Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins(AGPs)are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.In this study,we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes,38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.We further tested their expression patterns using RT-PCR and RT-qPCR.Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads(RPKM)values during pollen tube growth,implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.Therefore,19 PbrAGPs(PbrAGP1 to PbrAGP19)were selected to test their influences on pollen tube growth.Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen,and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.Additionally,pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.In summary,this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

Introduction of herbicide resistant gene (bar) into maize (Zea mays L.) via pollen tube pathway

The pollen tube pathway method of transformation has been reported to be successful in most crops, but less successfu in maizc. DNA can be transferred by cutting the stigma following pollination and applying the DNA solution in a suitable period DNA presumably reaches the ovary by flowing down the pollen tube and then integrates into the just fertilized but undivided zygotic cells. To provide the molecular evidence for this procedure, the plasmids pGBIRC carrying a CaMV35S promoter-PPT acetyle transferase （bar） gene-nos terminator genc fusion construct were used. Total 3 276 seeds were produced from the ears trcated with DNA. It was found that 35 scedlings were GUS assay positive, but less intense than that of the positive controls, of which 17 were PCR amplification positive. But, only 13 of the seeds from the plants treated with DNA containing the bar gene were found to be resistant compared with the negative control. Less than 1.07% of progeny seedlings tested cxpressed a herbicide positive reaction and polymerase chain reaction （PCR） with seedling DNA did detect the bar genc. Morphological variation was observed in six plants. We succeed in obtain PPT-resistant maize inbred lines via pollen tube pathway

Role of Wall-bound β-Glucanases in Regulating Tip-growth of Lilium longiflorum Pollen Tubes

Glucanases were found in the cell wall of Lilium longiflorum Thunb. pollen tubes grown in vitro . The activity of β_glucanases was, in a certain extent, decreased by nojirimycin, an inhibitor of glucosidase. Pollen germination percentage reduced dramatically when nojirimycin was applied in the culture medium. In case that nojirimycin was added at 0 or 1 h after the onset of incubation, the inhibition rate was 99.6% and 91.4%, respectively. When 3 mmol/L of nojirimycin was applied in the liquid medium at 0, 1, 1.5 and 2 h after the onset of incubation, the growth of pollen tubes was interrupted, which resulted in the morphological change of the pollen tubes such as the newly grown portion of pollen tubes being bent, curved and swollen. Tracing the growth pattern of the individual pollen tube grown in semi_solid medium by video microscopy, the authors demonstrated that pollen tube growth rate was strongly inhibited by nojirimycin at concentrations ranged from 0.003 to 3 mmol/L. Moreover, the cytoplasmic arrangement and the morphology of the pollen tubes were also affected by nojirimycin. The growth inhibition brought about by nojirimycin was reversible. These results indicated that β_glucanases, which degrade 1,3_β_glucan and/or 1,4_β_glucan or 1,3:1,4_β_glucan constructed in the cell wall, are involved in pollen germination and pollen tube growth. It provides new insight into an understanding of the contribution of β_glucanases to the cell wall extensibility and the crucial role of cell wall in regards to the regulation of pollen tube growth.

Pollen Grain Germination and Pollen Tube Growth in Pistil of Rice

The germination of pollen grain in vitro and the growth of pollen tube in the pistil of rice were observed with a microscope. The stigma was removed at different time points after pollination to study its effect on seed setting rate. The rice pollen grain started to germinate at 2 min after pollination and the pollen tube penetrated stigma into style in 5-10 min, 30 min later the end of pollen tube reached the bottom of ovary, and only some pollen tubes arrived at embryo sac at 40 min after pollination. Meanwhile, a small amount of callose began to deposit in the pollen tubes, a great deal of callose was observed at 50 min after pollination, whereas the pollen grain began to shrink. The growing rates of pollen tube in the rice stigma, style and ovary were 1500, 5000, and 5400 μm/h, respectively. The seed setting rate was quite low when the stigma was removed at about 10-15 min after pollination, gradually increased when it removed at 20 min to 50 min after pollination, and over 60% when it removed at 50 min after pollination and finally tended to be stable.

Effects of Ion Implantation on in Vitro Pollen Germination and Cellular Organization of Pollen Tube in Pinus thunbergii Parl. (Japanese Black Pine)

Low-energy ion implantation, as a new technology to produce mutation in plant breeding, has been widely applied in agriculture in China. But so far there is a little understanding of the underlying mechanisms responsible for its biological effects at the cellular level. Here we report the biological effects of a nitrogen ion beams of 30 keV on the pollen grains of Pinus thunbergii Parl. In general, ion implantation inhibited pollen germination. The dose-response curve presented a particular saddle-like pattern. Ion implantation also changed the dimension of the elongated tubes and significantly induced tip swelling. Confocal microscopy indicated that the pollen tube tips in P. thunbergii contained an enriched network of microtubules. Ion implantation led to the disruption of microtubules especially in swollen tips. Treatment with colchicine demonstrated that tip swelling was caused by the disruption of microtubules in the tip, indicating a unique role for microtubules in maintaining the tip integrality of the pollen tube in conifer. Our results suggest that ion implantation induce the disruption of microtubule organization in pollen and pollen tubes and subsequently cause morphological abnormalities in the pollen tubes. This study may provide a clue for further investigation on the interaction between low-energy ion beams and pollen tube growth.

Ion Implantation Hampers Pollen Tube Growth and Disrupts Actin Cytoskeleton Organization in Pollen Tubes of Pinus thunbergii

Pollen grains of Pinus thunbergii Parl. （Japanese black pine） were implanted with 30 keV nitrogen ion beams and the effects of nitrogen ion implantation on pollen tube growth in vitro and the organization of actin cytoskeleton in the pollen tube cell were investigated using a confocal laser scanning microscope after fluorescence labeling. Treatment with ion implantation significantly blocked pollen tube growth. Confocal microscopy showed that ion implantation disrupted actin filament cytoskeleton organization in the pollen tube. It was found that there was a distinct correlation between the inhibition of pollen tube growth and the disruption of actin cytoskeleton organization, indicating that an intact actin cytoskeleton is essential for continuous pollen tube elongation in Pinus thunbergii. Although the detailed mechanism for the ion-implantation-induced bioeffect still remains to be elucidated, the present study assumes that the cytoskeleton system in pollen grains may provide a key target in response to ion beam implantation and is involved in mediating certain subsequent cytological changes.

Screening of Transgenic Soybean Transformed by Means of Pollen-tube Using Kanamycin

Kanamycin was used to screen To seeds of the variety Dongnong 46 transformed by means of pollen-tube method. The results showed that 400 mg·L^-1 kanamycin could inhibit growth of non-transgenic plants, and 2 positive plants were gotten combined with Gus dyeing and PCR detection. It is proved that this method is economic and effective in preliminary screening the transgenic plants.

Inositol (1,4,5)-triphosphate Modulates Pollen Tube Polar Growth

In this article, the functions of D-myo-inositol-1,4,5-trisphosphate (Ins [1,4,5] P3) in regulating pollen tube polar growth were investigated by application of caged version of the phosphoinositides. To increase the intracellular Ins（1,4,5）P3 concentration at the apical region of pollen tube, the caged Ins（1,4,5）P3 loaded by osmotic shock was activated by 10 s 360 nm UV flash at this domain （10 μm from the tip）. More than 70% pollen tubes were induced swelling at apex and/or growth axis reorientation, accompanying temporarily growth arrest, by localized increase of Ins（1,4,5）P3 concentration （n=21）. While, pollen tubes without being loaded with caged Ins（1,4,5）P3 had not response to the same dosage UV flash. With FM 1-43 fluorescent staining, it was found that growth perturbation by UV activated caged Ins（1,4,5）P3 had tight link with membrane trafficking at the apical zone of pollen tubes. Upon UV pulse, the apical V-shaped bright area where was full of secretory vesicles spread to a much broader region, which implied that actin filaments at the apical region were remodeled. It was also observed that the FM 1-43 fluorescence intensity at tip remarkably increased than that before UV flash, which demonstrated that more secretory vesicles were accumulated at this region. To estimate the role of Ins（1,4,5）P3 in modulating intracellular calcium concentration and distribution, dextran conjugated fluorescent dye Calcium Green-1 and Rhodamine B were microinjected into pollen tubes together with caged Ins（1,4,5）P3. The results showed that calcium concentration at the subapical region increased upon UV released Ins（1,4,5）P3. Consequently, the tip-focused calcium gradient under the apical dome of pollen tube was disturbed. Simultaneously, the pollen tube bulged at the apical region and its growth rate decreased. As the tip-focused calcium gradient was reestablished, the pollen tube morphology and growth recovered to the normal level. Therefore, Ins（1,4,5）P3 can modulate pollen tube growth rate and direction through mobilizing intracellular calcium and regulating vesicle trafficking during pollen tube finding its way to the ovary.

Pollen Germination and Pollen Tube Elongation of Tomato （Lycopersicum esculentum L.） Regulated by Cell Wall Invertase through Sucrose Hydrolysis

Sucrose is a sugar required for pollen germination and pollen tube elongation. It is little known on the regulation mechanism. As such, this research was conducted to reveal mechanism pathway of the sugar in regulating pollen germination and pollen tube elongation by cell wall invertase. The pollen grains, respectively originated from wild type and transgenic tomato plants, which had been silenced their inhibitor gene （INVINH1） of the cell wall invertase were used in this study. The pollen grains were cultured in media containing glucose, fi＇uctose or sucrose. Results of the study showed that percentage of the pollen germination derived from transgenic plants was much higher than that from wild type plants. Moreover, pollen tube elongation was longer in transgenic plants compared with wild type plants. Interestingly, these results were observed in medium containing sucrose only, but not in glucose and fructose. This result suggests that cell wall invertase activity regulates pollen germination and pollen tube elongation through sucrose hydrolysis.

Regulation of Petunia Pollen Tube Growth by Phytohormones： Identification of Their Potential Targets

It is known that cytoskeleton-dependent trafficking of cell wall and membrane components to apical plasma membrane （PM） coupled with ion transport across pollen PM is crucial for maintaining polar pollen tube growth. To elucidate whether plant hormones are involved in these processes, the effects of exogenous phytohormones, indole-3-acetic acid （IAA）, abscisic acid （ABA）, gibberellin A3 （GA3） and cytokinin （kinetin） on the growth, PM polarization, actin cytoskeleton （AC） organization and cytoplasmic pH （pile） of in vitro 4 h-growing petunia pollen tubes were investigated. IAA, ABA and GA3 displayed the growth-stimulating effects and these were accompanied by orthovanadate-sensitive hyperpolarization of the PM. Fluorescent labeling the enzyme with H＋-ATPase antibodies exhibited IAA- and ABA-induced lateral PM redistribution of it into the subapical zone of pollen tube PM. Pollen cultivation on the medium with latrunculin B, the inhibitor of actin polymerization, resulted in inhibition of pollen tube growth and simultaneously in the drop of endogenous IAA content. The IAA-growth stimulating effect was correlated with increased content of actin filaments （AF） in both apical and subapical zones of tubes, while ABA and GA3 exerted the same effect but it was accompanied by redistributing F-actin only to apical zone. In contrast, kinetin decreased the total F-actin content and inhibited pollen tube growth. It has been shown that the pHe of growing pollen tubes is sensitive to the plant hormones. In the case of male gametophyte growing for 1, 2 and 4 h, IAA induced alkalinization of the cytosol, while ABA and GA3 exerted qualitatively similar effect only after its growth for 1 h and 4 h, respectively. Kinetin, in contrast, resulted in acidification of the cytosol. All these results, taken together, indicate, for the first time, potential targets of the phytohormone action in pollen tubes.

In situ Localization and Isolation of Actin Filaments from Pollen Tubes of Amaryllis vittata Ait

Actin filaments (AFs) in un-fixed pollen tubes of Amaryllis vittata Ait were visualized after TRITC-phalloidin staining with DMSO as a permeabilising agent. Typically, strands or hundles of microfilaments (Mfs) were distributed in the extreme tip as well as pollen tubes in a form of network.Fluorescent granules or circles of various sizes were frequently found that continued with the filamentous structures. In addition, a more brightly stained structure, possibly Mf organizing center, was observed. Treatment of pollen tubes with cytochalasin D(CD)for increasing time intervals (5-40 minutes) caused gradual reduction of strands until flurescent granules filled up the pollen tubes. Mcanwhile, cytoplasmie streaming was inhibited completely. Though closely associated with vegetative nuclei (VN) and generative cells (GC), AFs were not found in the cytoplasm of GC.Mg++concentration greatly affected the isolated Mfs.

Regulation of Bacterial, Yeast and Plant Polysaccharide Synthases: Implications for the Regulation of Pollen_tube Callose Synthase

多糖作为结构和能量贮存分子是植物重要的组成成分。植物细胞壁主要成分为多糖。细胞壁在确定细胞生长、形状方面起重要作用,细胞壁还参与细胞的营养吸收、信息传递,也是防止外源对细胞不良影响的第一道防线。不同植物细胞壁的多糖成分可作为食品、建筑及造纸的原料,具有广泛的工业价值。通过描述细菌、酵母及植物多糖合成酶的机制,推断花粉管胼胝质合成酶的可能调控机制。

不同培养基组分对“台农一号”杧花粉萌发和花粉管生长特性的影响

为筛选“台农一号”杧花粉体外培养最适培养基和培养时间,探讨不同培养基组分和培养时间对其花粉体外萌发及花粉管生长的影响,以15%蔗糖+25%聚乙二醇-4000+0.015%硼酸+0.03%钙+0.02%镁+0.01%钾为基础培养基,分别设置1、2、3、4、5、6 h培养,不同蔗糖、硼酸、聚乙二醇-4000、钙、镁、钾浓度梯度单因素及蔗糖、硼酸、聚乙二醇-40003个因素正交试验,统计比较不同处理的花粉萌发率和花粉管生长长度。结果表明,培养时间,培养基中蔗糖、聚乙二醇-4000、硼酸对花粉萌发及花粉管生长均有显著影响,而钙、镁、钾影响不显著。培养2~3 h即可统计花粉萌发率及测量花粉管长度。随着培养基中蔗糖、聚乙二醇-4000、硼酸浓度增加,花粉萌发率及花粉管长度均呈现先增加后降低的趋势,适宜浓度的3个组分能够促进花粉萌发及花粉管生长,浓度过高则起抑制作用。3个因素之间存在明显的互作效应,蔗糖与聚乙二醇-4000的互作效应突出。说明培养基中蔗糖、聚乙二醇-4000、硼酸是花粉体外萌发的必需成分,钙、镁、钾为非必需成分。“台农一号”杧花粉体外萌发及花粉管生长的最适培养基为15%蔗糖+20%聚乙二醇-4000+0.015%硼酸+0.03%钙+0.02%镁+0.01%钾,适宜培养时间为3 h,此条件下花粉萌发率达到83.40%。

报春苣苔属植物杂交亲和性分析及杂交障碍

【目的】探究报春苣苔属属内杂交亲和性及杂交障碍的原因。【方法】以4个报春苣苔原生种、2个栽培品种为材料进行常规杂交,通过花粉活力、柱头可授性筛选父母本后开展杂交工作,初步确定杂交组合亲和性关系,并对授粉后的子房发育跟踪观察。【结果】1)6种报春苣苔属植物的花粉活力和柱头可授性强弱与其杂交亲和性反应没有绝对的因果关系。2)16个杂交组合的子房膨大率均在65.00%以上,结实率有明显差别,以牛耳朵Primulina eburnea、尖萼报春苣苔P. pungentisepala、大根报春苣苔P. macrorhiza、蚂蝗七P. fimbrisepala作为母本时,各杂交组合的平均结实率分别达到了75.55%、78.44%、46.67%、57.78%,表现出了较高的亲和性,而以‘北林之春’报春苣苔P.‘Spring of Beilin’、‘四季’报春苣苔P.‘Four Season’为母本的组合,结实率最高也仅有6.67%,亲和性较弱。3)对5个亲和性较弱的杂交组合进行花粉管行为荧光观察,发现均存在一定程度的受精前障碍,表现为花粉管胼胝质反应、花粉管末端膨大、扭曲断裂,但各组合最终均有花粉管伸入胚珠,从到达胚珠的花粉管数量来看,牛耳朵等3个原生种的花粉数量均可满足胚珠受精需要,但结实率仍较低。【结论】报春苣苔属属内杂交具有一定的亲和性,但部分组合未获得种子,并非由受精前的花粉萌发和花粉管生长所限制,可能是由于受精卵或幼胚的后期发育过程中出现代谢紊乱而导致败育。

Influence of Plant Growth Regulators on Pollen Germination and Growth of Xinjiang Apricots

[Objective] This study aimed to investigate the influence of different plant growth regulators on apricot pollen germination and pollen tube growth. [Method] Pollens of six kinds of Xinjiang apricots were cultured in solid media supplemented with five plant growth regulators （GA3 , NAA, 2, 4-D, 6-BA, IAA）. Then the rate of pollen germination and the length of pollen tube were respectively measured. [Result] In a certain concentration range, GA3 most significantly promoted the pollen germination and the pollen tube growth of Shushanggan, Kalayulvke, Dayoujia, Yiliakeyulvke and Kabakehuanna; NNA had the strongest improvement function on Kumaiti’s pollen germination and pollen tube growth. [Conclusion] All the five plant growth regulators promoted the pollen germination and the pollen tube growth of apricots at low concentration but inhibited them at high concentration.

水稻花粉管生长及其对非生物逆境胁迫的响应机理研究进展

水稻是我国乃至全世界最为重要的粮食作物之一。全球气候变暖背景下,高温、干旱、低温寡照等极端天气频繁发生,水稻产量品质形成受到不同程度的影响,严重威胁我国的粮食安全。花粉管在雌蕊组织中的生长是水稻生殖生长最为关键的过程,高温等非生物逆境胁迫可严重干扰花粉管生长的信号传递和能量代谢,进而导致水稻受孕失败。因此,研究高温等逆境胁迫影响水稻花粉管生长及其调控机制具有重要意义。本文综述了水稻花粉管生长的基本过程,包括信号传递以及能量代谢,同时阐述了高温、干旱、低温寡照、重金属等逆境影响花粉管生长致使小穗败育的作用机制及其栽培调控技术,为减缓高温等逆境胁迫干扰水稻花粉管生长,提高水稻授粉受孕能力的栽培技术提供理论参考,并对今后的研究方向进行展望。

金柑杂交授粉生物学特性研究

【目的】掌握金柑花粉活力与柱头可授性的变化情况,寻找最佳授粉时期,探明金柑杂交授粉的生物学特性,提高杂交成功率。【方法】对23份金柑材料进行胚型的统计。选择不同胚数材料,采用TTC染色法和联苯胺-过氧化氢法分别对小蕾期、大蕾期、初开期和盛开期的花粉活力和柱头可授性进行研究。对金柑进行去雄不授粉、自交授粉和杂交授粉处理,比较自交和杂交花粉管的生长情况及3种处理下的坐果率。【结果】罗纹种胚数最少,适合作母本,其次是山金柑、金弹和罗浮,而长寿金柑的胚数最多;花粉活力和柱头可授性均在4个时期呈先增后减的趋势,并均在初开期达到了最高活性;金柑杂交花粉管和部分的自交花粉管均进入了子房;除宁波金弹和宁波罗纹CS外,其他5种金柑种质杂交授粉比自交授粉的坐果率更高,并呈极显著性差异;母本的柱头活性越高,坐果率越高,种内杂交坐果率高于种间,并呈极显著性差异;金柑去雄不授粉下仍能得到果实,具有单性结实的特性。【结论】金柑杂交中应选择单胚型母本材料;金柑属于雌雄同熟,采集花粉最佳时期和授粉最佳时期均为初开期;金柑杂交亲和性比自交亲和性更强,有利于金柑的杂交育种。

攸县油茶远缘杂交亲和性的解剖学研究

【目的】为了探究攸县油茶(Camellia yuhsienensis)远缘杂交亲和性的生物学机制。【方法】研究以石果毛蕊山茶[C.mairei var.lapidea(Y.C.Wu)Sealy](SG)、海南油茶‘海油3号’(C.hainanica‘Haiyou 3’)(HN-3)和普通油茶‘德油6号’(C.oleifera‘Deyou 6’)(DY-6)为父本,采用常规授粉和混合授粉方式对攸县油茶(YX)进行自交和杂交授粉。采用流式细胞仪测定亲本倍性,通过离体萌发法测定父本花粉活力,运用显微技术观察花粉萌发及花粉管生长动态以及早期果实和胚珠发育动态,并田间调查各授粉组合的座果率和落果率。【结果】(1)YX为六倍体,SG为二倍体,HN-3为八倍体,DY-6为六倍体。(2)4种油茶花粉具有较强的活力和育性,其中花粉活力最高的是DY-6,为70.13%;花粉活力最低的是YX,为40.68%。(3)自交和杂交所有组合的花粉均能在柱头上正常萌发,且花粉管在授粉后12 h均生长到花柱基部,但自交和YX×SG的花粉管进入子房后生长受阻,无法到达胚珠;YX×HN-3和YX×DY-6的花粉管则在授粉后72h到达胚珠。(4)自交和杂交授粉后100 d内,YX×DY-6的果实横径、纵径最大,YX×SG的果实横径、纵径最小;YX×DY-6的败育胚珠数最少,平均为3.83枚/果;YX×SG的败育胚珠数最多,平均为6.00枚/果。(5)自交和杂交授粉后100 d内,YX×DY-6的座果率最高,为68.0%;YX×SG的座果率最低,为4.0%;YX×HN-3的座果率为16.0%,其落果高峰主要集中在授粉后0~30 d和40~70 d;混合授粉组合YX×SD的座果率为22.0%。【结论】自交和YX×SG的杂交障碍主要为受精前障碍,YX×HN-3的杂交障碍为受精前和受精后障碍;攸县油茶远缘杂交亲和性也与亲本倍性有关,其亲和性大小为YX(六倍体)×DY-6(六倍体)>YX×HN-3(八倍体)>YX×YX>YX×SG(二倍体);混合授粉方式能在一定程度上提高攸县油茶远缘杂交亲和性。研究结果为攸县油茶远缘杂交亲和性机理研究提供了细胞学基础,也为油茶远缘杂交育种工作提供理论参考。

钐钴永磁漂移管出气及质谱分析

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