玉米花粉形成过程不同阶段特异蛋白变化规律及酯酶同工酶研究

利用薄层胶电泳技术对玉米花粉形成过程不同阶段的酯酶同工酶及可溶性蛋白种类和含量变化进行了研究。结果表明在玉米花粉形成过程中的不同阶段酯酶同工酶的种类和活性均有不同。四分体时期出现两种小分子量酯酶同工酶到小孢子阶段即很快消失,早期小孢子阶段酯酶同工活性及种类达到高峰,以后则逐步递减。玉米花粉可溶性蛋白电泳显示玉米花粉形成过程中不同发育时期蛋白类和含量有很大变化。四分体时期存在有特异性蛋白（ＴＰ）,它     

苜蓿组织培养中球形胚发生时特异蛋白质和同工酶分析

试验在苜蓿组织培养中,对球形胚形成过程中特异蛋白质表达的模式、过氧化物酶及酯酶同工酶酶谱变化进行研究,结果表明：苜蓿组织培养中从胚性愈伤组织到球形胚发育的进程中,顺序消失和出现了11种中小分子量多肽;过氧化物酶同工酶酶谱发生了显著的变化;酯酶同工酶酶谱变化不大,但其总活力对于维持体细胞胚胎发生是必须的。     

小麦与玉米及鸭茅状摩擦禾远缘杂交的胚发育过程中同工酶和蛋白质的变化

通过比较小麦与玉米及鸭茅状摩擦禾属间杂交获得的胚与小麦正常自交的胚之间在不同发育时期过氧化物酶和酯酶的同工酶谱,发现过氧化物酶同工酶表现出时空顺序的特异性变化。在同一发育时期,远缘杂交的具胚子房和无胚子房之间存在过氧化物酶同工酶谱的差异,这可能涉及到与胚发育相关的同工酶的出现。远缘杂交的具胚子房和正常自交的小麦子房之间也有一定的酶谱差异。同时,同一材料还表现出不同发育时期的过氧化物酶酶谱差别。在远缘杂交后的胚发育期间,酯酶同工酶的时空表达不如过氧化物酶显著。此外,对远缘杂交后的胚中的水溶性蛋白质进行了SD S-PAGE分析,初步的分析结果表明,可能存在与胚发育相关的蛋白质。     

两种光合细菌生物转化槲寄生培养液菌体中几种同工酶的变化

采用聚丙烯酰胺凝胶电泳法对两种光合细菌的生物转化槲寄生培养液中菌体的蛋白质和几种同工酶进行研究,并以纯光合细菌培养液中菌体作对照。结果表明,光合细菌生物转化槲寄生过程中,两种光合细菌的蛋白质、酯酶同工酶和过氧化物酶同工酶均发生改变,某些蛋白质、酯酶和过氧化物酶的合成受到抑制,并有新的蛋白质、酯酶和过氧化物酶生成;超氧化物歧化酶的表达未明显改变。由此可见,槲寄生能诱导光合细菌合成新的酯酶和过氧化物酶,这些诱导酶可能参与了槲寄生的生物转化。为光合细菌生物转化槲寄生转化机理的研究及槲寄生在抗肿瘤领域的进一步应用奠定了基础。     

马唐体细胞胚胎发生过程中生理变化的研究

本文通过对马唐体细胞胚胎发生过程中生理变化的研究后发现,球形胚游离氨基酸种类最少、浓度最低;其过氧化物酶、酯酶和淀粉酶同工酶活性较高、种类较多;其可溶性蛋白质相对浓度最高,并且出现至少两种新带。这说明球形胚已开始分子水平的分化;在这一时期合成的蛋白质(酶),对于马唐体细胞胚胎发生中胚体细胞水平的分化有着重要作用。     

玉米酯酶同工酶和过氧化物酶同工酶的分子量研究

本试验利用聚丙烯酰胺凝胶梯度电泳分步染色法直接对玉米苗期酯酶同工酶和过氧化物酶同工酶各酶带的分子量进行了比较测定。酯酶同工酶 E_1、E_2、E_3~F、E_3~S、a、b、c 各酶带的分子量分别为<20000,35200、33000、38500、29900、28500、34000道尔顿过氧化物酶同工酶 PX_4~F和 PX_4~S酶带的分子量分别为131000和149000道尔顿。根据酶带在均匀胶和梯度胶中的位置变化对各酶带的生化性质作了初步分析,发现 E_3~F和 E_3~S、PX_4~F 和 PX_4~S 在迁移率上的差异主要是分子量的差异。本文为同工酶的分子量测定提供了一个简便的方法。     

中华绒螯蟹不同发育时期胚胎及流产胚胎的同工酶变化

采用聚丙烯酰胺凝胶电泳技术,对中华绒螯蟹(Erlocheir sinensis)不同发育时期胚胎及流产胚胎的6种同工酶(乳酸脱氢酶、醇脱氢酶、苹果酸脱氢酶、酯酶、淀粉酶和过氧化物酶)进行了研究。结果表明,不同发育时期胚胎的乳酸脱氢酶、醇脱氢酶、苹果酸脱氢酶、酯酶及淀粉酶酶谱表现出一定差异。受精卵中未检测到乳酸脱氢酶同工酶活性,卵裂期和囊胚期出现4条酶带,无节幼体及溞状幼体期只有2条酶带;醇脱氢酶同工酶在中华绒螯蟹胚胎发育的各阶段均有表达,但表达的酶带数和活性有区别;在受精卵和溞状幼体期无苹果酸脱氢酶酶带显示,卵裂期酶带数最多,酶活性相对也最强,以后随着发育的进行,酶带数和酶活性都有减弱的现象;酯酶同工酶的变化较为复杂,特别是囊胚期,酯酶酶带突然全部消失;淀粉酶有两种类型：α-淀粉酶和R-淀粉酶。从受精卵发育到幼体其酶带数不增反减。不同发育阶段均检测不到过氧化物酶的活性。第二次抱卵胚胎的酶活性和酶带数低于或有别于第一次抱卵的卵裂期胚胎。流产胚胎中醇脱氢酶、乳酸脱氢酶、淀粉酶与第一次抱卵胚胎不同发育时期的酶谱相比略有变化,而苹果酸脱氢酶、酯酶酶带数迅速减少。     

红花的愈伤组织诱导及其与过氧化物酶和酯酶同工酶的关系

红花外植体培养于附加不同激素的 MS培养基中,根、胚轴、子叶均可大量形成愈伤组织和根。5个红花品种间过氧化物酶和酯酶同工酶酶谱的差异小于不同器官间的差异。随外植体子叶脱分化过程的进行,过氧化物酶同工酶酶带逐渐增多,而酯酶同工酶酶带则逐渐减少,并出现特异的诱导酶带。     

高粱的杂种优势与同工酶

Schwarcz 在玉米胚乳中发现有酯酶同工酶的“杂种酶带”;chiang Pai 等在栽培稻和野生稻的杂种第一代中,也发现有过氧化物酶的“杂种酶带”;Beckman 在授粉后16天的玉米胚乳中发现有亮氨酰肽酶同工酶的“互补酶带”;Gupta 在水稻胚乳中发现酯酶同工酶与粒重优势有关。李继耕发现玉米胚乳中有与杂种优势有关的四种过氧化物酶酶谱类型。本试验的目的是研究高梁杂种同工酶酶谱型与产量优势的关系。     

沙田柚花粉发育过程中同工酶的变化研究

分析了沙田柚花粉在发育过程中３ 种酶的同工酶的活力及类型的变化。结果表明, 过氧化物酶活力在单核期达到最大, 在四分体期与单核期所具有的一条染色较深的快速酶带在双核期消失。α－ 淀粉酶活力在成熟期达到最大, 谱带上也表现出在成熟期有酶带的增多。分析酯酶酶谱, 可见其在各个发育时期均有明显的特征带, 这些特征带可作为花粉发育各时期的生化指标。     

Distinct chromatin environment associated with phosphorylated H3S10 histone during pollen mitosis I in orchids

Pollen developmental pathway in plants involving synchronized transferal of cellular divisions from meiosis (microsporogenesis) to mitosis (pollen mitosis I/II) eventually offers a unique “meiosis-mitosis shift” at pollen mitosis I. Since the cell type (haploid microspore) and fate of pollen mitosis I differ from typical mitosis (in meristem cells), it is immensely important to analyze the chromosomal distribution of phosphorylated H3S10 histone during atypical pollen mitosis I to comprehend the role of histone phosphorylation in pollen development. We investigated the chromosomal phosphorylation of H3S10 histone during pollen mitosis I in orchids using immunostaining technique. The chromosomal distribution of H3S10ph during pollen mitosis I revealed differential pattern than that of typical mitosis in plants, however, eventually following the similar trends of mitosis in animals where H3S10 phosphorylation begins in the pericentromeric regions first, later extending to the whole chromosomes, and finally declining at anaphase/early cytokinesis (differentiation of vegetative and generative cells). The study suggests that the chromosomal distribution of H3S10ph during cell division is not universal and can be altered between different cell types encoded for diverse cellular processes. During pollen development, phosphorylation of histone might play a critical role in chromosome condensation events throughout pollen mitosis I in plants.     

Isolation of viable wheat male gametophytes of different stages of development and variations in their protein patterns

Procedures have been designed to isolate viable immature male gametophytes from wheat (Triticum aestivum L.) anthers of different stages of development. Maceration of anthers by a micro-blender allowed for the release of numerous intact vacuolate microspores. Tris-buffered media prevented tricellular pollen grains from bursting during the isolation procedure. Proteins from the undamaged male gametophytes have been analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. A set of new proteins was detected at the onset of the second pollen grain mitosis. The results demonstrate the opportunity for studies on haploid gene expression at the translational level.     

Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development

Reversibly glycosylated polypeptides (RGPs) have been implicated in polysaccharide biosynthesis. To date, to our knowledge, no direct evidence exists for the involvement of RGPs in a particular biochemical process. The Arabidopsis (Arabidopsis thaliana) genome contains five RGP genes out of which RGP1 and RGP2 share the highest sequence identity. We characterized the native expression pattern of Arabidopsis RGP1 and RGP2 and used reverse genetics to investigate their respective functions. Although both genes are ubiquitously expressed, the highest levels are observed in actively growing tissues and in mature pollen, in particular. RGPs showed cytoplasmic and transient association with Golgi. In addition, both proteins colocalized in the same compartments and coimmunoprecipitated from plant cell extracts. Single-gene disruptions did not show any obvious morphological defects under greenhouse conditions, whereas the double-insertion mutant could not be recovered. We present evidence that the double mutant is lethal and demonstrate the critical role of RGPs, particularly in pollen development. Detailed analysis demonstrated that mutant pollen development is associated with abnormally enlarged vacuoles and a poorly defined inner cell wall layer, which consequently results in disintegration of the pollen structure during pollen mitosis I. Taken together, our results indicate that RGP1 and RGP2 are required during microspore development and pollen mitosis, either affecting cell division and/or vacuolar integrity.     

Ultrastructural studies on plastids of generative and vegetative cells inLiliaceae 3. Plastid distribution during the pollen development inGasteria verrucosa (Mill.) Duval

Summary This paper describes the development of pollen grains ofGasteria verrucosa from the late microspore to the mature two-cellular pollen grain. Ultrastructural changes and the distribution of plastids as a result of the first pollen mitosis have been investigated using light and electron microscopy. The microspores as well as the generative and the vegetative cell contain mitochondria and other cytoplasmic organelles during all of the observed developmental stages. In contrast, the generative cell and the vegetative cell show a different plastid content. Plastids are randomly distributed within the microspores before pollen mitosis. During the prophase of the first pollen mitosis the plastids become clustered at the proximal pole of the microspore. The dividing nucleus of the microspore is located at the distal pole of the microspore. Therefore, the plastids are not equally distributed into both the generative and the vegetative cell. The possible reasons for the polarization of plastids within the microspore are briefly discussed. The lack of plastids in the generative cell causes a maternal inheritance of plastids inGasteria verrucosa.     

The coexistence of bicellular and tricellular pollen in Annona cherimola (Annonaceae): Implications for pollen evolution

Most angiosperms release bicellular pollen. However, in about one-third of extant angiosperms, the second pollen mitosis occurs before anthesis such that pollen is tricellular upon release. The shift from bicellular to tricellular development has occurred several times independently, but its causes are largely unknown. In this work, we observed the coexistence of both kinds of pollen at anther dehiscence in Annona cherimola, a species that belongs to the basal angiosperm family Annonaceae. Examination of pollen cell number during anther development showed that this coexistence was due to a late mitosis starting shortly before pollen shedding. Both types of pollen germinated equally well over the course of development. Because variable proportions of bicellular and tricellular pollen were observed at different sampling times, we tested the role of temperature by performing field and growth chamber experiments, which showed that higher temperatures near anthesis advanced the time of pollen mitosis II. The results show that selection could favor the production of tricellular pollen under certain environmental circumstances that prime rapid pollen germination and provide evidence of a system in which developmental variation persists, but that can be modified by external factors such as temperature.     

Pollen performance, cell number, and physiological state in the early-divergent angiosperm Annona cherimola Mill. (Annonaceae) are related to environmental conditions during the final stages of pollen development

Pollen performance is an important determinant for fertilization success, but high variability in pollen behavior both between and within species occurs in different years and under varying environmental conditions. Annona cherimola, an early-divergent angiosperm, is a species that releases a variable ratio of bicellular and tricellular hydrated pollen at anther dehiscence depending on temperature. The presence of both bi- and tricellular types of pollen is an uncommon characteristic in angiosperms and makes Annona cherimola an interesting model to study the effect of varying environmental conditions on subsequent pollen performance during the final stages of pollen development. In this work, we study the influence of changes in temperature and humidity during the final stages of pollen development on subsequent pollen performance, evaluating pollen germination, presence of carbohydrates, number of nuclei, and water content. At 25?°C, which is the average field temperature during the flowering period of this species, pollen had a viability of 60-70?%, starch hydrolyzed just prior to shedding, and pollen mitosis II was taking place, resulting in a mixture of bi- and tricellular pollen. This activity may be related to the pollen retaining 70?% water content at shedding. Temperatures above 30?°C resulted in a decrease in pollen germination, whereas lower temperatures did not have a clear influence on pollen germination, although they did have a clear effect on starch hydrolysis. On the other hand, slightly higher dehydration accelerated mitosis II, whereas strong dehydration arrested starch hydrolysis and reduced pollen germination. These results show a significant influence of environmental conditions on myriad pollen characteristics during the final stages of pollen development modifying subsequent pollen behavior and contributing to our understanding of the variability observed in pollen tube performance.     

Unique actin and microtubule arrays co-ordinate the differentiation of microspores to mature pollen in Nicotiana tabacum

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development. Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle. Actin is localized within the region of the mitotic spindle and in the phragmoplast. Following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.     

LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.