The effects of extracellular calmodulin on initiation of Hippeastrum rutilum pollen germination and tube growth

The effects of anti-calmodulin (CaM) serum, the CaM antagonist W7-agarose, the Ca²⁺ chelator ethyleneglycol-bis-(β-aminoethyl)-N,N,N′,N′-tetraacetic acid (EGTA) and exogenous pure CaM on pollen germination and tube growth of Hippeastrum rutilum Herb were studied. Pollen germination and tube growth were inhibited or completely stopped by anti-CaM serum in a dose-dependent manner, while the same amount of preimmune serum had no effect on either process. Pollen germination and tube growth were also inhibited or completely stopped by the CaM antagonist W7-agarose and the Ca²⁺ chelator EGTA. The addition of exogenous pure CaM enhanced pollen germination and tube growth, whereas the same amount of bovine serum albumin had no effect. The inhibitory effects caused by anti-CaM serum, W7-agarose and EGTA-washing could be reversed completely by the addition of exogenous pure CaM. These results indicate that extracellular CaM initiates pollen germination and tube growth, whereas exogenous CaM enhances the above processes, and may provide a novel view for understanding the control of pollen germination and tube growth. Received: 12 December 1996 / Accepted: 15 January 1997     

Does aluminum inhibit pollen germination via extracellular calmodulin?

The effect of aluminum (Al) on pollen germination and its mechanism of action were investigated. Pollen germination and pollen tube elongation were inhibited by Al at pH 4.5. This inhibitory effect was reversed by the addition of purified calmodulin (CaM), whereas neither the calcium binding-protein S-100 nor Al chelator citric acid at the same concentrations had any obvious effect on Al-inhibited pollen germination. The presence of either the membrane-impermeable CaM inhibitor anti-CaM antiserum or Ca2+ chelator EGTA completely suppressed the effect of exogenous CaM. These results indicate the involvement of extracellular calmodulin in the short-term effects of Al on pollen germination and pollen tube elongation.     

Apoplastic calmodulin promotes self-incompatibility pollen tube growth by enhancing calcium influx and reactive oxygen species concentration in Pyrus pyrifolia

Key message

This study indicated that Ca ²⁺ , ROS and actin filaments were involved with CaM in regulating pollen tube growth and providing a potential way for overcoming pear self-incompatibility.

Abstract

Calmodulin (CaM) has been associated with various physiological and developmental processes in plants, including pollen tube growth. In this study, we showed that CaM regulated the pear pollen tube growth in a concentration-dependent bi-phasic response. Using a whole-cell patch-clamp configuration, we showed that apoplastic CaM induced a hyperpolarization-activated calcium ion (Ca²⁺) current, and anti-CaM largely inhibited this type of Ca²⁺ current. Moreover, upon anti-CaM treatment, the reactive oxygen species (ROS) concentration decreased and actin filaments depolymerized in the pollen tube. Interestingly, CaM could partially rescue the inhibition of self-incompatible pear pollen tube growth. This phenotype could be mediated by CaM-enhanced pollen plasma membrane Ca²⁺ current, tip-localized ROS concentration and stabilized actin filaments. These data indicated that Ca²⁺, ROS and actin filaments were involved with CaM in regulating pollen tube growth and provide a potential way for overcoming pear self-incompatibility.     

花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响

以烟草为材料,通过半体内实验,就花柱和花粉胞外钙调素对花粉萌发和花粉管伸长的影响进行了观察。发现用EGTA及钙调素抗血清处理柱头或花粉均可抑制花粉在柱头上的萌发;向花柱引导组织中显微注射纯化钙调素可促进花粉管束伸长,而注射钙调素抗血清可抑制花粉管束伸长;同时证实玉米花柱和花粉细胞壁中均存在钙调素及钙调素结合蛋白,而且花粉和花柱细胞壁中钙调素结合蛋白的种类有差异。结果表明存在于花粉和花柱细胞外的钙调素对花粉萌发和花粉管伸长均有促进作用。     

肌醇磷脂信号途径参与胞外钙调素启动花粉萌发和花粉管伸长

高等植物有性生殖是植物发育生物学研究的重要内容之一,而作为雄配子体的花粉在雌蕊柱头上萌发及花粉管在花柱内的持续生长是有性生殖实现的关键。已有许多研究表明Ca2 在花粉萌发和花粉管生长过程中起重要作用。最近,我室在多年细胞外钙调素（calinodulin,CaM）存在。性质及生物学功能研究（孙大业等1995;Sun等1994,1995;Tang等1996）的基础上,通过不过膜的大分子CaM拈抗剂或抗体并结合恢复实验证实细胞外CaM对花粉的萌发和花粉管的伸长具有启动作用（马力耕和孙大业1996）,并发现G蛋白、质膜Caz”通道及胞内依赖Caz”的蛋白…     

Analysis of the tip-to-base gradient of CaM in pollen tube pulsant growth using in vivo CaM–GFP system

Ca²⁺-CaM signaling is involved in pollen tube development. However, the distribution and function of CaM and the downstream components of Ca²⁺-CaM signal in pollen tube development still need more exploration. Here we obtained the CaM–GFP fusion protein transgenic line of Nicotiana tobacum SRI, which allowed us to monitor CaM distribution pattern in vivo and provided a useful tool to observe CaM response to various exogenous stimulations and afforded solid evidences of the essential functions of CaM in pollen tube growth. CaM–GFP fusion gene was constructed under the control of Lat52-7 pollen-specific promoter and transformed into Nicotiana tobacum SRI. High level of CaM–GFP fluorescence was detected at the germinal pores and the tip-to-base gradient of fluorescence was observed in developing pollen tubes. The distribution of CaM at apical dome had close relationship with the pulsant growth mode of pollen tubes: when CaM aggregated at the apical dome, pollen tubes stepped into growth state; When CaM showed non-polarized distribution, pollen tubes stopped growing. In addition, after affording exogenous Ca²⁺, calmidazolium (antagonism of CaM) or Brefeldin A (an inhibitor of membrane trafficking), CaM turned to a uniform distribution at the apical dome and pollen tube growth was held back. Taken together, our results showed that CaM played a vital role in pollen tube elongation and growth rate, and the oscillation of tip-to-base gradient of CaM was required for the normal pulsant growth of pollen tube.     

The presence of a heterotrimeric G protein and its role in signal transduction of extracellular calmodulin in pollen germination and tube growth

The role of heterotrimeric G proteins in pollen germination, tube growth, and signal transduction of extracellular calmodulin (CaM) was examined in lily pollen. Two kinds of antibodies raised against animal Gzalpha, one against an internal sequence and the other against its N terminus, cross-reacted with the same 41-kD protein from lily pollen plasma membrane. This 41-kD protein was also specifically ADP ribosylated by pertussis toxin. Microinjection of the membrane-impermeable G protein agonist GTP-gamma-S into a pollen tube increased its growth rate, whereas microinjection of the membrane-impermeable G protein antagonist GDP-beta-S and the anti-Galpha antibody decreased pollen tube growth. The membrane-permeable G protein agonist cholera toxin stimulated pollen germination and tube growth. Anti-CaM antiserum inhibited pollen germination and tube growth, and this inhibitory effect was completely reversed by cholera toxin. The membrane-permeable heterotrimeric G protein antagonist pertussis toxin completely stopped pollen germination and tube growth. Purified CaM, when added directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in plasma membrane vesicles, and this increase in GTPase activity was completely inhibited by pertussis toxin and the nonhydrolyzable GTP analogs GTP-gamma-S and guanylyl-5'-imidodiphosphate. The GTPase activity in plasma membrane vesicles was also stimulated by cholera toxin. These data suggest that heterotrimeric G proteins may be present in the pollen system where they may be involved in the signal transduction of extracellular CaM and in pollen germination and tube growth.     

钙调素对花粉萌发和花粉管生长的效应

牛脑和玉米胚ＣａＭ能显著促进花粉萌发和花粉管生长（图１）,而ＣａＭ抑制剂ＴＦＰ、ＣＰＺ及另外两个专一性更强的抑制剂Ｃｏｍｐｏｕｎｄ４８／８０和Ｗ７均严重抑制甚至阻止花粉的萌发（图２,３）。用对ＣａＭ亲和性较低的Ｗ７同系物Ｗ５,在与Ｗ７同样浓度下,对花粉萌发和花粉管生长无明显影响。此外,Ｗ７对花粉萌发和花粉管生长的抑制效应可被外源ＣａＭ所消除（图４）。在花粉萌发过程中,其内源ＣａＭ含量显著上升,在花粉萌发率接近最大值时,花粉ＣａＭ含量达最高水平（图５）。上述结果表明ＣａＭ对花粉萌发和花粉管生长的调控起重要作用。     

Ca2+对韭兰花粉萌发和花粉管生长的影响

采用离体花粉培养技术,研究不同浓度Ca²⁺对韭兰花粉萌发和花粉管生长的影响。结果表明,较低浓度(10^-3 mol/L)的Ca²⁺对花粉萌发具明显地促进作用,并促进花粉管较快伸长;而过高或过低浓度则起不到促进的作用。     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.     

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.     

Fast pollen tube growth in Conospermum species

BACKGROUND AND AIMS: An unusual form of pollen tube growth was observed for several Conospermum species (family Proteaceae). The rate of pollen tube growth, the number of tubes to emerge and the ultrastructure of these tubes are given here. METHODS: Pollen was germinated in vitro in different sucrose concentrations and in the presence of calcium channel blockers, and tube emergence and growth were recorded on a VCR. Measurements were taken of the number of tubes to emerge and rate of tube emergence. Pollen behaviour in vivo was also observed. The ultrastructure of germinated and ungerminated pollen was observed using TEM. RESULTS: After 10 s to 3 min in germination medium, up to three pollen tubes emerged and grew at rates of up to 55 micro m s(-1); the rate then slowed to around 2 micro m s(-1), 30 s after the initial growth spurt. Tubes were observed to grow in pulses, and the pulsed growth continued in the presence of calcium channel blockers. Optimal sugar concentration for pollen germination was 300 g L(-1), in which up to 81 % of pollen grains showed fast germination. Germination and emergence of multiple tubes were observed in sucrose concentrations of 100-800 g L(-1). The vegetative and generative nuclei moved into one of the tubes. Multiple tubes from a single grain were observed on the stigma. Under light microscopy, the cytoplasm in the tube showed a clear region at the tip. The ultrastructure of C. amoenum pollen showed a bilayered exine, with the intine being very thick at the pores, and elsewhere having large intrusions into the plasma membrane. The cytoplasm was dense with vesicles packed with inner tube cell wall material. Golgi apparatus producing secretory vesicles, and mitochondria were found throughout the tube. The tube wall was bilayered; both layers being fibrous and loosely packed. CONCLUSIONS: It is proposed that, for Conospermum, initial pollen tube wall constituents are manufactured and stored prior to pollen germination, and that tube extension occurs as described in the literature for other species, but at an exceptionally fast rate.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

G蛋白调节剂对梨花粉萌发及花粉胞内Ca2+浓度变化的影响

用激光共聚焦技术研究了异三聚体G蛋白活性调节剂对梨花粉萌发、花粉管生长及花粉细胞内游离钙离子浓度动态的影响。结果表明：异三聚体G蛋白激活剂霍乱毒素(CTX)可促进梨花粉萌发与花粉管生长,而其抑制剂百日咳毒素(PTX)则抑制花粉萌发与花粉管生长;霍乱毒素处理后,花粉细胞内产生特异性的“钙瞬变”信号,而百日咳毒素处理后则引起花粉细胞内游离钙离子浓度的持续下降。这表明：异三聚体G蛋白可能参与了梨花粉萌发与花粉管生长的调控过程,G蛋白的活性变化对花粉萌发的效应可能是通过调控花粉细胞内游离Ca^2 浓度的动态变化产生特异性的钙信号来实现的。     

Characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) from Lilium daviddi pollen

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.     

蚕豆下表皮细胞外钙调素的存在及其对气孔运动的调节

细胞外钙调素可能作为多肽第一信使,调节细胞增殖,花粉萌发,特定基因表达等生理过程,气孔能灵敏地对外界刺激作出反应,快速开闭,本文用免疫电镜和免疫荧光显微镜技术证明保卫细胞及其它表皮细胞胞外都存在钙调素;外源纯化钙调素能促进气孔关闭,抑制气孔开放,最适浓度为10^-8mol/L;不能透过质膜的大分子钙调素拮抗剂W—-agarose和钙调素抗血清都能抑制气孔关闭,促进开放,说明保卫细胞的内源胞外钙调素确实能促进气孔关闭,抑制开放。而且只能在细胞外起作用,推测在自然情况下,保卫细胞内源胞外钙调素可能作为胞外第一信使和其它信号分子一起调节气孔的开关运动,而且可能在环境刺激与细胞响应之间起重要作用。     

Pollen germination, tube growth and morphology, and microtubule organization after exposure to benomyl

The inhibitory effects of benomyl on pollen tube growth have received little attention, particularly at the microscopic and immunohistochemical levels. Pollen germination and tube growth in the presence of benomyl were evaluated in Tradescantia virginiana to investigate the effects of this fungicide on pollen germination rate, tube growth and morphology, and microtubule (Mt) organization. Benomyl was incorporated in germination media at 0,480, 600 or 720 mg 1⁻¹. Inhibition of pollen germination, cytoplasmic streaming, and tube elongation were associated with benomyl treatments. Benomyl also induced abnormal pollen tube morphology and Mt organization. Compared to controls, Mts in the treated tubes were characteristically fewer in number, fragmented, sinuous and increasingly disorganized. At the two highest benomyl concentrations, Mts were considerably fewer or absent in apical/subapical regions of the pollen tubes. This work verifies that benomyl incorporated into germination media at concentrations lower than recommended field rates inhibit pollen germination and tube growth, and that the effects are associated with alterations of Mt organization.     

In vitro germination and growth of lily pollen tubes is affected by calcium inhibitor with reference to calcium distribution

The calcium inhibitors A23187, EGTA and La³⁺ inhibit pollen grain germination and growth of pollen tubes of Lilium davidii var. unicolor at different concentrations. Treatment with 10⁻⁴ or 10⁻⁵ M ionophores A23187 reduced germination rate and resulted in distortion of pollen tube. Addition of 2 or 10 mM of the chelator EGTA disturbed the direction of pollen tube growth and extended the diameter of pollen tube as observed by light and confocal microscopy. The Ca²⁺-channel blocker lanthanum chloride (La³⁺) restrained germination or markedly caused transformation of pollen tube. Furthermore, all treatments led to disappearance of any calcium gradient. Calcium distribution in pollen grain and pollen tube was altered as shown by confocal microscopy for each treatment. This indicates that the inhibitors influence pollen development by affecting the calcium gradient which may play a critical role in germination and tube growth. Fourier transform infrared (FTIR) spectra indicated slight increases in contents of amide I and a substantial decrease in the content of aliphatic esters and saturated esters in treated pollen tubes compared with normal pollen tubes. The FTIR analysis confirmed that EGTA and La³⁺ weakened the accumulation of ester in pollen tubes, which may be associated with an increased content of amide I.