中国特有属牛筋条属的花粉形态与其系统位置

从发育的角度研究了中国特有单种属DichotomanthesKurz及与其系统学研究有关的外类群Prinsepiautilis的花粉形态 ,扫描电镜观察显示Dichotomanthes花粉粒自脱离四分体胼胝质膜开始至成熟二核花粉粒不同发育时期 ,花粉形态和外壁纹饰未见变化 ,仅花粉体积随成熟度增加而有所增大。而Prinsepiautilis ,其花粉粒刚脱离四分体时形状和成熟花粉明显不同 ,成熟花粉极面观为三裂圆形 ,赤道面观为圆形 ,外壁具清晰的平行条纹 ,但幼嫩花粉粒的形状很特别 ,极面观为深三裂圆形 ,赤道面观亦见花粉在两条沟之间下陷而沟部外突 ,明显为角萌发孔花粉 ,且花粉体积较成熟者小 ,而外壁纹饰同成熟者相比无根本性差异。前述两种植物花粉在不同成熟期体积有明显差异 ,而外壁纹饰在不同成熟期不存在质的变化并相对稳定 ,说明花粉外壁纹饰这一性状在蔷薇科中具有较为重要的分类学意义。DichotomanthesKurz具典型Rosaceae花粉的三孔沟结构 ,外壁具条纹 -穴状纹饰。将其孢粉学特征同Rosaceae 4个亚科有关类群的同类资料相比较 ,并结合其它形态解剖与细胞学等研究结果 ,支持将Dichotoman thes置入Maloideae下而不赞同将其另立亚科或置于Prunoideae之下。此外 ,由于Prinsepiautilis的花粉在其发育初期具角萌发孔花粉 ,与Cunoniacea     

Cytochemistry of pollen development in Brachypodium distachyon

Brachypodium distachyon is a widely recognized model plant belonging to subfamily Pooideae with a sequenced genome. To gain a better understanding of the male reproductive development in B. distachyon we examined pollen morphology and cytochemical changes of microspore cytoplasm from pollen mother cell stage to mature pollen using light, fluorescent and scanning electron microscopy. Our results show that B. distachyon exhibits a typical monocot-type pollen ontogeny. Meiosis in the pollen mother cells is accomplished by successive cytokinesis generating isobilateral tetrads. Cytochemical examination indicated that microspore cytoplasm contains variable amounts of insoluble carbohydrates and proteins at different developmental stages. Deposition of starch in the cytoplasm of microspores starts at the bicellular stage and continues till the mature pollen stage. The formation of the exine wall progresses by the deposition of sporopollenin from the tapetum layer of the anther. The mature pollen is trinucleate, spheroidal in shape and possesses a single pore with an annulus and operculum. The exine pattern is smooth and of granular type.     

Monitoring by epifluorescence microscopy of organelle DNA fate during pollen development in five angiosperm species

The fates of mitochondrial and plastid nucleoids during pollen development in six angiosperm species (Antirrhinum majus, Glycine max, Medicago sativa, Nicotiana tabacum, Pisum sativum, and Trifolium pratense) were examined using epifluorescence microscopy after double staining with 4',6-diamidino-2- phenylindole (DAPI) to stain DNA and with a potentiometric dye (either DiOC7 or rhodamine 123) for visualization of metabolically active mitochondria. From the pollen mother cell stage to the microspore stage of pollen development, mitochondria and plastids both contained DNA detectable by DAPI staining. However, during the further maturation preceding anthesis, mitochondrial DNA became undetectable cytologically in either the generative or the vegetative cell of mature pollen; even in germinated pollen tubes containing hundreds of metabolically active mitochondria undergoing cytoplasmic streaming, vital staining with DAPI failed to reveal mitochondrial DNA. By the mature pollen stage, plastid DNA also became undetectable by DAPI staining in the vegetative cell. However, in the generative cell of mature pollen the timing of plastid DNA disappearance as detected by DAPI varied with the species. Plastid DNA remained detectable only in the generative cells of pollen grains from species known or suspected to have biparental transmission of plastids. The apparent absence of cytologically detectable organelle genomes in living pollen was further examined using molecular methods by hybridizing organelle DNA-specific probes to digests of total DNA from mature pollen and from other organs of A. majus and N. tabacum, both known to be maternal for organelle inheritance. Mitochondrial DNA was detected in pollen of both species; thus the cytological alteration of mitochondrial genomes during pollen development does not correspond with total mtDNA loss from the pollen. Plastid DNA was detectable with molecular probes in N. tabacum pollen but not in A. majus pollen. Since the organelle DNA detected by molecular methods in mature pollen may lie solely in the vegetative cell, further study of the basis of maternal inheritance of mitochondria and plastids will require molecular methods which distinguish vegetative cell from reproductive cell organelle genomes. The biological effect of the striking morphological alteration of organelle genomes during later stages of pollen development, which leaves them detectable by molecular methods but not by DAPI staining, is as yet unknown.     

A structural study on the mode of action of CHATM Chemical Hybridizing Agent in wheat

Summary This study has compared mature pollen grains while still in the anther, as well as post-pollination responses, from untreated and CHA^TM Chemical Hybridizing Agent-treated wheat plants using light (bright-field, phase-contrast, and fluorescence), scanning, and transmission electron microscopy. The chemical, azetidine-3-carboxylic acid (A3C), was applied at three treatment levels of 0.75, 1.0 and 1.5 kg/ha for the mature pollen investigations. It was found that the major effect of A3C on mature pollen is an alteration of the wall precursor vesicles (wp-vesicles), which form a high proportion of the contents of the mature grass pollen grain. The degree of deformation of the wp-vesicles is dose dependent. There is some evidence that increased aggregations of ribosomes are formed in treated pollen cytoplasm. Pollination studies (all at a treatment level of 1.0 kg/ha) show that, in most cases, the treated pollen does not germinate, and a high percentage of the pollen grains burst (60% burst grains in treated material compared to 28% in controls). In about 20% of the cases from treated plants, a short pollen tube forms, but no tubes were seen to grow far enough to enter the stigma hairs of the pistil. Thus, A3C does not act by preventing pollen formation, but by the prevention of normal pollen tube growth. There appears to be a specific targeting of the wp-vesicles such that, even in cases where the ultra-structure of the vesicles is not altered, the normal course of events leading to the incorporation of their contents into the extending tube wall is arrested. Further studies must be undertaken to determine the significance of the effect of CHA^TM Chemical Hybridizing Agent on wp-vesicle composition.     

Developmental accumulation of hydroxyproline and hydroxyproline-containing proteins in Zea mays pollen

The hydroxyproline content of developing Zea mays (maize) pollen was determined. The level of hydroxyproline in uninucleate microspores early in pollen development was low (0.004% of dry weight). In contrast, mature pollen is enriched for this amino acid (0.1% of dry weight). In mature pollen, 90% of the hydroxyproline is in the soluble fraction. Upon in vitro pollen germination, hydroxyproline associated with the insoluble fraction increased from 10% to 26% of the total hydroxyproline. Antibodies specific to extensins and arabinogalactan proteins (AGPs), two major classes of hydroxyproline-containing proteins, recognized two distinct groups of proteins in maize pollen by Western analysis. The two types of pollen hydroxyproline-containing proteins could also be distinguished based on their behavior upon anion exchange chromatography.     

Elimination of hop latent viroid upon developmental activation of pollen nucleases

Hop latent viroid (HLVd) is not transmissible through hop generative tissues and seeds. Here we describe the process of HLVd elimination during development of hop pollen. HLVd propagates in uninucleate hop pollen, but is eliminated at stages following first pollen mitosis during pollen vacuolization and maturation. Only traces of HLVd were detected by RT-PCR in mature pollen after anthesis and no viroid was detectable in in vitro germinating pollen, suggesting complete degradation of circular and linear HLVd forms. The majority of the degraded HLVd RNA in immature pollen included discrete products in the range of 230-100 nucleotides and therefore did not correspond to siRNAs. HLVd eradication from pollen correlated with developmental expression of a pollen nuclease and specific RNAses. Activity of the pollen nuclease HBN1 was maximal during the vacuolization step and decreased in mature pollen. Total RNAse activity increased continuously up to the final steps of pollen maturation. HBN1 mRNA, which is abundant at the uninucleate microspore stage, encodes a protein of 300 amino acids (34.1 kDa, isoeletric point 5.1). Sequence comparisons revealed that HBN1 is a homolog of S1-like bifunctional plant endonucleases. The developmentally activated HBN1 and pollen ribonucleases could participate in the mechanism of HLVd recognition and degradation.     

Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen

Proteins, acyl-lipids and polysaccharides from cell walls of mature and germinated olive pollen were studied. In general, hemicelluloses are the most abundant polysaccharides, arabinose in mature and glucose in germinated pollen being the main components of these macromolecules. Protein content and its amino acid composition are very similar in walls from mature and germinated pollen, these compounds showing a weak acid character. Free-fatty acids are the most abundant lipid molecules in mature and germinated pollen walls and a decrease in acyl-lipids, especially in polar lipids, as well as a higher unsaturation of their fatty acid components are observed after germination.     

The reversible inhibition of pollen germination of Nicotiana tabacum L.: an entry into a transformation system

Summary The hydrodynamics of mature pollen rehydration in Nicotiana tabacum was used to study reversible inhibition of pollen germination in vitro. Tobacco pollen was incubated for various times in media containing calcium, potassium and magnesium salts, boric acid, and exhibiting different osmotic pressures as a function of sucrose concentration. Total inhibition of germination with complete viability preservation was achieved for 56 h by keeping the grains in medium with 80% sucrose, since typical percentages of germination and pollen tube lengths were recovered after this treatment. These effects were considered as consequences of natural osmoregulation of rehydration/germination in mature pollen. The possibility of applying these findings to the incubation of pollen with Agrobacterium tumefaciens to develop a pollen transformation method is discussed.     

A simple method for staining nuclei of mature and germinated maize pollen

A sugar acetocarmine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocarmine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.     

Confocal Microscopic Observations on Actin Filament Distribution in Lily Pollen Protoplasts

Actin filaments (F-actin) were localized in the isolated pollen protoplasts of lily using TRITC-phalloidin probe and confocal microscopy. Two kinds of pollen protoplasts were examined: one from pollen grains of non-dehiscent anthers(referred to as ‘nearly mature’ pollen); and the other from pollen grains of just dehiscent anthers(referred to as ‘just mature’ pollen). In the cytoplasm of the pollen protoplasts of the ‘nearly mature’ pollen there was a very well organized actin network made up of thick actin bundles. Two types of bundle connections were seen in the network; namely ‘branch’ connections and 'junction' connections. The ‘branch’ connection (or branching points) was formed due to branching or merging of bundies. The ‘junction’ connection (or 'junction' point) had two or more bundles associated with it. Some of the ‘junction’ points might be actin filament organization: centres. The generative cell in iht pollen protoplasts of the ‘nearly mature’ pollen also contained an actin network. But this network was structurally quite loose and the pundles made up the network were short and thick. In the cytoplasm of the pollen protoplasts of the ‘just mature’ pollen the actin net work was more densely packed. The bundles made up the network were also thinner. The actin network in the generative cell was, however, less densely packed. If the pollen protoplasts from both the ‘nearly mature’ and the 'just mature' pollen grains were transferred from a B5 medium into a Brewbaker and Kwack medium supplemented with sucrose, protoplasts rapidly (i.e. within 2 to 3 hours) developed vacuoles and transvacuolar strand. In these va cuolated protoplasts the vegetative nucleus andthe generative cell became tightly surrounded by a new actin network. In the transvacuolar strands there were numerous actin bundles. The “ends” of some of these bundles appeared to be tightly attached to the protoplast membrane indicating that some kind of structures might be present in the protoplast membrane for actin filament attachment.     

Detection and release of allergenic proteins in Parietaria judaica pollen grains

Summary. Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains. Correspondence and reprints: Department of Plant Biology, Faculty of Biology, University of León, Campus de Vegazana, 24071 León, Spain.     

Cytopathology of Anthers and Pollen From Lettuce Plants Infected by Lettuce Mosaic Virus

Thin sections of mature anthers and pollen grains from three lettuce (Lactuca sativa) plants infected with lettuce mosaic potyvirus (LMV) were studied by immunogold labelling. Labelled LMV particles were present externally on the exine of pollen grains from all plants, but were observed internally in the pollen grains from only one plant. Within mature pollen grains the virus particles were associated with the cytoplasmic bundle inclusions typical of infection by potyviruses. The tapetal plasmodium and the epidermal and endothecial layers of mature anthers from all infected plants also contained labelled virus particles, together with pinwheel and bundle inclusions.     

ULTRASTRUCTURE OF NICOTIANA ALATA POLLEN,ITS GERMINATION AND EARLY TUBE FORMATION

Both internal anatomy and external morphology of the mature pollen grain of Nicotiana alata have been studied, together with pollen activation and tube emergence and its cytological zonation. A peculiar feature of mature pollen is the occurrence in the vegetative cell cytoplasm of a large quantity of stacked cisterns of rough endoplasmic reticulum, which then dissociate during pollen activation. Pollen tube emergence generally occurs 2 hr 30 min after hydration, while cytological zonation is complete 4 hr 30 min after hydration when the first callosic plug is formed.     

Comparison of different techniques for gene transfer into mature and immature tobacco pollen

The particle gun, cocultivation withAgrobacterium tumefaciens, and imbibition in DNA solutions were compared as methods to transfer DNA into mature and immature pollen ofNicotiana tabacum. Bombardment of mature pollen with the β-glucuronidase gene cloned behind the pollen-specific PA2 promoter of the chalcone isomerase gene ofPetunia hybrida resulted in the expression of the β-glucuronidase gene in 0.025% of the pollen grains. Bombardment of younger stages followed byin vitro maturation also resulted in the formation of mature pollen that expressed β-glucuronidase, although at a lower frequency. Cocultivation of pollen duringin vitro maturation orin vitro germination withAgrobacterium tumefaciens did not yeild β-glucuronidase-expressing pollen. In these cases, an intron-containing β-glucuronidase gene was used which effectively prevented β-glucuronidase expression in the bacteria. Imbibition of mature, dry pollen in various DNA solutions of the same constructs also did not lead to the formation of β-glucuronidase expressing pollen.     

A Simple Method for Staining Nuclei of Mature and Germinated Maize Pollen

A sugar acetocannine staining technique has been developed for staining the sperm and vegetative nucleus of mature and germinated maize pollen grains. This procedure is simple, stable and highly repeatable. The physiological properties of the mature maize pollen grains are first adjusted by using an in vitro germinating culture solution. This solution is 15% sucrose and contains 360 ppm calcium chloride dihydrate, and 120 ppm boric acid. One part fresh pollen grains is uniformly mixed with nine parts of the solution and left at room temperature for at least 5 hr. One part of this solution is then mixed with two parts of regular acetocannine stain and left overnight. The color of this mixture is pinkish red or raspberry. The sugar in the mixture helps to increase color contrast between the pollen cytoplasm (light pink) and the nuclei (reddish purple), decreases the frequency of burst pollen, increases pollen expansion, stabilizes pollen figures and automatically seals the coverglass.     

胡萝卜花药发育的组织化学研究

胡萝卜四分体时期的花药在药室内壁和绒毡层细胞中积累淀粉粒,随着花药的发育,花粉先出现大液泡,同时药室内壁和绒毡层细胞中淀粉粒消失;以后花粉中的大液泡消失,在花粉细胞质中出现淀粉粒。伴随着花粉的发育,绒毡层细胞退化,在细胞中积累较多的脂类物质,同时花粉中脂类物质含量也明显增加。胡萝卜成熟花粉粒的储存物主要为脂滴,也有少部分淀粉颗粒。胡萝卜花药在特定时间和特定部位积累营养储存物的过程也是其发育的一个特征。