Cdi gene is required for pollen germination and tube growth in Arabidopsis

Pollen germination and tube growth are of essential importance to sexual reproduction of flowering plants. Several biological processes including cell wall biosynthesis and modification are known to be involved in pollen tube growth, though the underlying molecular mechanisms remain largely to be investigated. Here we report the identification and functional characterization of the Arabidopsis gene Cdi, which encodes a putative nucleotide-diphospho-sugar transferase. Cdi is preferentially expressed in pollen grains and pollen tubes. Transient expression of Cdi:GFP fusion protein showed that CDI is localized in the cytosol. Mutation in Cdi impaired pollen germination and pollen tube growth thus leading to disrupted male transmission. These results suggest that Cdi is an essential gene required for pollen germination and tube growth.     

Auxin abolishes inhibitory effects of methylcyclopropen and amino oxyacetic acid on pollen grain germination,pollen tube growth,and the synthesis of ACC in petunia

As established by us earlier, ethylene behaves as a regulator of germination, development, and growth of male gametophyte during the progamic phase of fertilization. However, the mechanisms of the regulation of these processes remain so far unstudied. It is believed that the main factor providing variety of the ethylene responses is its interaction with other phytohormones. According to our working hypothesis, ethylene controls germination of pollen grains (PGs) and growth of pollen tubes (PTs) by interacting with auxin, which, as the available data indicate, is likely a key regulator of plant cell polarization and morphogenesis and one of the factors modulating the biosynthesis of ethylene at the level of ACC-synthase gene expression. In the present work, on germinating in vitro male gametophyte and the pollen-stigma system for petunia (Petunia hybrida L.) effects of phytohormones (ethylene and IAA) and known blockers repressing ethylene reception (1-methylcyclopropene, 1-MCP), the synthesis of ACC (amino oxyacetic acid, AOA) and transport IAA (triyodbenzoynaya acid, TYBA) on PGs germination, PTs growth and the synthesis of ACC were investigated. According to the data obtained, exogenous ethylene and IAA stimulated both PGs germination and PTs growth. 1-MCP and TYBA completely inhibited the first process, whereas IAA abolished the inhibitory action of 1-MCP and AOA on both the above processes. Etrel only partially weakened the inhibitory effect of TYBA. Examination of ACC synthesis modulation with AOA showed that IAA does not affect the level of ACC in germinating in vitro male gametophyte and nonpollinated stigmas, while this phytohormone insignificantly raised the level of ACC and abolished the inhibitory effect of AOA on its synthesis in the pollenstigma system. Pollination of stigmas with the pollen preliminarily treated with 1-MCP led to 2.5-fold decline in both the rate of PT growth and the level of ACC. At the same time, IAA abolished the inhibitory action of 1-MCP recovering the synthesis of ACC and growth of PTs to the control values. All these results, taken together, provide evidence for the interaction of the signal transduction pathways of ethylene and auxin at the level of ACC biosynthesis in the course of germination and growth of petunia male gametophyte during the progamic phase of fertilization.     

Gibberellins are required for seed development and pollen tube growth in Arabidopsis

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

Proteomic analyses of Oryza sativa mature pollen reveal novel proteins associated with pollen germination and tube growth

As a highly reduced organism, pollen performs specialized functions to generate and carry sperm into the ovule by its polarily growing pollen tube. Yet the molecular genetic basis of these functions is poorly understood. Here, we identified 322 unique proteins, most of which were not reported previously to be in pollen, from mature pollen of Oryza sativa L. ssp japonica using a proteomic approach, 23% of them having more than one isoform. Functional classification reveals that an overrepresentation of the proteins was related to signal transduction (10%), wall remodeling and metabolism (11%), and protein synthesis, assembly and degradation (14%), as well as carbohydrate and energy metabolism (25%). Further, 11% of the identified proteins are functionally unknown and do not contain any conserved domain associated with known activities. These analyses also identified 5 novel proteins by de novo sequencing and revealed several important proteins, mainly involved in signal transduction (such as protein kinases, receptor kinase-interacting proteins, guanosine 5'-diphosphate dissociation inhibitors, C2 domain-containing proteins, cyclophilins), protein synthesis, assembly and degradation (such as prohibitin, mitochondrial processing peptidase, putative UFD1, AAA+ ATPase), and wall remodeling and metabolism (such as reversibly glycosylated polypeptides, cellulose synthase-like OsCsLF7). The study is the first close investigation, to our knowledge, of protein complement in mature pollen, and presents useful molecular information at the protein level to further understand the mechanisms underlying pollen germination and tube growth.     

Arabidopsis FIMBRIN5, an actin bundling factor, is required for pollen germination and pollen tube growth

Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures.     

Signalling pathways in pollen germination and tube growth

Summary. Signalling is an integral component in the establishment and maintenance of cellular identity. In plants, tip-growing cells represent an ideal system to investigate signal transduction mechanisms, and among these, pollen tubes (PTs) are one of the favourite models. Many signalling pathways have been identified during germination and tip growth, namely, Ca²⁺, calmodulin, phosphoinositides, protein kinases, cyclic AMP, and GTPases. These constitute a large and complex web of signalling networks that intersect at various levels such as the control of vesicle targeting and fusion and the physical state of the actin cytoskeleton. Here we discuss some of the most recent advances made in PT signal transduction cascades and their implications for our future research. For reasons of space, emphasis was given to signalling mechanisms that control PT reorientation, so naturally many other relevant works have not been cited. Correspondence and reprints: Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal.     

Pollen germination and pollen tube growth in Fraxinus pennsylvanica

With regard to adaptation of green ash (Fraxinus pennsylvanica Marshall) to ecological conditions in Croatia, pollen germination and pollen tube length after 2, 4 and 6 hours were examined in vitro at 10, 15, 20 and 25°C during two years 2001 and 2002. Narrow leaved ash (F. angustifolia Vahl) pollen served as a control in 2002. The year, time and temperature, and the interaction between time and temperature were significant for both germination percentage and pollen tube length. Interactions year × temperature and year × time were significant for pollen tube length only. The highest germination percentage (17.86% in 2001 and 19.40% in 2002) of green ash pollen was at 15°C after 6 hours. The pollen tube length was greatest at 20°C (393.46 μm) in 2001 and 25°C (899.50 μm) in 2002 after 6 hours. Narrow leaved ash pollen had the highest germination percentage (19.22%) at 20°C after 6 hours and was significantly reduced at 25°C. The pollen tube length was greatest at 25°C (518.90 μm) after 6 hours. It can be concluded that green ash pollen has satisfactory germination in ecological conditions in Croatia and that the optimum temperature for pollen germination is higher than 20°C.     

Genetic variation for in vitro sesame pollen germination and tube growth

  In vitro pollen germination and tube length studies are valuable in elucidating mechanisms (germination capacity and rate, tube growth rate) possibly associated with genetic differences in male transmission. On each of two collection dates, the percentage germination and tube length of the binucleate pollen grains from five diverse sesame (Sesamum indicum L.) genotypes were determined at eight times (30, 60, 90, 120, 150, 180, 240, 300 min) after inoculation on a semisolid medium containing 10% (100 g l^-1) sucrose (C₁₂H₂₂O₁₁), 0.4% (4 g l^-1) purified agar (Fisher Lot 914409), 0.1% (1 g l^-1) calcium nitrate [Ca(NO₃)₂ ⋅ 4H₂O] and 0.01% (100 mg l^-1) boric acid (H₃BO₃). Before heating, the pH of the medium was adjusted to 7.0 with a 0.1 N potassium hydroxide (KOH) solution. Over the five genotypes, 5% germination was found 30 min after inoculation and a maximum of 37% germination 120 min after inoculation with no significant changes thereafter. As indicated by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which germination was initiated and maximum germination attained. Over all five genotypes, the tube length was 91 μm 30 min after inoculation, reaching a maximum of 1000 μm 300 min after inoculation. As shown by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which tube length was observed and the maximum tube length was attained. Little or no relationship between percent germination and tube length was observed among the genotypes. For both percent germination and tube length, the statistical significance of collection date and its interactions with genotype and time after inoculation indicated that environment in the form of collection date was also an influencing factor. These results indicated that genetic differences among genotypes were present for in vitro germination capacity, germination rate and tube growth rate and that these factors singly or in combination could alter male transmission of genetic elements. Received: 5 February 1997 / Accepted: 23 June 1997     

Effect of toluidine blue on pollen germination and pollen tube growth

Toluidine blue is known to induce gynogenic haploids in significant numbersin Populus]. Because the efficacy of a chemical in inducing gynogenesis depends largely on its effeot on pollen germination, on pollen tube growth, and on male gamete formation, the effect of toluidine blue (0, 1, 10 and 100 mgl-1) on these processes was studied in treated pistils ofSolatium nigrum (4 X), as well as on cultured pollen grains ofS. nigrum andTrigonella foenumgraecum. Irrespective of the time of application, toluidine blue (1 and 10 mg I-1) had no effect on pollen germination or pollen tube growth in pistils ofS. nigrum; at 100 mg I-1 it invariably inhibited both the processes. Almost similar responses were elicited by cultured pollen grains. InT. foenum-graecum toluidine blue had no effeot on pollen germination and suppressed tube growth. Gamete formation was inhibited, to various degrees, at all the concentrations tested; at 100 ing I-1 hardly any pollen tube showed gamete formation. Based on our results, and those on other systems, the potentiality of toluidine blue as an inducer of gynogenesis has been analysed.     

Membrane potential changes during pollen germination and tube growth

Using methods of quantitative fluorescent microscopy, we studied membrane potential changes during pollen germination and in growing pollen tubes. Two voltage-sensitive dyes were used, i.e., DiBAC₄(3), to determine the mean membrane potential values in pollen grains and isolated protoplasts, and Di-4-ANEPPS, to map the membrane potential distribution on the surfaces of the pollen protoplast and pollen tube. We have shown that the activation of the tobacco pollen grain is accompanied by the hyperpolarization of the vegetative cell plasma membrane by about 8 mV. Lily pollen protoplasts were significantly hyperpolarized (−108 mV) with respect to the pollen grains (−23 mV) from which they were isolated. We have found the polar distribution of the membrane potential along the protoplast surface and the longitudinal potential gradient along the pollen tube. In the presence of plasma membrane H⁺-ATPase inhibitor sodium orthovanadate (1 mM) or its activator fusicoccin (1 μM), the longitudinal voltage gradient was modified, but did not disappear. Anion channel blocker NPPB (40 μM) fully discarded the gradient in pollen tubes. The obtained results indicate the hyperpolarization of the plasma membrane during pollen germination and uneven potential distribution on the pollen grain and tube surfaces. An inhibitory analysis of the distribution of the potential in the tube has revealed the involvement of the plasma membrane H⁺-ATPase and anion channels in the regulation of its value.     

Serine esterases are required for pollen tube penetration of the stigma in Brassica

. We have investigated the diversity of serine esterases in pollen and stigma tissues of Brassica napus and the role of these enzymes in pollen germination and pollen tube penetration of the stigma. The serine esterase-specific inhibitor diisopropyl fluorophosphate was used as a probe in a tritiated form, [³H]-DIPF, to determine the number and diversity of serine esterases in crude protein extracts from pollen and stigma. Seven serine esterases were identified in pollen and at least seven serine esterases were identified in stigma. The most abundant enzymes had molecular weights of 30-50 kDa. In the pollen extract a serine esterase was detected with the same molecular weight, 22 kDa, as an esterase previously shown to be a cutinase. Only one serine esterase (40 kDa) appeared to be shared between pollen and stigma extracts. Butyrate esterase activity in pollen and stigma extracts was assayed using p-nitrophenyl butyrate (PNB), an ester substrate frequently used in 'cutinase' assays. Total PNBase activity in pollen and stigma extracts was shown to be significantly reduced by the serine esterase inhibitors DIPF and ebelactone B. When DIPF and ebelactone B were applied to stigmas prior to pollination, pollen germination was not significantly affected but, at the highest inhibitor concentrations, up to 70% of germinating pollen tubes failed to penetrate the stigma surface. These data demonstrate that serine esterases, most probably cutinase(s), are required for pollen tube penetration of the dry cuticularised Brassica stigma.     

Reversible protein tyrosine phosphorylation affects pollen germination and pollen tube growth via the actin cytoskeleton

Summary. Phenylarsine oxide (PAO) and genistein are two well-known specific inhibitors of tyrosine phosphatases and kinases, respectively, that have been used in the functional analysis of the status of protein phosphotyrosine in different cell types. Our experiments showed that both PAO and genistein arrested pollen germination and pollen tube growth and led to the malformation of the pollen tubes, although genistein had a lesser effect. The malformations of the pollen tubes caused by PAO and genistein were, however, quite different. In addition, it was found that the rate of pollen germination and tube growth recovered to a certain extent when phalloidin was present during PAO treatment, but not when it was present during genistein treatment. Furthermore, PAO treatment also had a great effect on the dynamic organization of filamentous actin in the pollen grain and pollen tube, while genistein only caused reorganization of actin at the turning point of the pollen tube. Our results suggest that reversible protein tyrosine phosphorylation is a crucial step in pollen germination and pollen tube growth, but that tyrosine kinases and phosphatases may have different effects which may function through the reorganization of the actin cytoskeleton. Correspondence and reprints: Key Laboratory of Cell Proliferation and Regulation Biology of the Ministry of Education, College of Life Science, Beijing Normal University, Beijing 100875, People’s Republic of China.     

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。     

Latrunculin B has different effects on pollen germination and tube growth

The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton.     

Free IAA in stigmas and styles during pollen germination and pollen tube growth of Nicotiana tabacum

Although many studies have emphasized the importance of auxin in plant growth and development, the thorough understanding of its effect on pollen–pistil interactions is largely unknown. In this study, we investigated the role of free IAA in pollen–pistil interactions during pollen germination and tube growth in Nicotiana tabacum L. through using histo and subcellular immunolocalization with auxin monoclonal antibodies, quantification by HPLC and ELISA together with GUS staining in DR5::GUS -transformed plants. The results showed that free IAA in unpollinated styles was higher in the apical part and basal part than in the middle part, and it was more abundant in the transmitting tissue (TT). At the stage of pollen germination, IAA reached its highest content in the stigma and was mainly distributed in TT. After the pollen tubes entered the styles, the signal increased in the part where pollen tubes would enter and then rapidly declined in the part where pollen tubes had penetrated. Subcellular localization confirmed the presence of IAA in TT cells of stigmas and styles. Accordingly, a schematic diagram summarizes the changing pattern of free IAA level during flowering, pollination and pollen tube growth. Furthermore, we presented evidence that low concentration of exogenous IAA could, to a certain extent, facilitate in vitro pollen tube growth. These results suggest that IAA may be directly or indirectly involved in the pollen–pistil interactions. Additionally, some improvements of the IAA immunolocalization technique were made.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains containing either theWx,wx,Su ₁,Su ₁,Sh ₂orsh ₂alleles were stored for 0, 1, 2, 3, 4 or 5 days at 2 °C. After each storage period, a portion of each genotype was cultured on a 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid medium, while another portion was placed on receptive silks, the number of kernels produced being a measure of fertilization ability. Regardless of the allele present in the pollen grain, 1 day of storage greatly increased the germination percentage and significantly increased pollen tube length. After 4 days of storage, there was noin vitro germination but some fertilization ability was found. The experiment was designed so that comparisons free from genetic background effects could be made between alleles at each locus. Significant differences at each storage period and a differential response to storage were obtained at some loci for germination percentage, ruptured percentage, pollen tube length and fertilization ability. A relationship between dominance of the allele and response to storage was detected only for fertilization ability. Since alleles at these loci affect the biochemical composition of pollen grains containing them, the results suggest that differences inin vitro germination characteristics and fertilization ability may be associated with biochemical composition.Journal Series Paper No. 3950, Florida Agricultural Experiment Station.     

In vitro germination and pollen tube growth of maize (Zea mays L.) pollen

Summary Pollen grains from two hybrids, WF9xH55 (W) and K64xK55 (K) were collected and a sample from each was cultured immediately (0 h). The remainder was subdivided and stored at 2, 20, and 35° C. At 3, 6, 12, 24, 36, 48, 72, 96, and 120 h, a sample was cultured. The culture medium contained 15% sucrose, 0.6% bacto-agar, 0.03% calcium nitrate and 0.01% boric acid. Storage at 2° C resulted in a large increase in germination percentage in both W and K reaching a maximum at 24 h and then slowly decreasing with additional storage. No germination was observed at 96 h with W and at 120 h with K. The complete loss of germination occurred during a 24 h period and was very abrupt. At 20° C, a similar but less pronounced pattern was observed. However, after 24 h, aggregates of 50–1 000 pollen grains developed during storage in both W and K. Storage of W at 35° C slightly decreased the germination percentage at 3 h and eliminated it at 6 h. Storage of K at 35° C substantially increased the germination percentage at 3 h with further increases in storage periods resulting in the aggregation of grains. This general pattern of an increase at shorter storage periods followed by a gradual decrease as the storage period was extended, was found for pollen tube length and growth rate. In vitro germination characteristics can be substantially altered by the temperature and length of storage and the response to storage is associated with pollen source.Journal Series Paper No. 4566, Florida Agricultural Experiment Station.