GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 （gnl2-1） and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

Annexin5 Is Essential for Pollen Development in Arabidopsis

Pollen development is a post-meiotic process that produces mature pollen grains from microspores and can be regarded as an ideal model for the study of important plant physiological processes such as reproduction, cellular differentiation, cell fate determination, signal transduction, membrane transport, and fusion and polar growth. The regulation of pollen development is a complicated biological process that is crucial for sexual reproduction in flowering plants （Yamamoto et al.,     

芫花的花粉形态观察及生活力和萌发率分析

Pollen morphology of Daphne genkwa Sieb.et Zucc.was observed by scanning electron microscope(SEM),and its viability and germination rate were measured by TTC and sucrose in vitro culture methods.The results show that pollen is stenopalynous type with a diameter of 15.6-21.6 μm.Per pollen has 10-16 apertures which is irregular circular with a diameter of 1.4-2.0 μm.Surface ornamentation of pollen is rough reticulate pattern which is circular polygon(tetragon-heptagon,mostly pentagon-hexagon).Pollen viability is 51% by TTC.Sucrose of different concentrations has a significant effect on pollen germination rate during pollen culture.And pollen germination rate is the highest with a percentage of 27.0% in medium containing 50 g·L-1 sucrose,while pollen could not germinate in medium containing sucrose over 250 g·L-1.Otherwise,there is the phenomenon of "multi-aperture germination" during pollen germinating.     

Arabidopsis shaker pollen inward K~+ channel SPIK functions in SnRK1 complex-regulated pollen hydration on the stigma

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plantspecific subunit of the SNF1-related protein kinase 1 complex(SnRK1 complex). In pollen of the Arabidopsis kinbg mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K~+ channel SPIK was down-regulated in the kinβγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro.Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma,indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression,which functions in determining pollen hydration on the stigma.     

An Examination of the Function of Male Flowers in an Andromonoecious Shrub Capparis spinosa

The pollen donor and pollinator attractor hypotheses are explanations for the functions of the male flowers of andromonoecious plants. We tested these two hypotheses in the andromonoecious shrub Capparis spinosa L. （Capparaceae） and confirmed that pollen production and cumulative volume and sugar concentration of nectar do not differ between male and perfect flowers. However, male flowers produced larger anthers, larger pollen grains and smaller ovaries than perfect flowers. Observations on pollinators indicated that two major pollinators （Xylocopa valga Gerst and Proxylocopa sinensis Wu） did not discriminate between flower morphs and that they transferred pollen grains a similar distance. However, there were more seeds per fruit following hand pollination with pollen from male flowers than from perfect flowers. Individuals of C. spinosa with a larger floral display （i.e. bearing more flowers） received more pollen grains on the stigma of perfect flowers. Female reproductive success probably is not limited by pollen. These results indicate that male flowers of C. spinosa save resources for female function and that they primarily serve to attract pollinators as pollen donors.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Organelle trafficking,the cytoskeleton,and pollen tube growth

The pollen tube is fundamental for the reproduction of seed plants. Characteristically, it grows relatively quickly and uni‐directionally("polarized growth") to extend the male gametophyte to reach the female gametophyte. The pollen tube forms a channel through which the sperm cells move so that they can reach their targets in the ovule. To grow quickly and directionally, the pollen tube requires an intense movement of organelles and vesicles that allows the cell's contents to be distributed to sustain the growth rate. While the various organelles distribute more or less uniformly within the pollen tube, Golgi‐released secretory vesicles accumulate massively at the pollen tube apex, that is, the growing region. This intense movement of organelles and vesicles is dependent on the dynamics of the cytoskeleton,which reorganizes differentially in response to external signals and coordinates membrane trafficking with the growth rate of pollen tubes.     

Pollen and Resource Limitation in Veratrum nigrum L. （Liliaceae）, an Andromonoecious Herb

Pollen limitation and resource limitation were invoked to account for the pattern that flowering plants produce more flowers and ovules than fruits and seeds. This study aimed to determine their relative importance in Veratrum nigrum, a self-compatible, perennial, andromonoecious herb. In order to determine whether female production was limited by pollen grains on stigmas or by available resources, we performed supplemental hand pollination in three populations, male-flower-bud removal in three other populations, and emasculation of hermaphroditic flowers in still another population, resulting in a total of seven populations experimentally manipulated. Across the three populations, supplemental hand pollination did not significantly increase fruit set, seed number per fruit, and total seed production per individual, nor did emasculation of hermaphroditic flowers. Taken together, our results suggest that pollen grains deposited on stigmas were abundant enough to fertilize all the ovules. Male-flower-bud removal significantly increased the mean size of hermaphroditic flowers in all three populations. Female reproductive success was increased in one population, but not in the other two populations possibly due to heavy flower/seed predation. We concluded that the female reproductive success of V. nigrum was not limited by pollen grains but by available resources, which is consistent with Bateman＇s principle. Furthermore, the female reproduction increase of male-flower-bud removal individuals might suggest a trade-off between male and female sexual functions.     

四倍体和二倍体盾叶薯蓣的花粉活力及柱头可受性分析

盾叶薯蓣（Dioscorea zingiberens C．H．Wright）叶片呈盾形,叶面有不规则的黄白色斑块;蒴果干燥后呈蓝黑色,表面常附有白色粉状物;染色体数20;花粉两端略尖,外壁具有明显条纹。盾叶薯蓣根茎横生,俗称黄姜,可用于治疗皮肤感染、软组织损伤、蜂蜇虫咬及各种外科炎症。盾叶薯蓣根茎所含的薯蓣皂苷元（皂素,diosgenin）是合成甾体激素药物的主要原料,除具有抗炎、避孕等作用外,还可月季于合成镇痛药、杀虫剂及治疗冠心病的药物。     

七叶树花粉活力和柱头可授性变化的研究

采用花粉离体萌发法研究不同蔗糖浓度、硼酸浓度和不同温度对七叶树花粉萌发的影响及花粉活力测定,用联苯胺-过氧化氢法测定柱头可授性.结果表明:七叶树花粉萌发的最佳培养基是12％蔗糖+30 mg/L硼酸,花粉萌发的最适温度为25℃.雄花在开花当天花粉活力最高达75.69％,并在开花当天的上午10:00时,花粉活力最强,10:00～16:00花粉活力保持较高活力,是最佳授粉时段.两性花的柱头可授期持续时间较长,为8～9 d,开花3d达到最强,开花1～4 d柱头可授性保持较高水平,为授粉的最佳时间段.因此,从七叶树的花部特征、花粉活力与柱头可授性及花粉萌发的条件看,在长期的自然选择下七叶树在花部结构和开花生理上都是相配合的,以保障生殖成功.     

外来入侵植物意大利苍耳的传粉生态学特性

从开花物候、花粉活力、柱头可受性、传粉媒介、花粉散布距离、雄性、雌性功能以及繁育系统等方面系统地研究了意大利苍耳的传粉生态学特性,旨在为揭示该物种成功入侵的机理提供科学依据。研究结果显示:意大利苍耳种群的花期较长,雌花花期可达40d,雄花花期可达30d。其雄性和雌性功能都很强,意大利苍耳单株雄花序和花粉量分别高达3847个/株和37903037粒/株,单株雌花序和单株胚珠数高达3847个/株和7694枚/株,种群平均结实率高达80.59%,即平均每株个体能形成6200枚成熟的果实。花粉活力的日变化呈单峰型曲线,早晨和傍晚的花粉活力最低,14:00时的活力高达99%。雌蕊柱头一经伸出总苞即具有可受性,可受期长达7d,开花后的3—4d柱头的可受性最强。意大利苍耳为风媒传粉植物,在4.5km/h的微风条件下,其花粉的散布距离可达45m,大量的花粉集中分布在距花粉源0—20m的范围内,这对于往往高密度连片分布的意大利苍耳种群来说无疑是一种高效的传粉策略。意大利苍耳的交配系统灵活多样,盛花期自然结实率达到100%,套袋试验结果表明该植物自交亲和,自株自然授粉的结实率高达93%。表明较长的花期、大量的雌雄花序及花粉数量、较高的花粉活力、较长的柱头可授期、较远的花粉风媒散布距离、混合交配系统、以及较高的结实率是意大利苍耳繁殖成功的重要保障,也是其成功入侵的重要原因。     

Changes in Florets’ Vertical Direction within Inflorescence Affects Pollinator Behavior,and Fitness in <i>Trifolium repens</i>

Ecological interactions between flowers and pollinators greatly affect the reproductive success. To facilitate these interactions, many flowers are known to display their attractive qualities, such as scent emission, flower rewards and floral vertical direction, in a rhythmic fashion. However, less is known about how plants regulate the relationship between these flower traits to adapt to pollinator visiting behavior and increase reproduction success. Here we investigated the adaptive significance of the flower bending from erect to downward in Trifolium repens. We observed the flowering dynamic characteristics (changes of vertical direction of florets, flowering number, pollen grain numbers, pollen viability and stigma receptivity over time after blossom) and the factors affecting the rate of flower bending in T. repens. Then we altered the vertical direction of florets in inflorescence of different types (upright and downward), and compared the pollinator behaviors and female reproductive success. Our results showed that florets opened sequentially in inflorescence, and then bend downwards slowly after flowering. The bending speed of florets was mainly influenced by pollination, and bending angle increased with the prolongation of flowering time, while the pollen germination rate, stigma receptivity and nectar secretion has a rhythm of “low-high-low” during the whole period with the time going. The visiting frequency of all the four species of pollinators on upward flowers was significantly higher than that of downward flowers, and they especially prefer to visit flowers with a bending angle of 30°–60°, when the flowers was exactly of the highest flower rewards (nectar secretion and number of pollen grains), stigma receptivity and pollen germination rate. The seed set ratio and fruit set ratio of upward flowers were significantly higher than downward flowers, but significantly lower than unmanipulated flowers. Our results indicated that the T. repens could increase female and male fitness by accurate pollination. The most suitable flower angle saves pollinators’ visiting energy and enables them to obtain the highest nectar rewards. This coordination between plants and pollinators maximizes the interests of them, which is a crucial factor in initiating specialized plant-pollinator relationships.     

濒危植物蒙古扁桃花粉活力和柱头可授性研究

用过氧化氢酶法测定蒙古扁桃花粉的活力和寿命,用联苯胺-过氧化氢法测定柱头可授性。结果表明,蒙古扁桃花粉活力在散粉后第2天最高,3d后活力迅速下降,三种类型的植株的花粉活力下降速度存在差异,其活力可持续30d左右。短柱头植株柱头在开花后1d可分泌黏液,长柱头和中柱头花植株的柱头在开花后2d才分泌黏液,短柱头分泌黏液的持续时间较后者长。柱头可授性持续时间同种之间也存在差异,长柱头和中柱头植株可达6d,而短柱头花植株可持续10d左右。     

桂林唇柱苣苔和百寿唇柱苣苔的开花动态及花粉活力和柱头可授性的比较

对桂林唇柱苣苔的两个居群(YZ居群和DB居群)和百寿唇柱苣苔的开花动态、花粉活力和柱头可授性进行了比较研究。结果表明,桂林唇柱苣苔的两个居群花期、单花持续期存在明显差异,DB居群的花期较YZ居群晚,但花期长于后者。YZ居群的花期、单花持续期与百寿唇柱苣苔的花期、单花持续期相近。三者开花过程相似。花粉活性和柱头可授性达最大的时间、持续时间在桂林唇柱苣苔的两个居群以及百寿唇柱苣苔之间具有明显差异。     

簇枝补血草花的二态性及繁育系统研究

白花丹科被列为异型花柱较多的科,该文以补血草属簇枝补血草为材料,通过对野外居群的花部特征、花期物候和繁育系统等研究,探讨花部形态多态性及其对生殖的影响。簇枝补血草具有两种类型植株,具玉米状柱头和A型花粉的植株称为C型植株;具乳头状柱头和B型花粉的植株称为P型植株,且A/B型花粉的大小、外壁纹饰不同。单花发育过程中两型植株的花萼、雌蕊大小差异明显,随着花的成熟差异逐渐消失。两型植株都为雌蕊高于雄蕊,柱头探出式雌雄异位;C型植株的花期较P型植株长约2 d。一天当中,A型花粉活力高峰期为13:00,B型花粉在11:00和15:00都表现为高峰期;玉米状柱头可授性最强时期在13:00～15:00,乳头状柱头则在15:00。两型花粉可育性在盛花期最高,终花期时B型花粉可育性明显高于A型。荧光染色观察发现花粉萌发和花粉管的生长速度都为:异型授粉＞同型授粉,B型花粉在乳头状柱头上不能完成受精。此外,人工授粉实验也表明异型授粉结实率显著高于同型,P型植株经同型授粉结实现象并不明显,存在少量无融合生殖。研究表明簇枝补血草花部具二态性,繁育系统为兼性异交,其中乳头状柱头对A型花粉亲和,对B型花粉不亲和,但玉米状柱头却对A/B型花粉都亲和,这打破了该科的异型自交不亲和的例证。     

Pollination, Pollen Viability and Pistil Receptivity in Cucurbita pepo

Cucurbita pepo carries male and female flowers on the same plant,and is pollinated by nectar-collecting bees. The nectaries aredimorphic in the two sexes and pollen is loaded and unloadedas the bees gain access to the nectar. Both types of flowerare open for only 6 h (from 0600 h to 1200 h); male flowersopen and close half an hour earlier than female flowers. Thelatter produce more nectar and are visited more often by thebees than the male flowers. Pollen viability determined by fluoresceindiacetate (fluorochromatic reaction) decreases by 20% duringanthesis and more rapidly after the flower closes. This decreaseis due to dehydration of the grain, especially around the porewhere the intine is exposed. An unusual feature of this speciesis that the grains do not dehydrate before anther dehiscence.Female receptivity has two aspects, that of the stigma lasting4 d, and that of the ovules lasting 2 d. The receptivity ofthe two sexes and the short period of anthesis are discussedin the light of the reproductive ecology of the species.Copyright1993, 1999 Academic Press Entomophilous pollination, anthesis, nectars, pollen viability, female receptivity, bees, pollinator efficiency, courgette Cucurbita pepo, Cucurbitaceae     

峨眉拟单性木兰的开花生物学特性与繁育系统

为阐明峨眉拟单性木兰(Parakmeria omeiensis)在自然条件结实率低的原因和确定最佳人工授粉时期,该文通过观察峨眉拟单性木兰的开花动态,采用杂交指数估算、花粉胚珠比、花粉活力及柱头活性检测、人工授粉试验等方法对其繁育系统进行了研究。结果表明:(1)植物园保育的峨眉拟单性木兰花期在4月底到5月中下旬,持续17～23 d,雄株始花期比雌株早3～4 d,但两者花期可遇。(2)两性花经检测雄蕊败育,实为功能上的雌性,部分雄株个体的雄花上残留1～2个心皮,其性别分化是通过雌、雄蕊选择性败育形成的,为隐性雌雄异株(cryptic dioecy)。(3)雄花、两性花开放经历佛焰苞开裂、花被片开裂、展开、闭合、二次开放、凋落6个阶段,历时4 d。(4)雄花初次展开时花粉活力最高,达92.8%,开花2 d后活力显著下降;两性花柱头在花被片展开期可授性最强,盛开后柱头部分可授。(5)杂交指数为5,P/O比为2.14×10~4。(6)套袋试验表明,峨眉拟单性木兰不能进行自花传粉,人工异花授粉的结实率和出种数显著高于自然授粉,且不存在无融合生殖。这说明峨眉拟单性木兰繁育系统为专性异交,传粉过程需要传粉媒介,自然条件下结实率低,主要是受传粉昆虫和柱头可授期短的限制。     

宁夏枸杞传粉生态学初步研究

田间观察检测了宁夏枸杞(Lycium barbarum L.)9个品系的开花物候、开花动态、花粉活力、柱头可授性及其繁育系统,并对花粉萌发、花粉管生长以及访花者的种类和访花行为进行了初步研究.结果表明,宁夏枸杞整个居群有长达5个月的花期,单花的花期仅为3～4 d,花粉活力可保持15 d左右,柱头可授性在72 h左右,雌雄生殖单位在持续时间上有较长的相遇期;花粉数量与胚珠比(P/O)为4 000左右,杂交指数(OCI)介于3或4,繁育系统为异交型,需要传粉者;荧光显微观察表明,参试的3个品系的花粉均能在柱头上萌发,2个有自交不亲和现象,其位置发生在花柱的上半部分和子房内.访花昆虫有17种,分属双翅目、膜翅目和鳞翅目,花的结构和开花式样适合以意大利蜜蜂和食蚜蝇为主的多种昆虫传粉.