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.

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.

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.

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.

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

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.

SEC1A and SEC6 synergistically regulate pollen tube polar growth

Pollen tube polar growth is a key physiological activity for angiosperms to complete double fertilization, which is highly dependent on the transport of polar substances mediated by secretory vesicles.The exocyst and Sec1/Munc18(SM) proteins are involved in the regulation of the tethering and fusion of vesicles and plasma membranes, but the molecular mechanism by which they regulate pollen tube polar growth is still unclear. In this study, we found that loss of function of SEC1A, a member of the SM protein family in Arabidopsis thaliana, resulted in reducing pollen tube growth and a significant increase in pollen tube width. SEC1A was diffusely distributed in the pollen tube cytoplasm, and was more concentrated at the tip of the pollen tube. Through coimmunoprecipitation-mass spectrometry screening,protein interaction analysis and in vivo microscopy,we found that SEC1A interacted with the exocyst subunit SEC6, and they mutually affected the distribution and secretion rate at the tip of the pollen tube. Meanwhile, the functional loss of SEC1A and SEC6 significantly affected the distribution of the SNARE(soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex member SYP125 at the tip of the pollen tube, and led to the disorder of pollen tube cell wall components. Genetic analysis revealed that the pollen tube-related phenotype of the sec1a sec6 double mutant was significantly enhanced compared with their respective single mutants. Therefore, we speculated that SEC1A and SEC6 cooperatively regulate the fusion of secretory vesicles and plasma membranes in pollen tubes, thereby affecting the length and the width of pollen tubes.

Arabidopsis pollen-specific glycerophosphodiester phosphodiesterase-like genes are essential for pollen tube tip growth

In angiosperms,pollen tube growth is critical for double fertilization and seed formation.Many of the factors involved in pollen tube tip growth are unknown.Here,we report the roles of pollenspecific GLYCEROPHOSPHODIESTER PHOSPHO DIESTERASE-LIKE(GDPD-LIKE)genes in pollen tube tip growth.Arabidopsis thaliana GDPD-LIKE6(At GDPDL6)and At GDPDL7 were specifically expressed in mature pollen grains and pollen tubes and green fluorescent protein(GFP)-At GDPDL6 and GFP-At GDPDL7 fusion proteins were enriched at the plasma membrane at the apex of forming pollen tubes.Atgdpdl6 Atgdpdl7 double mutants displayed severe sterility that was rescued by genetic complementation with At GDPDL6 or At GDPDL7.This sterility was associated with defective male gametophytic transmission.Atgdpdl6 Atgdpdl7 pollen tubes burst immediately after initiation of pollen germination in vitro and in vivo,consistent with the thin and fragile walls in their tips.Cellulose deposition was greatly reduced along the mutant pollen tube tip walls,and the localization of pollen-specific CELLULOSE SYNTHASE-LIKE D1(CSLD1)and CSLD4 was impaired to the apex of mutant pollen tubes.A rice pollen-specific GDPD-LIKE protein also contributed to pollen tube tip growth,suggesting that members of this family have conserved functions in angiosperms.Thus,pollen-specific GDPDLIKEs mediate pollen tube tip growth,possibly by modulating cellulose deposition in pollen tube walls.

Rice pollen-specific OsRALF17 and OsRALF19 are essential for pollen tube growth

Pollen tube growth is essential for successful double fertilization, which is critical for grain yield in crop plants. Rapid alkalinization factors(RALFs) function as ligands for signal transduction during fertilization. However, functional studies on RALF in monocot plants are lacking.Herein, we functionally characterized two pollen-specific RALFs in rice(Oryza sativa) using multiple clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated protein 9-induced loss-of-function mutants,peptide treatment, expression analyses, and tag reporter lines. Among the 41 RALF members in rice, OsRALF17 was specifically expressed at the highest level in pollen and pollen tubes.Exogenously applied OsRALF17 or OsRALF19peptide inhibited pollen tube germination and elongation at high concentrations but enhanced tube elongation at low concentrations, indicating growth regulation. Double mutants of OsRALF17 and OsRALF19(ralf17/19) exhibited almost full male sterility with defects in pollen hydration, germination, and tube elongation,which was partially recovered by exogenous treatment with OsRALF17 peptide. This study revealed that two partially functionally redundant OsRALF17 and OsRALF19 bind to Oryza sativa male-gene transfer defective 2(OsMTD2) and transmit reactive oxygen species signals for pollen tube germination and integrity maintenance in rice. Transcriptomic analysis confirmed their common downstream genes, in osmtd2 and ralf17/19. This study provides new insights into the role of RALF, expanding our knowledge of the biological role of RALF in regulating rice fertilization.

Acquisition of LURE-Binding Activity at the Pollen Tube Tip of Torenia fournieri

Pollen tube guidance is controlled by multiple complex interactions with the female tissues. Here, we show that pollen tubes of Torenia fournieri are regulated by a stylar tissue in a length-dependent manner to receive and respond to attractant LURE peptides secreted from synergid cells. We developed an immunostaining method to visualize LURE peptides bound at the plasma membrane of the tip region of the pollen tube. Using this method, we found that LURE peptides bound specifically to pollen tubes growing through a cut style. The peptides also bound to pollen tubes growing through a shorter style, which were not competent to respond to these peptides. These observations suggested a possibility that acquisition of the LURE peptide reception ability and acquisition of full competency are separable pro- cesses. RNA-Seq suggested that the transcription profile of pollen tubes was affected by both the length of the style and the cultivation period, consistently with physiological changes in binding activity and LURE response ability. The data- base generated from de novo RNA-Seq of Torenia pollen tubes was shown to be useful to identify pollen tube proteins by mass spectrometry. Our studies provide insight and an effective platform for protein identification to understand pollen tube guidance.

Screening for High-Temperature Tolerant Cotton Cultivars by Testing In Vitro Pollen Germination,Pollen Tube Growth and Boll Retention

With radical global climate change and global warming, high temperature stress has become one of major factors exerting a major Influence on crop production, In the cotton （Gossyplum hirsutum L.）-growlng areas of China, especially in the Yangtze River valley, unexpected periodic episodes of extreme heat stress usually occur In July and August, the peak time of cotton flowering and boll loading, resulting In lower boll set and lint yield. Breeding programs for screening high temperature-tolerant cotton germplasm and cultlvars are urgent In order to stabilize yield in the current and future warmer weather conditions. In the present study, 14 cotton cultivars were quantified for in vitro pollen germination and pollen tube growth in response to temperatures ranging from 10 to 50 ℃ at 5 ℃ intervals. Different cotton genotypes varied In their in vitro pollen germination and pollen tube length responses to the different temperatures. Maximum pollen germination and pollen tube length ranged from 25.2% to 56.2% and from 414 to 682 pro, respectively. The average cardinal temperatures （Tmin,, Topt, and Tmax） also varied among the 14 cultivars and were 11.8, 27.3, and 42.7 ℃ for pollen germination and 11.8, 27.8, and 44.1 ℃ for maximum pollen tube length. Variations In boll retention and boll numbers per plant in field experiments were found for the 14 cotton cultivars and the boll retention and boll retained per plant on 20 August varied considerably In different years according to weather conditions. Boll retention on 20 August was highly correlated with maximum pollen germination （R^2 = 0.84） and pollen tube length （R^2=0.64）. A screening method based on principle component analysis of the combination of pollen characterlatics In an in vitro experiment and boll retention testing In the field environment was used In the present study and, as a result, the 14 cotton cultlvars could be classified as tolerant, moderately tolerant, moderately susceptible and susceptible to high temperature.

The Calcineurin B-Like Ca2＋ Sensors CBL1 and CBL9 Function in Pollen Germination and Pollen Tube Growth in Arabidopsis

Ca2＋ has been established as an important second messenger regulating pollen germination and tube growth. However, to date, only a few signaling components have been identified to decode and relay Ca2＋ signals in growing pollen tubes. Here, we report a function for the calcineurin B-like （CBL） Ca2＋ sensor proteins CBL1 and CBL9 from Arabidopsis in pollen germination and tube growth. Both proteins are expressed in mature pollen and pollen tubes and impair pollen tube growth and morphology if transiently overexpressed in tobacco pollen. The induction of these phenotypes requires efficient plasma membrane targeting of CBL1 and is independent of Ca2＋ binding to the fourth EF-hand of CBL1. Overexpression of CBL1 or its closest homolog CBL9 in Arabidopsis renders pollen germination and tube growth hypersensitive towards high external K＋ concentrations while disruption of CBL1 and CBL9 reduces pollen tube growth under low K~ conditions. Together, our data identify a crucial function for CBL1 and CBL9 in pollen germination and tube growth and suggest a model in which both proteins act at the plasma membrane through regulation of K＋ homeostasis.

Application of Non-invasive Microsensing System to Simultaneously Measure Both H^＋ and O2 Fluxes Around the Pollen Tube

Various Ionic and molecular activities in the extraceUular environment are vital to plant cell physiological processes. A noninvasive microsensing system （NMS） based on either the scanning ion-selective electrode technique （SIET） or the scanning polarographlc electrode technique （SPET） is able to obtain information regarding the transportation of various Ions/molecules in Intact samples under normal physiological conditions. The two-probe simultaneous test system （2STS） Is an Integrated system composed of SIET, SPET, and a Xu-Kunkel sampling protocol. In the present study, 2STS was able to simultaneously measure fluxes of H^＋ and O2 of the Uly （Lillum Iongiflorum Thunb. cv. Ace） pollen tube while avoiding interference between the two probes. The results Indicate that the proton fluxes were effluxes, whereas the oxygen fluxes were Influxes, and they were closely correlated to each other surrounding the constitutive alkaline band region. Specifically, when the proton effluxes increased, the oxygen Influxes also increased. Therefore, the hypothesis of condensed active mitochondria existing in the alkalized area of the pollen tube proposed by Hepler＇s group is supported.

Targeting of Pollen Tubes to Ovules Is Dependent on Nitric Oxide （NO） Signaling

The guidance signals that drive pollen tube navigation inside the pistil and micropyle targeting are still, to a great extent, unknown. Previous studies in vitro showed that nitric oxide （NO） works as a negative chemotropic cue for pollen tube growth in lily （Lilium Iongiflorum）. Furthermore, Arabidopsis thaliana Atnosl mutant plants, which show de- fective NO production, have reduced fertility. Here, we focus in the role of NO in the process of pollen-pistil communication, using Arabidopsis in-vivo and lily semi-vivo assays. Cross-pollination between wild-type and Atnosl plants shows that the mutation affects the pistil tissues in a way that is compatible with abnormal pollen tube guidance. Moreover, DAF- 2DA staining for NO in kanadi floral mutants showed the presence of NO in an asymmetric restricted area around the micropyle. The pollen-pistil interaction transcriptome indicates a time-course-specific modulation of transcripts of AtNOS1 and two Nitrate Reductases （nrl and nr2）, which collectively are thought to trigger a putative NO signaling pathway. Semivivo assays with isolated ovules and lily pollen further showed that NO is necessary for micropyle targeting to occur. This evidence is supported by CPTIO treatment with subsequent formation of balloon tips in pollen tubes facing ovules. Activation of calcium influx in pollen tubes partially rescued normal pollen tube morphology, suggesting that this pathway is also dependent on Ca^2＋ signaling. A role of NO in modulating Ca^2＋ signaling was further substantiated by direct imaging the cytosolic free Ca^2＋ concentration during NO-induced re-orientation, where two peaks of Ca^2＋ occur--one during the slowdown/stop response, the second during re-orientation and growth resumption. Taken together, these results provide evidence for the participation of NO signaling events during pollen-pistil interaction. Of special relevance, NO seems to directly affect the targeting of pollen tubes to the ovule＇s micropyle by modulating the action of its diffusible factors.

Arabidopsis Galacturonosyltransferase （GAUT） 13 and GAUT14 Have Redundant Functions in Pollen Tube Growth

Cell wall biosynthesis is indispensable for pollen tube growth. Despite its importance to sexual reproduction, the molecular mechanisms of pollen tube wall biosynthesis remain poorly understood. Here, we report functional characterization of two putative Arabidopsis galacturonosyltransferase genes, GAUT13 and GAUT14, which are essential for pollen tube growth. GAUT13 and GAUT14 encode the proteins that share a high amino acid sequence identity and are located in the Golgi apparatus. The T-DNA insertion mutants, gaut13 and gaut14, did not exhibit any observable defects, but the gaut13 gaut14 double mutants were defective in pollen tube growth; 35.2-37.3% pollen tubes in the heterozygous double mutants were swollen and defective in elongation. The outer layer of the cell wall did not appear distinctly fibrillar in the double mutant pollen tubes. Furthermore, distribution of homogalacturonan labeled with JIM5 and JIM7 in the double mutant pollen tube wall was significantly altered compared to wild-type. Our results suggest that GAUT13 and GAUT14 function redundantly in pollen tube growth, possibly through participation in pectin biosynthesis of the pollen tube wall.

Influences of Cross Pollination on Pollen Tube Growth and Fruit Set in Zuili Plums (Prunus salicina)

Zuili plum （Prunus salicina L.） trees usually set fruit poorly, although they produce high quality fruit. To elucidate the causes of the poor fruit set, pollen tube growth into pistils and fruit set percentage were investigated after cross-, self- and open-pollination. Ovule development in Zuili pistils was also investigated. Pollen tube penetration into the ovules via the obturator and micropyle was best when Zuili pistils were pollinated by cv. Black Amber （P. domestica） pollen grains, although cross-pollinations with Hongxinli and Miili （P. salicina） pollen were more effective than self- and open-pollination. The fruit set percentage was also highest in pistils pollinated with Black Amber pollen grains. Morphological observation of Zuili pistils revealed that the trees produce ＂double pistils＂, developing two ovaries from a basal pistil, at a rate as high as 28%. In such abnormal pistils, most ovules were lacking an embryo sac or were entirely degenerated. The percentage of normally developed ovules was 24.3% and 8.9% in normal and double pistils, respectively. From these results, we conclude that the main causes of poor fruit set of Zuili plums are a lack of effective cross-pollination and the production of high percentages of double pistils in which normally developed ovules are scarcely formed.

Cytoskeleton in Pollen and Pollen Tubes of Ginkgo biloba L.

The distribution of F-actin and microtubules was investigated in pollen and pollen tubes of Ginkgo biloba L. using a confocal laser scanning microscope after fluorescence and immunofluorescence labeling. A dense F-actin network was found in hydrated Ginkgo pollen. When Ginkgo pollen was germinating, F-actin mesh was found under the plasma membrane from which the pollen tube would emerge. After pollen germination, F-actin bundles were distributed axially in long pollen tubes of G. biloba. Thick F-actin bundles and network were found in the tip of the Ginkgo pollen tube, which is opposite to the results reported for the pollen tubes of some angiosperms and conifers. In addition, a few circular F-actin bundles were found in Ginkgo pollen tubes. Using immunofluorescence labeling, a dense microtubule network was found in hydrated Ginkgo pollen under confocal microscope. In the Ginkgo pollen tube, the microtubules were distributed along the longitudinal axis and extended to the tip. These results suggest that the cytoskeleton may have an essential role in the germination of Ginkgo pollen and tube growth.

Regulation of Actin Dynamics in Pollen Tubes:Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.