Calcium ionophore A 23187 affects localized wall secretion in the tip region of pollen tubes of Lilium longiflorum

The effects of the calcium inonophore A 23187 on growing pollen tubes of Lilium longiflorum Thunb. cv. Ace were investigated with the light and electron microscope. Tip growth is slowed down and stopped within 20 min after application of 5x10^-5 M ionophore A 23187. The main effects are the disappearance of the clear zone at the pollen tube tip and a thickening of the cell wall at the tip and at the pollen tube flanks. This effect on cell wall formation is confirmed under the electron microscope: The vesicular zone in treated pollen tubes is reduced, numerous vesicular contents are irregularly integrated in the pollen tube wall not only in the tip, but over a long distance of the pollen tube wall. In addition, effects on mitochondria and dictyosomes are observed. These results are interpreted as a disorientation of the Ca²⁺-based orientation mechanism of exocytosis after equilibration of the Ca²⁺-gradient     

Visualization of the Ca2+-gradient in growing pollen tubes ofLilium longiflorum with chlorotetracycline fluorescence

Summary Epifluorescence microscopy with chlorotetracycline (CTC) fluorescence was used to visualize the Ca²⁺ distribution in germinating pollen grains and growing pollen tubes ofLilium longiflorum. The intensity of the fluorescence shows a gradient with the highest fluorescence at the growing tip. Added external Ca²⁺ influences the intensity of the gradient in germinating grains. Ionophore A 23187 treated pollen tubes do not show the fluorescence gradient with CTC. These results are interpreted as evidence for the role of a Ca²⁺ gradient in pollen tube tip growth.     

Broad-range effects of ionophore X-537A on pollen tubes of Lilium longiflorum

The effects of the broad-range cationophore X-537A on pollen tubes of Lilium longiflorum were investigated, using both light and electron microscopy. Pollen tube growth is completely inhibited within 30 min after the application of 5·10^-5 M ionophore X-537A; cytoplasmic streaming is stopped only after 60 min of ionophore treatment. Ultrastructurally, X-537A effects are a vacuolation of Golgi cisternae and a general vacuolation. The wall is thickened at the very tip. Coated vesicles and coated regions are enriched close to and at the plasma membrane. The results indicate that pollen tube tip growth needs a specific ion distribution.Abbreviations CTC chlorotetracycline - DMSO dimethylsulfoxide     

The 135 kDa actin-bundling protein fromLilium longiflorum pollen is the plant homologue of villin

Summary Actin microfilaments, which are essential for cell growth and cytoplasmic streaming in pollen tubes, are closely dependent on actin-binding proteins for their organization and regulation. We have purified the plant 135 kDa actin-bundling protein (P-135-ABP) fromLilium longiflorum pollen and determined that its amino acid composition is highly similar to members of the villin-gelsolin family of proteins. We used antibodies against P-135-ABP to probe an expression cDNA library ofL. longiflorum pollen and isolated a full-length clone (ABP135) that corresponds to a 106 kDa polypeptide. The deduced amino acid sequence ofABP135 shows homology with members of the villin-gelsolin family of proteins and contains the characteristic six repeats of this family, as well as an extended carboxy-terminal domain that includes the villin headpiece preceded by a highly variable region. Using two-dimensional polyacrylamide gel electrophoresis we detected at least 5 isoforms of P-135-ABP, with isoelectric points (pI) ranging between 5.6 to 5.9. The most abundant P-135-ABP isoform has a pI of 5.8, closely approximating the pI predicted from the deducedABP135 amino acid sequence. These data, together with the partial amino acid sequence from a proteolytic peptide of the protein, indicate that P-135-ABP is a plant villin. Immuno-detection of Lilium villin in rapidly frozen pollen tubes localized it to actin bundles. Lilium villin is also ubiquitously expressed in all tissues tested. Since villins, like gelsolins, are also Ca²⁺-dependent severing, capping, and nucleating proteins, Lilium villin may participate in F-actin fragmentation and nucleation in the apex of the pollen tube where there is steep Ca²⁺ gradient.Abbreviations BMM butyl methyl-methacrylate - PPI polyphos-phoinositides - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

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.     

Ca2+-induced fragmentation of actin filaments in pollen tubes

Summary To control the intracellular free Ca²⁺ concentration from the cell exterior, pollen tubes ofLilium longiflorum were treated with a Ca²⁺ ionophore, A23187. Cytoplasmic streaming was inhibited when the free Ca²⁺ concentration of the external medium ([Ca²⁺]) was raised to 5×10^–6 M or higher. At [Ca²⁺] below 1×10^–6 M, the rhodamine-phalloidin stained actin filaments appeared straight and thin. However, at [Ca²⁺] which inhibited cytoplasmic streaming, the actin filaments appeared fragmented. In pollen tubes, Ca²⁺ regulation of cytoplasmic streaming may be linked not only to myosin (Shimmen 1987) but also to actin.Abbreviations ATP adenosine-5-triphosphoric acid - [Ca²⁺] concentration of free Ca²⁺ - EGTA ethyleneglycol-bis-(-aminoethylether)N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - Rh-ph rhodamine-conjugated phalloidin     

Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum

Summary Pollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium²⁺-concentration was obtained using the intensity ratio R=I₃₄₀/I₃₆₀. The analysis exhibits a [Ca²⁺] of nearly 10^–7mol·l^–1 in the tip region and about 2·10^–8 mol·l^–1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length.     

Release of an acid phosphatase activity during lily pollen tube growth involves components of the secretory pathway

Summary. An acid phosphatase (acPAse) activity was released during germination and tube growth of pollen of Lilium longiflorum Thunb. By inhibiting components of the secretory pathway, the export of the acPase activity was affected and tube growth stopped. Brefeldin A (1 μM) and cytochalasin D (1 μM), which block the production and transport of secretory vesicles, respectively, inhibited the acPase secretion. The Ca²⁺ channel blocker gadolinium (100 μM Gd³⁺) also inhibited acPase secretion and tube growth, whereas 3 mM caffeine, another Ca²⁺ uptake inhibitor, stimulated the acPase release, while tube growth was inhibited. The Yariv reagent (β-D-glucosyl)₃ Yariv phenylglycoside stopped tube growth by binding to arabinogalactan proteins of the tube tip cell wall but did not affect acPase secretion. A strong correlation between tube growth and acPase release was detected. The secreted acPase activity had a pH optimum at pH 5.5, a K _M of 0.4 mM for p-nitrophenyl phosphate, and was inhibited by zinc, molybdate, phosphate, and fluoride ions, but not by tartrate. In electrophoresis gels the main acPase activity was detected at 32 kDa. The conspicuous correlation between activity of the secretory pathway and acPase secretion during tube elongation strongly indicates an important role of the acPase during pollen tube growth and the secreted acPase activity may serve as a useful marker enzyme assay for secretory activity in pollen tubes Received July 25, 2001 Accepted January 15, 2002     

K+ Channels in the Plasma Membrane of Lily Pollen Protoplasts

Using the patch-clamp technique K⁺ channels could be observed in the plasma membrane of protoplasts from pollen grains of Lilium longiflorum. With depolarizing membrane potentials the open probability of the different K⁺ channels increased. Two K⁺ channel populations occurring occasionally had a single channel conductance of 120 pS and 42 pS, respectively. The most often observed K⁺ channel had a single channel conductance of 19 pS which showed an increase of channel activity with increasing free cytoplasmic Ca²⁺ concentration. This channel population might be involved in the pathway of endogenous transcellular K⁺ currents which are activated during pollen tube tip extension.     

Identification and Characterization of Higher Plant Myosins Responsible for Cytoplasmic Streaming

in vitro using these myosins and of localization studies using antiserum raised against each heavy chain, we suggested that both myosins are molecular motors for generating the motive force for cytoplasmic streaming in higher plant cells. The 170-kDa myosin is expressed not only in somatic cells but also in germinating pollen. In contrast, the 175-kDa myosin is distributed only in somatic cells. In the tip region of growing pollen tubes, it has been demonstrated that a tip-focused Ca²⁺ gradient is indispensable for growth and tube orientation. Cytoplasmic streaming in this region has been shown to be inactivated by high concentrations of Ca²⁺. The motile activity in vitro of 170-kDa myosin is suppressed by low (μM) levels of Ca²⁺ through its CaM light chain, suggesting that this suppression is one of the mechanisms for inactivating cytoplasmic streaming near the tip region of pollen tubes. The motile activity in vitro of 175-kDa myosin is also inhibited by Ca²⁺ at concentrations higher than 10⁻⁶M. It has been revealed that the elevation of cytosolic Ca²⁺ concentrations causes the cessation of cytoplasmic streaming even in somatic cells. Therefore, Ca²⁺-sensitivity of the motile activity of myosin appears to be a general molecular basis for Ca²⁺-induced cessation of cytoplasmic streaming. Received 6 September 2000/ Accepted in revised form 7 October 2000     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.     

Demonstration of polar zinc distribution in pollen tubes ofLilium longiflorum with the Heidelberg proton microprobe

Summary The zinc distribution in pollen tubes ofLilium longiflorum was analysed by proton-induced X-ray emission (PIXE) with the Heidelberg proton microprobe. A very high zinc concentration was measured in the pollen tube tip region.On leave from Shanghai Institute of Nuclear Research, Academia Sinica, Shanghai, China     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Boron deficiency alters cytosolic Ca2+ concentration and affects the cell wall components of pollen tubes in Malus domestica

• Boron (B) is essential for normal plant growth, including pollen tube growth. B deficiency influences various physiological and metabolic processes in plants. However, the underlying mechanism of B deficiency in pollen tube growth is not sufficiently understood. In the present research, the influence of B deficiency on apple (Malus domestica) pollen tube growth was studied and the possible regulatory mechanism evaluated.
• Apple pollen grains were cultured under different concentrations of B. Scanning ion‐selective electrode technique, fluorescence labelling and Fourier‐transform infrared (FTIR) analysis were used to detect calcium ion flux, cytosolic Ca²⁺ concentration ([Ca²⁺]cyt), actin filaments and cell wall components of pollen tubes.
• B deficiency inhibited apple pollen germination and induced retardation of tube growth. B deficiency increased extracellular Ca²⁺ influx and thus led to increased [Ca²⁺]cyt in the pollen tube tip. In addition, B deficiency modified actin filament arrangement at the pollen tube apex. B deficiency also altered the deposition of pollen tube wall components. Clear differences were not observed in the distribution patterns of cellulose and callose between control and B deficiency treated pollen tubes. However, B deficiency affected distribution patterns of pectin and arabinogalactan proteins (AGP). Clear ring‐like signals of pectins and AGP on control pollen tubes varied according to B deficiency. B deficiency further decreased acid pectins, esterified pectins and AGP content at the tip of the pollen tube, which were supported by changes in chemical composition of the tube walls.
• B appears to have an active role in pollen tube growth by affecting [Ca²⁺]cyt, actin filament assembly and pectin and AGP deposition in the pollen tube. These findings provide valuable information that enhances our current understanding of the mechanism regulating pollen tube growth.

     

Uncoupling secretion and tip growth in lily pollen tubes: evidence for the role of calcium in exocytosis

Cytoplasmic calcium concentration ([Ca²⁺]_i) and extracellular calcium (Ca²⁺_o) influx has been studied in pollen tubes of Lilium longliflorum in which the processes of cell elongation and exocytosis have been uncoupled by use of Yariv phenylglycoside ((β-D-Glc)₃). Growing pollen tubes were pressure injected with the ratio dye fura-2 dextran and imaged after application of (β-D-Glc)₃, which binds arabinogalactan proteins (AGPs). Application of (β-D-Glc)₃ inhibited growth but not secretion. Ratiometric imaging of [Ca²⁺]_i revealed an initial spread in the locus of the apical [Ca²⁺]_i gradient and substantial elevations in basal [Ca²⁺]_i followed by the establishment of new regions of elevated [Ca²⁺]_i on the flanks of the tip region. Areas of elevated [Ca²⁺]_i corresponded to sites of pronounced exocytosis, as evidenced by the formation of wall ingrowths adjacent to the plasma membrane. Ca²⁺_o influx at the tip of (β-D-Glc)₃-treated pollen tubes was not significantly different to that of control tubes. Taken together these data indicate that regions of elevated [Ca²⁺]_i, probably resulting from Ca²⁺_o influx across the plasma membrane, stimulate exocytosis in pollen tubes independent of cell elongation.     

Addition of Phenylboronic Acid to Malus domestica Pollen Tubes Alters Calcium Dynamics,Disrupts Actin Filaments and Affects Cell Wall Architecture

A key role of boron in plants is to cross-link the cell wall pectic polysaccharide rhamnogalacturonan-II (RG-II) through borate diester linkages. Phenylboronic acid (PBA) can form the same reversible ester bonds but cannot cross-link two molecules, so can be used as an antagonist to study the function of boron. This study aimed to evaluate the effect of PBA on apple (Malus domestica) pollen tube growth and the underlying regulatory mechanism. We observed that PBA caused an inhibition of pollen germination, tube growth and led to pollen tube morphological abnormalities. Fluorescent labeling, coupled with a scanning ion-selective electrode technique, revealed that PBA induced an increase in extracellular Ca²⁺ influx, thereby elevating the cytosolic Ca²⁺ concentration [Ca²⁺]c and disrupting the [Ca²⁺]c gradient, which is critical for pollen tube growth. Moreover the organization of actin filaments was severely perturbed by the PBA treatment. Immunolocalization studies and fluorescent labeling, together with Fourier-transform infrared analysis (FTIR) suggested that PBA caused an increase in the abundance of callose, de-esterified pectins and arabinogalactan proteins (AGPs) at the tip. However, it had no effect on the deposition of the wall polymers cellulose. These effects are similar to those of boron deficiency in roots and other organs, indicating that PBA can induce boron deficiency symptoms. The results provide new insights into the roles of boron in pollen tube development, which likely include regulating [Ca²⁺]c and the formation of the actin cytoskeleton, in addition to the synthesis and assembly of cell wall components.     

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     

Involvement of a Ca2+-calmodulin interaction in the yeast-mycelial (Y-M) transition of Candida albicans

A yeast-mycelium (Y-M) transition of Candida albicans (3153A) was induced by 1.5 mM CaCl₂ · 2H₂O in defined liquid medium, pH 7, at 25 °C. Germ tube formation was detected after approximately 8 h and peaks of maximum germination occurred at approximately 20 h in all experimental treatments. Non-toxic concentrations of the calmodulin inhibitor R24571 almost completely suppressed germ tube formation whereas trifluoperazine (TFP) and the Ca²⁺ ionophore A23187 were only about half as effective. Further Ca²⁺ addition failed to reverse the inhibitory effect of R24571 and induced only about 10% of the cells inhibited by TFP or A23187 to germinate.     

Pollen tube NAD(P)H oxidases act as a speed control to dampen growth rate oscillations during polarized cell growth

Reactive oxygen species (ROS) produced by NAD(P)H oxidases play a central role in plant stress responses and development. To better understand the function of NAD(P)H oxidases in plant development, we characterized the Arabidopsis thaliana NAD(P)H oxidases RBOHH and RBOHJ. Both proteins were specifically expressed in pollen and dynamically targeted to distinct and overlapping plasma membrane domains at the pollen tube tip. Functional loss of RBOHH and RBOHJ in homozygous double mutants resulted in reduced fertility. Analyses of pollen tube growth revealed remarkable differences in growth dynamics between Col–0 and rbohh–1 rbohj–2 pollen tubes. Growth rate oscillations of rbohh–1 rbohj–2 pollen tubes showed strong fluctuations in amplitude and frequency, ultimately leading to pollen tube collapse. Prior to disintegration, rbohh–1 rbohj–2 pollen tubes exhibit high‐frequency growth oscillations, with significantly elevated growth rates, suggesting that an increase in the rate of cell‐wall exocytosis precedes pollen tube collapse. Time‐lapse imaging of exocytic dynamics revealed that NAD(P)H oxidases slow down pollen tube growth to coordinate the rate of cell expansion with the rate of exocytosis, thereby dampening the amplitude of intrinsic growth oscillations. Using the Ca²⁺ reporter Yellow Cameleon 3.6, we demonstrate that high‐amplitude growth rate oscillations in rbohh–1 rbohj–2 pollen tubes are correlated with growth‐dependent Ca²⁺ bursts. Electrophysiological experiments involving double mutant pollen tubes and pharmacological treatments also showed that ROS influence K⁺ homeostasis. Our results indicate that, by limiting pollen tube growth, ROS produced by NAD(P)H oxidases modulate the amplitude and frequency of pollen tube growth rate oscillations.     

Annexin5 Plays a Vital Role in Arabidopsis Pollen Development via Ca2+-Dependent Membrane Trafficking

The regulation of pollen development and pollen tube growth is a complicated biological process that is crucial for sexual reproduction in flowering plants. Annexins are widely distributed from protists to higher eukaryotes and play multiple roles in numerous cellular events by acting as a putative “linker” between Ca²⁺ signaling, the actin cytoskeleton and the membrane, which are required for pollen development and pollen tube growth. Our recent report suggested that downregulation of the function of Arabidopsis annexin 5 (Ann5) in transgenic Ann5-RNAi lines caused severely sterile pollen grains. However, little is known about the underlying mechanisms of the function of Ann5 in pollen. This study demonstrated that Ann5 associates with phospholipid membrane and this association is stimulated by Ca²⁺ in vitro. Brefeldin A (BFA) interferes with endomembrane trafficking and inhibits pollen germination and pollen tube growth. Both pollen germination and pollen tube growth of Ann5-overexpressing plants showed increased resistance to BFA treatment, and this effect was regulated by calcium. Overexpression of Ann5 promoted Ca²⁺-dependent cytoplasmic streaming in pollen tubes in vivo in response to BFA. Lactrunculin (LatB) significantly prohibited pollen germination and tube growth by binding with high affinity to monomeric actin and preferentially targeting dynamic actin filament arrays and preventing actin polymerization. Overexpression of Ann5 did not affect pollen germination or pollen tube growth in response to LatB compared with wild-type, although Ann5 interacts with actin filaments in a manner similar to some animal annexins. In addition, the sterile pollen phenotype could be only partially rescued by Ann5 mutants at Ca²⁺-binding sites when compared to the complete recovery by wild-type Ann5. These data demonstrated that Ann5 is involved in pollen development, germination and pollen tube growth through the promotion of endomembrane trafficking modulated by calcium. Our results provide reliable molecular mechanisms that underlie the function of Ann5 in pollen.