kinase activity downstream of ABA.     

Analysis of bean (Phaseolus vulgaris L.) proteins affected by copper stress

The effect of excess copper on the expression of soluble proteins in 10-day old Phaseolus vulgaris seedlings was studied with two-dimensional electrophoresis and mass spectrometry, to find sensitive biochemical markers of exposure. Despite major differences in root Cu contents, both 15 and 50 microM Cu treatments resulted in equal enhancements of Cu in the primary leaves. Three proteins, apparently reacting in a dose-dependent manner to Cu exposure, were identified from roots. The levels of an intracellular pathogenesis-related protein and a newly identified protein homologous to PvPR1, PvPR2, were increased with increasing Cu concentration. The level of a newly identified PR-10 protein decreased in a dose-dependent manner. No significant difference was observed in the leaf protein pattern between controls and 15 microM Cu-treated plants. However, at 50 microM Cu exposure, the appearance of PvPR1 and a homologue of Arabidopsis thaliana thylakoid lumenal 17.4kDa protein was observed. Another protein slightly enhanced by Cu treatment had sequence homology to a mitochondrial precursor of glycine cleavage system H protein of Flaveria pringlei.     

Secretory tapetum of Brassica oleracea L.: polarity and ultrastructural features

Summary The ultrastructure of the secretory, binucleate tapetum of Brassica oleracea in the micro spore mother cell (MMC) stage through to the mature pollen stage is reported. The tapetal cells differentiate as highly specialized cells whose development is involved in lipid accumulation in their final stage. They start breaking down just before anther dehiscence. Nuclei with dispersed chromatin, large nucleoli and many ribosomes in the cytoplasm characterize the tapetal cells. The wall-bearing tapetum phase ends at the tetrade stage. The dissolution of tapetal walls begins from the inner tangential wall oriented towards the loculus and proceeds gradually along the radial walls to the outer tangential one. The plasmodesmata transversing the radial walls between tapetal cells persist until the mature microspore, long after loss of the inner tangential wall. After wall dissolution, the tapetal protoplasts retain their integrity and position within the anther locule. The tapetal cell membrane is in direct contact with the exine of the microspores/pollen grains and forms tubular evaginations that increase its surface area and appear to be involved in the translocation of solutes from the tapetal cells to the microspores/ pollen grains. The tapetal cells exhibit a polarity expressed by spatial differentiation in the radial direction.     

Immunological Characterization of a Tapetal Protein in Developing Anthers of Lilium longiflorum

Antiserum was raised in rabbits against a lily (Lilium longiflorum) anther-specific protein (LLA-15). Monospecific anti-LLA-15 antibodies were prepared to investigate the distribution of LLA-15 during anther development in a variety of flowering plants. Immunoblot analyses of total protein from floral and vegetative organs confirmed that LLA-15 or LLA-15-like proteins accumulated to detectable levels only in a discrete stage of anther development. In situ localization using anti-rabbit immunoglobulin G conjugated with gold particles confirmed that LLA-15 was specifically localized in the tapetal tissue of lily anthers. The maximal level of LLA-15 was strictly coincident with the peak of tapetal secretory functions. Immunoblots of two-dimensional polyacrylamide gels of lily anther proteins indicated that the seven LLA-15 isoforms ranged from isoelectric point 5.6 to 6.1. In vitro translation of lily anther mRNAs showed that four of these isoforms were primary products, the additional three being a result of posttranslational processing of the primary translation products.     

芝麻核雄性不育系ms86-1微粉发生的细胞学观察

芝麻(Sesamum indicum)核雄性不育系ms86-1姊妹交后代表现为可育、部分不育(即微粉)及完全不育(简称不育)3种类型。不同育性类型的花药及花粉粒形态差异明显。Alexander染色实验显示微粉植株花粉粒外壁为蓝绿色, 内部为不均一洋红色, 与可育株及不育株花粉粒的染色特征均不相同。为探明芝麻微粉发生机理, 在电子显微镜下比较观察了可育、微粉、不育类型的小孢子发育过程。结果表明, 可育株小孢子母细胞减数分裂时期代谢旺盛, 胞质中出现大量脂质小球; 四分体时期绒毡层细胞开始降解, 单核小孢子时期开始出现乌氏体, 成熟花粉时期花粉囊腔内及花粉粒周围分布着大量乌氏体, 花粉粒外壁有11–13个棱状凸起, 表面存在大量基粒棒, 形成紧密的覆盖层。不育株小孢子发育异常显现于减数分裂时期, 此时胞质中无脂质小球出现, 细胞壁开始积累胼胝质; 四分体时期绒毡层细胞未见降解; 单核小孢子时期无乌氏体出现; 成熟花粉时期花粉囊腔中未发现正常的乌氏体, 存在大量空瘪的败育小孢子, 外壁积累胼胝质, 缺乏基粒棒。微粉株小孢子在减数分裂时期可见胞质内有大量脂质小球, 四分体时期部分绒毡层发生变形, 单核小孢子时期有部分绒毡层开始降解; 绒毡层细胞降解滞后为少量发育进程迟缓的小孢子提供了营养物质, 部分小孢子发育为正常花粉粒; 这些花粉粒比较饱满, 表面有少量颗粒状突起, 但未能形成覆盖层, 花粉囊腔中及小孢子周围存在少量的乌氏体。小孢子形成的育性类型与绒毡层降解是否正常有关。     

Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth

Vacuolar sorting receptors (VSRs) are type‐I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type‐IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans‐Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP‐VSR/GFP‐LIVSR is located throughout the pollen tubes, excepting the apical clear‐zone region, whereas GFP‐LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.     

扁豆绒毡层发育的超微结构研究

应用透射电镜对扁豆绒毡层发育过程进行了研究,主要结果如下：１）首次发现扁豆绒毡层在发育过程中,经历了二交胞质重组（第一次始于减数分裂末期Ⅱ,第二次始于小孢子发育早期）,使绒毡层细胞的活动呈现３个高峰期（即小孢子母细胞减数分裂期、小孢子四分体期一小孢子早期、小孢子晚期－二胞花粉中期）。２绒毡层细胞的分泌作用有３种形式（渗透分泌、胞吐分泌和自溶）。３．首次观察到绒毡层细胞的内切向壁和径向壁经历了两个周     

Ultrastructure of microsporogenesis and microgametogenesis inArabidopsis thaliana (L.) Heynh. ecotype Wassilewskija (Brassicaceae)

Summary The process of microsporogenesis and microgametogenesis was studied at the ultrastructural level in wild-typeArabidopsis thaliana ecotype Wassilewskija to provide a basis for comparison with nuclear male-sterile mutants of the same ecotype. From the earliest stage studied to mature pollen just prior to anther dehiscence, microsporocyte/microspore/pollen development follows the general pattern seen in most angiosperms. The tapetum is of the secretory type with loss of the tapetal cell walls beginning at about the time of microsporocyte meiosis. Wall loss exhibits polarity with the tapetal protoplasts becoming located at a distance from the inner tangential walls first, followed by an increase in distance from the radial walls beginning at the interior edge and progressing outward. The inner tangential and radial tapetal walls are completely degenerated by the microspore tetrad stage. Unlike other members of the Brassicaceae that have been studied, the tapetal cells ofA. thaliana Wassilewskija also lose their outer tangential walls, and secretion occurs from all sides of the cells. Exine wall precursors are secreted from the tapetal cells in a process that appears to involve dilation of individual endoplasmic reticulum cisternae that fuse with the tapetal cell membrane and release their contents into the locule. Following completion of the exine, the tapetal cell plastids develop membranebound inclusions with osmiophilic and electron-transparent regions. The plastids undergo ultrastructural changes that suggest breakdown of the inclusion membranes followed by release of their contents into the locule prior to the complete degeneration of the tapetal cells.