H-ATPase in the plasma membrane of Arabidopsis pollen cells is involved in extracellular calmodulin-promoted pollen germination

In this paper, the role of the plasma membrane (PM) H⁺-ATPase in extracellular calmodulin (CaM)-promoted pollen germination and in tube growth of Arabidopsis thaliana was investigated. Pollen germination, pollen tube growth rate, free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) and Ca²⁺ channel activity in the PM of pollen cells were measured. In response to fusicoccin or CaM treatment, in vitro pollen germination and pollen tube growth rate, [Ca²⁺]_cyt and activity of a hyperpolarization-activated Ca²⁺-permeable channel increased. Sodium vanadate inhibited the promotion of these parameters by extracellular CaM. The results suggest that H⁺-ATPase may be involved in extracellular CaM-regulated pollen germination and in tube growth by modulation of the hyperpolarization-activated Ca²⁺ channel in the PM of A. thaliana pollen cells.     

Extracellular calmodulin acceleratesrbcS-GUS expression in suspension-cultured transgenic tobacco cell

The role of extracellular calmodulin in regulating expression of rbcS in darkness was examined. A suspension-cultured cell line was generated from the transgenic rbcS-GUS tobacco. It was demonstrated that purified calmodulin added to the media enhanced rbcS-GUS expression. The time course of expression of rbcS-GUS and that of the secretion of calmodulin in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum calmodulin secretion preceding maximum GUS expression. The addition of membrane-impermeable calmodulin antagonist W7-agarose inhibited the expression of rbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified calmodulin. These results provide the first evidence that extracellular calmodulin accelerates rbcS gene expression.     

疏花疏果剂对苹果花粉萌发及花粉管生长的影响

以华美苹果为材料,采集铃铛花的花粉,采用固体培养法研究了TMN-6和6-BA+ NAA对苹果花粉萌发和花粉管生长的影响.结果表明,TMN-6对花粉的萌发及花粉管生长有抑制作用,且抑制程度随TMN-6的浓度增加而增强,浓度为1 mg/L的处理花粉的萌发几乎完全被抑制.NAA+BA各浓度处理对花粉的萌发抑制作用显著,对花粉...     

Characterization of an endo-1,4-β-glucanase from Lilium longiflorum that functioned in pollen germination and pollen tube growth

Endo-β-glucanases play vital roles in the regulation of pollen tube growth. Here, a previously identified endo-l,4-β-glucanase from Lilium Iongiflorum （lily）, named LlpCell, was expressed in Escherichia coli, purified, and further investigated for its physiological function. The recombinant LlpCell protein hydrolyzed carboxy-methylcellulose （CMC） and exhibited activity towards laminarin from Eisenia. arborea and 1,3：l,4-β-glucan of barley. The pH for the optimum activity was 6.0 and the value of Km calculated from CMC was 5.0 mg/mL. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca^2＋. Western blotting analysis showed that LlpCell protein was present in pollen grains and rehydrated pollen grains, and the amount of the protein was increased during pollen germinating, but not in the pollen tube. Consistently, the immunofluorescence labeling study with the antibody against LIpCell also indicated the presence of LIpCell at the begin-ning of germination, but not in the elongating pollen tube. Furthermore, incubation of LlpCell with pollen at the beginning of pollen germination increased the germination percentage and the length of pollen tube. All of these results suggested that LlpCell could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.     

红花罂粟花粉萌发的研究

以新鲜红花罂粟花粉为试材,比较研究了培养基中蔗糖浓度及硼酸和Ca^2＋、赤霉素浓度对红花罂粟花粉萌发和花粉管生长的影响。结果表明：过高过低的蔗糖、硼酸浓度抑制红花罂粟花粉萌发。低浓度Ca^2＋和赤霉素促进了花粉萌发和花粉管的生长,高浓度Ca、赤霉素抑制了花粉萌发和花粉管的生长。红花罂粟花粉萌发的最佳培养基是琼脂20g＋蔗糖110g＋硼酸110mg／L＋200mg／LCa^2＋＋80mg／LGA3。     

油菜花粉钙结合蛋白的研究

以油菜花粉(Brassica campestris)为材料纯化了钙调素(CaM),并得到一种新钙结合蛋白(NCBP)。经SDS-PAGE、PAGE和等电聚焦电泳(IEF)鉴定,这两种蛋白质均表现均一。CaM分子量为18.8kD, NCBP为16.3kD,等电点分别为3.6和4.2。研究证明花粉CaM具有与其他来源CaM所特有的性质,对环核苷酸磷酸二酯酶(简称PDE)有明显的激活作用,而花粉NCBP不明显。花粉CaM在PAGE和SDS-PAGE中电泳行为有Ca²⁺效应,而NCBP仅表现在PAGE中。经氨基酸组成分析表明两种蛋白质氨基酸组成不同,但均含有较多的酸性氨基酸,不含Trp, 含1个Cys。CaM和NCBP N-端均为封闭,CaM C-端为-Met-Ala-Lys-COOH。两种蛋白质具有不同的肽谱和CD谱。用DTNB修饰花粉CaM Cys残基,则激活PDE的能力明显下降。     

IAA对黄瓜花粉管伸长及花粉内源cAMP含量的影响

研究不同浓度的ＩＡＡ对黄瓜花粉管伸长及内源ｃＡＭＰ含量的影响。结果表明：５ｍｇ／ＬＩＡＡ明显促进花粉管伸长，而１００ｍｇ／ＬＩＡＡ则完全抑制花粉萌发。     

自交不亲和甘蓝的花粉萌发与花柱内保护酶活性变化

研究了柱头提取液对自交不亲和甘蓝花粉萌发、花粉管生长以及自花、异花授粉过程中花柱内过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)活性动态变化.结果表明:自花柱头提取液能显著抑制花粉萌发和花粉管的生长,而异花柱头提取液能提高花粉萌发率和花粉管的生长;授粉过程中花柱内SOD,POD和CAT活性都显著上升,而自花授粉上升幅度明显低于异花授粉.花柱内蛋白质含量在授粉过程中都会上升,但自花授粉在授粉后最初3～5min出现一个含量峰值.     

Involvement of heterotrimeric G protein in signal transduction of extracellular calmodulin in regulatingrbcS expression

The role of heterotrimeric G protein in signal transduction pathway of extracellular calmodulin in regulating rbcS expression was examined in suspension-cultured cells of transgenic tobacco. Pharmalogical experiments indicated that G protein agonist cholera toxin enhanced rbcS expression and heterotrimeric G protein antagonist pertussis toxin inhibited rbcS expression in transgenic tobacco cells. Pertussis toxin also inhibited the enhancement effect caused by exogenous purified calmodulin on rbcS expression, whereas cholera toxin completely reversed the inhibitory effects caused by anti-calmodulin serum on rbcS expression. The right side-out vesicles from tobacco cell membrane were purified, which contained all of substrates for fluometric assay of GTPase activity. Exogenous purified calmodulin, when adding directly to the medium of plasma membrane vesicles, significantly activated GTPase activity in the right side-out plasma membrane vesicles, and this increase in GTPase activity was completely inhibited both by heterotrimeric G proteins antagonist pertussis toxin and nonhy-drolyzable GTP analogs GMP-PCP. These results provided the evidence that heterotrimeric G proteins may be involved in signal transduction pathways of extracellular calmodulin to regulate rbcS gene expression.     

慈姑雌花在自然居群中的受粉过程--花粉粒落置、萌芽及花粉管生长的荧光显微观察

用荧光显微术观察了慈姑（Ｓａｇｉｔｔａｒｉａ ｔｒｉｆｏｌｉａ Ｌｉｎｎ）不同时期的雌花柱头上花粉粒的落置、萌发及花粉管的生长过程与途径。结果表明：该种雌花开花２ｈ 柱头受粉率为０,开花４ｈ 和８ｈ受粉率分别为５３８％ 和９８３％ ,柱头上花粉萌发率达１００％ ;花粉管沿雌蕊之向心一侧的组织中穿行,至子房基部后部分花粉管转向胚珠,由珠孔进入珠心。本研究发现该种花粉管常穿过子房基部至花托组织,并可进入其他雌蕊中,作者认为这种行为可能对保证雌花的结实率有一定意义     

紫薇受粉习性及花粉管生长的研究

用脱色苯胺蓝对紫薇花粉和花粉管进行染色，在荧光显微镜下观察其自然条件下的受粉习性及花粉管生长行为，以确定花粉管通道的形成时期．结果表明，盛花期的紫薇在开花后1.5h内完成受粉，花粉落量多，受粉率为100％；开花后24h左右进行受精；单果种子数为36～50粒，认为可以用花粉管通道法转化紫薇，导入外源DNA的最佳时机为开花后24h左右.     

Ca~（2＋）和亚精胺在莴苣种子萌发过程中的相互关系

莴苣（挂丝红品种）种子萌发过程中，内源亚精胺（Ｓｐｄ）含量逐渐增加．Ｃａ２＋通道抑制剂Ｃｏ２＋和Ｌ３＋、钙调素（ＣａＭ）拮抗剂氯两嗪（ＣＰＺ）处理抑制种子内源亚精胺含量的增加，Ｃａ２＋可部分逆转Ｃｏ２＋和Ｌａ３＋的抑制效应．外源亚精胺处理能部分逆转Ｃｏ２＋和Ｌａ３＋对萌发的抑制效应．表明Ｃａ２＋和ＣａＭ对种子萌发的调节作用与其对亚精胺含量的影响有关．     

盐胁迫对含笑花粉萌发的影响

以含笑(Micheliafigo)花粉为试验材料,采用花粉人工培养法研究了不同浓度NaCl对含笑花粉萌发特性的影响。结果表明:NaCl对含笑花粉萌发有抑制作用,且抑制程度随培养基中NaCl浓度的增大而加强,2 mmol·L-1NaCl就能显著(P<0.05)抑制含笑花粉萌发。耐盐程度分析表明,含笑花粉萌发的耐盐浓度、耐盐半致死浓度、耐盐极限浓度分别为1.86、15.80、38.11 mmol·L-1。     

Fertility analysis of the Arabidopsis transformed with antisense rice osRACD gene

The full length osRACD cDNA sequence was subcloned into the pBI121 plasmid in the antisense orientation under the control of the CaMV35S promoter to construct the expression vector pBID, and the constructs were introduced into Arabidopsis plants by using the vacuum infiltration method. The siliques of the transformants stopped growing after anthesis, and they turned yellow or died later; and the siliques from the control plants transformed by the pBI continued growing after anthesis and matured normally. In vitro pollen germination demonstrated that the growth and elongation process of the pollens of the transgenic plants was inhibited, the pollen tubes were shorter and slightly fatter than the tubes of the control plants, which grew normally with long cyclindrical tubes. The above results suggest the function of osRACD gene involved in regulation of the growth and elongation process of pollen tube, its encoding protein may be one of the important factors in regulation of fertility transition of the photoperiod-sensitive genic male-sterile rice Nongken 58S.     

Effects of nitrogen ion implantation on lily pollen germination and the distribution of the actin cytoskeleton during pollen germination

The effects of low energy nitrogen ion implantation on lily (Lilium davidii Duch.) pollen germination and the distribution of the actin cytoskeleton during pollen germination have been studied. Preliminary results showed that the ratio of pollen germination increased from (16.0± 1.6)% to (27.0±2.1)% when implanted with nitrogen ions by 100 keV and a dose of 10¹³ ions/cm². Further experiments were performed by staining the actin filaments in pollen with rhodamine-phalloidin and detected by using laser confocol microscopy. After hydration for 10 h, the actin filaments in ion implanted pollen grains tended to form thick bundles oriented in parallel or ring shape at the germinal furrow, indicating that the effect of nitrogen ion implantation on the germination of pollen might be mediated by reorganization of the actin cytoskeleton.     

芍药花粉离体萌发培养基组分的优化

以芍药花粉为材料,采用正交设计L^9（34）法研究蔗糖、硝酸钙、硼酸对芍药花粉离体萌发和花粉管生长的影响,筛选芍药花粉离体萌发最适宜的培养基,并研究了不同pH值对芍药花粉萌发的影响。结果表明,蔗糖对芍药花粉萌发率和花粉管生长影响最大,硼酸次之,硝酸钙影响最小;芍药花粉离体萌发最适宜培养基为,蔗糖20g·L^-1＋硼酸20mg·L^-1＋硝酸钙60mg·L^-1;其萌发率和花粉管长度分别为63．12％和183．33μm;适合芍药花粉萌发的pH为6～7。     

Effects of nitrogen ion implantation on Ca2+ concentration and membrane potential of pollen cell

The effects of low energy nitrogen ion implantation on Ca²⁺ concentration and membrane potential of lily (lilium davidii Duch) pollen cell have been studied. The results showed that the Ca²⁺ concentration was increased when pollen grain was implanted by nitrogen ion with energy 100 keV and dose 10¹³ ions/cm². However, the increase of Ca²⁺ concentration was partly inhibited by the addition of Ca²⁺ channel inhibitor depending on dose. And nitrogen ion implantation caused depolarization of pollen cell membrane potential. In other words, membrane potential was increased, but the effect decreased by adding Ca²⁺ channel inhibitor. However, it was still significantly higher than the membrane potential of control cells. It was indicated that the depolarization of cell membrane potential opened the calcium channel on the membrane that caused the increasing of intracellular calcium concentration. This might be an earlier step of the effect of low energy nitrogen ion implantation on pollen germination.     

External pH regulates the inward K+ channels in Brassica pollen protoplasts

Patch-clamp whole-cell recording techniques were used to investigate the regulation of the inward K* channels by external pH in Brassica pollen protoplasts. The inward K+ currents, conductance and voltage-independent maximum conductance were not significantly affected when the external pH was changed from 5.8 to 4.5. However, compared with the results obtained at pH 5.8, the inward K+ currents and conductance were significantly inhibited at pH 8 .5 . The activation kinetics of the inward K * channels was also significantly regulated by external pH. The channel activation time was shorter at the lower external pH than that at higher pH. Since external pH is a critical factor to regulate pollen germination, the results suggest that external pH regulation of the inward K+ channels may be one of potential physiological mechanisms for the regulation of pollen germination.     

Actin and myosin during pollen germination

Actin and myosin from pollen tubes of Lilium davidii were studied by using immunoblotting, Dot_Blot and myosin Ca 2+_ATPase analysis. On immunoblotting of the total soluble pollen tube proteins, anti_α_actin antibody labelled a polypeptide approximately 43 ku, which is considered to be the actin of lily. The mRNA encoding actin in ungerminated pollen and germinated pollen were both undetectable in our experiments. A myosin exhibited Ca 2+_ATPase activity, with a native molecular weight of 460 ku has been identified by using immunoblotting. A polypeptide of about 205 ku and a polypeptide of about 20 ku were the heavy chain and a set of light chain of the myosin, which can crossreact with anti_skeletal muscle myosin heavy chain monoclonal antibody and anti_skeletal muscle myosin light chain (20 ku) monoclonal antibody, respectively. The Ca 2+_ATPase activities of myosin in crude extracts of germinated pollen were positively related to the growth rates of pollen tubes.