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 ...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.展开更多
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 c...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.展开更多
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 che...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.展开更多
In angiosperms, initiation of ovule enlargement represents the start of seed development, the molecular mechanism of which is not yet elucidated. It was previously reported that pollen tube contents, rather than doubl...In angiosperms, initiation of ovule enlargement represents the start of seed development, the molecular mechanism of which is not yet elucidated. It was previously reported that pollen tube contents, rather than double fertilization, can trigger ovule enlargement. However, it remains unclear whether the signal(s) to trigger the initiation of ovule enlargement are from the sperm cells or fromthe pollen tubes. Recently, we identified a mutant dropl- drop2-, which produces pollen tubes with no sperm cells. Taking advantage of this special genetic material, we conducted pollination assays, and found that the ovules pollinated with dropl- drop2- pollen could initiate the enlargement and exhibited significant enlarged sizes at 36h after pollination in comparison with those unpollinated ovules. However, the sizes of the ovules pollinated with drops- drop2- pollen are significantly smaller than those of the ovules pollinated with wildtype pollen. These results demonstrate that the pollen tube, rather than the sperm cells, release the signal to trigger the initiation of ovule enlargement, and that double fertilization is required for further enlargement of the seeds.展开更多
The Arabidopsis phosphoinositide kinases PI4Kβ1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kβ1 and PIP5K5 catalyze key consecuti...The Arabidopsis phosphoinositide kinases PI4Kβ1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kβ1 and PIP5K5 catalyze key consecutive steps of phosphoinositide conversion, and it appears obvious that phosphatidylinositol-4-phosphate formed by PI4Kβ1 might act as a substrate for phosphatidylinositol-4,5-bisphosphate formation by PIP5K5. However, this hypothesis has not been experimentally addressed and distinct localization patterns of PI4Kβ1, PIP5K5, and also PI-synthases (PIS) generating phosphatidylinositol suggest additional complexity. Here, the synergistic functionality of enzymes of phosphoinositide conversion was assessed. In tobacco and Arabidopsis pollen tubes, phosphoinositides influence the apical secretion of pectin, and increased pectin deposition results in characteristic morphological alterations. Catalytically active and dominant negative variants of PI4Kβ1 and PIP5K5 were systematically co-expressed in tobacco pollen tubes and the incidence of morphologies related to enhanced pectin secretion was evaluated. The data support a proposed functional interplay of PI4Kβ1 and PIP5K5 at the trans-Golgi network, mediating directional vesicle trafficking. Co-expression experiments additionally including PIS isoforms, PIS1 or PIS2, indicate that pectin secretion is synergistically mediated by PI4Kβ1 and PIPSK5 acting on Ptdlns formed by PIS2 rather than PIS1. Possible ramifications for the preferential channeling of phosphoinositide intermediates between particular isoforms of PI pathway enzymes are discussed.展开更多
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-act...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.展开更多
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 ...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.展开更多
Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some...Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell mem- brane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.展开更多
Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and i...Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.展开更多
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 responsi...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.展开更多
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 se...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.展开更多
The success of sexual plant reproduction is directly influenced by specific interactions between the pollen and pistil.Light,fluorescence and scanning electron microscopy techniques were used to evaluate the steps of ...The success of sexual plant reproduction is directly influenced by specific interactions between the pollen and pistil.Light,fluorescence and scanning electron microscopy techniques were used to evaluate the steps of pollination in sour passion fruit plants(Passiflora edulis Sims).In the compatible interaction,pollen tubes grow through stigma projections towards the ovary.The pollen grain surface was found to be spheroidal and to consist of heteroreticulate exine with six colpi.Furthermore,analysis in vivo of pollenpistil interactions indicated that stigmas of flowers 24 hours before anthesis are unable to discriminate compatible(genetically unrelated)and incompatible(genetically related)pollen grains.Taken together,these results provide insight into the cellular mechanisms underlying pollination in passion fruit plants.展开更多
SWEET transporters play a pivotal role in sugar transport in plants.However,their functions in pollen tube growth,especially in coniferous species remain unknown.Here,we used RT-qPCR to reveal that a SWEET1 gene was s...SWEET transporters play a pivotal role in sugar transport in plants.However,their functions in pollen tube growth,especially in coniferous species remain unknown.Here,we used RT-qPCR to reveal that a SWEET1 gene was specifically expressed in pollen and pollen tubes of Picea wilsonii.A pollen germination assay showed that PwSWEET1 was induced by H3BO3 but not by Ca^2+.In a sugar specificity experiment,sucrose(Suc)and glucose(Glc)were effective sugars for pollen germination and pollen tube growth.PwSWEET1 expression was induced most by Suc and Glc.Heterologous expression of PwSWEET1 in yeast showed that PwSWEET1 can restore the glucose absorption in yeast strain EBY.VW4000,which has a hexose absorption defect,and the absorption of glucose is pH-independent.This evidence supports the involvment of PwSWEET1 in boron-dependent glucose transport in pollen germination and pollen tube growth of Picea wilsonii.展开更多
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 s...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展开更多
We examined the influence of storage time on germinability and tube growth of freeze stored pollens collected from 25 wild male plants in Actinidia eriantha variety. Pollens were stored in freezer at - 20°C ...We examined the influence of storage time on germinability and tube growth of freeze stored pollens collected from 25 wild male plants in Actinidia eriantha variety. Pollens were stored in freezer at - 20°C for six months and one year periods to determine changing at germinability in time. In vitro germination was conducted in certain cultural medium defined for Actinidia genus. The results showed that the germination percentages and tube lengths of genotypes decreased at the end of storage period. MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72, MH74, MH55 and MH61 genotypes were evaluated as vigor genotypes, because they maintained their viability and germination capability displaying statistically insignificant decreasing although their tube lengths significantly decreased except MH67. This investigation provided to determine some robust wild male germplasm resources in A. eriantha in point of durability of pollens against long term conservation for using at future pollination and breeding programs.展开更多
It is generally agreed that many Vitis vinifera L.cultivars are self-fertile,where self-pollination often occurs before capfall in a process called cleistogamy.Therefore,it is difficult to identify the right time to r...It is generally agreed that many Vitis vinifera L.cultivars are self-fertile,where self-pollination often occurs before capfall in a process called cleistogamy.Therefore,it is difficult to identify the right time to remove stamens before self-pollination during the cross-breeding of grape.For this paper,we observed the process of grape flowering and measured the pollen viability and stigma receptivity of grape flowers of‘Shine Muscat’in order to identify the starting time of self-pollination before capfall and to provide useful information for improving the efficiency of cross-breeding.The results demonstrate that the anther is not cracked during the visible clusters and separated clusters stages.Meanwhile,in the separated floral buds,flowering begins,and full bloom stages,the pollen viability is 60.7%,73.2%and 80.3%,respectively;however,at the berry set stage,pollen viability drops to zero.The top of the mature stigma is composed of a layer of nearly cylindrical papillary cells,and the stigma receptivity for pollen changes with the development of flowers:in particular,no reaction was observed in the visible clusters stage;weak positive reaction at the separated clusters stage;strong positive reaction at the separated floral buds,flowering begins,and full bloom stages;and no reaction at the berry set stage.In the separated floral buds stage,pollen tubes were seen germinating in the style.In the flowering begins stage,more pollen tubes were observed at the entry of the ovary.During the full bloom stage,most pollen tubes elongated into the ovary base and some entered the pearl hole.At the berry set stage,newborn endosperm nucleus could be seen in the ovule.From the above,we can conclude that the initiation time of closed fertilization for‘Shine Muscat’grape can be judged as the separated floral buds stage,and it is best to discard the stamen before the separated floral buds stage when conducting cross-breeding.展开更多
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 ...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.展开更多
Mildew resistance locus O(MLO) is a plant-specific gene family that plays an important role in the growth and development of plants and their interactions with the environment. However, the available information on th...Mildew resistance locus O(MLO) is a plant-specific gene family that plays an important role in the growth and development of plants and their interactions with the environment. However, the available information on this gene family in pear is limited. Here, 24 PbrMLO genes were identified and divided into five subfamilies(I, II, III, IV and V). Wholegenome duplication(WGD) and dispersed duplication contributed to the expansion of the PbrMLO family. In addition, gene expression analysis revealed that PbrMLO genes were distributed in various pear tissues, suggesting their diverse functions. We selected PbrMLO23 for further functional analysis. Expression profile analysis by qRT-PCR showed that PbrMLO23 was highly expressed in pollen. Subcellular localization analysis showed that PbrMLO23 was located on the plasma membrane. When the expression level of PbrMLO23 was knocked down by using antisense oligonucleotides, pollen tube lengths increased, indicating that PbrMLO23 plays a functional role in inhibiting pollen tube growth. In summary, these results provide evolutionary insight into PbrMLO and its functional characteristics and lay a foundation for further analysis of the functions of PbrMLO members in pear.展开更多
The pollination methods and embryo rescue techniques of hybridization between I.ilium Oriental hybrids were studied in this paper. The results showed that : ( 1 ) in the combination of ' Justina' x ' Shala', po...The pollination methods and embryo rescue techniques of hybridization between I.ilium Oriental hybrids were studied in this paper. The results showed that : ( 1 ) in the combination of ' Justina' x ' Shala', pollen tubes were observed to grow downward in styles and target to ovules in ovaries when normal sti^na 13o1- lination method was used; pollen tubes were found to grow directionless to a different degree at stylar cuts and in ovaries when cut-style pollination method was used; the longer the styles were left, the more pollen tubes toward to ovules were observed and more plump seeds were produced; the maximum plump seeds were obtained via normal stigma pollination. (2) Hybridization experiments were performed for the 56 combinations of Lilium Oriental hybrids with normal stigma pollina- tion, and one combination generated no plump ovule and three combinations generated fewer than 10 plump ovules while the other 52 combinations generated plenty of plump ovules, which preliminarily indicated that normal stigma pollination method could be used as an universal pollination technique for hybridization between Lilium Oriental hybrids. The experiment results of different embryo rescue techniques showed that using cut-ovule inoculation about 70 d after pollination had a fas- ter germination speed and a higher germination rate of hybrid embryo than normal ovule inoculation, and embryo rescue efficiency of the former method was obviously higher than "embryo isolated inoculation" on 40 d after pollination. The research suggested that "cut-ovule inoculation" about 70 d after pollination could be regarded as an universal embryo rescue technique for hybridization between Lilium Oriental hybrids.展开更多
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 GLYCEROPHOSPHODIE...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.展开更多
基金supported by the earmarked fund for China Agriculture Research System(CARS-28-37)the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences,China(CXGC2022E21)+1 种基金the Youth Foundation of Shandong Institute of Pomology,China(GSS2022QN11)the Natural Science Foundation of Shandong Province,China(ZR2019BC075,ZR2020MC141,and ZR2021MC177)。
文摘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.
基金National Science Foundation of Fujian Province of China(No.B0610031)Science and Technology Program of Put-Jan Municipality of China(No.2006N17)
文摘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.
文摘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.
基金supported by National Natural Science Foundation of China(31620103903)partially by the 111 projectsupported by the Peking-Tsinghua Joint Center for Life Sciences
文摘In angiosperms, initiation of ovule enlargement represents the start of seed development, the molecular mechanism of which is not yet elucidated. It was previously reported that pollen tube contents, rather than double fertilization, can trigger ovule enlargement. However, it remains unclear whether the signal(s) to trigger the initiation of ovule enlargement are from the sperm cells or fromthe pollen tubes. Recently, we identified a mutant dropl- drop2-, which produces pollen tubes with no sperm cells. Taking advantage of this special genetic material, we conducted pollination assays, and found that the ovules pollinated with dropl- drop2- pollen could initiate the enlargement and exhibited significant enlarged sizes at 36h after pollination in comparison with those unpollinated ovules. However, the sizes of the ovules pollinated with drops- drop2- pollen are significantly smaller than those of the ovules pollinated with wildtype pollen. These results demonstrate that the pollen tube, rather than the sperm cells, release the signal to trigger the initiation of ovule enlargement, and that double fertilization is required for further enlargement of the seeds.
文摘The Arabidopsis phosphoinositide kinases PI4Kβ1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kβ1 and PIP5K5 catalyze key consecutive steps of phosphoinositide conversion, and it appears obvious that phosphatidylinositol-4-phosphate formed by PI4Kβ1 might act as a substrate for phosphatidylinositol-4,5-bisphosphate formation by PIP5K5. However, this hypothesis has not been experimentally addressed and distinct localization patterns of PI4Kβ1, PIP5K5, and also PI-synthases (PIS) generating phosphatidylinositol suggest additional complexity. Here, the synergistic functionality of enzymes of phosphoinositide conversion was assessed. In tobacco and Arabidopsis pollen tubes, phosphoinositides influence the apical secretion of pectin, and increased pectin deposition results in characteristic morphological alterations. Catalytically active and dominant negative variants of PI4Kβ1 and PIP5K5 were systematically co-expressed in tobacco pollen tubes and the incidence of morphologies related to enhanced pectin secretion was evaluated. The data support a proposed functional interplay of PI4Kβ1 and PIP5K5 at the trans-Golgi network, mediating directional vesicle trafficking. Co-expression experiments additionally including PIS isoforms, PIS1 or PIS2, indicate that pectin secretion is synergistically mediated by PI4Kβ1 and PIPSK5 acting on Ptdlns formed by PIS2 rather than PIS1. Possible ramifications for the preferential channeling of phosphoinositide intermediates between particular isoforms of PI pathway enzymes are discussed.
文摘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.
基金Supported by the Ministry of Science and Technology (2007CB947600)the National Natural Science Foundation of China (30771088 and30821007)the Chinese Academy of Sciences (Hundred talents program)
文摘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.
文摘Polarized organization of the cytoplasm of growing pollen tubes is maintained by coordinated function of actin filaments (AFs) and microtubules (MTs). AFs convey post-Golgi secretory vesicles to the tip where some fuse with specific domains of the plasma membrane (PM). Secretory activity is balanced by PM retrieval that maintains cell mem- brane economy and regulates the polarized composition of the PM, by dividing lipids/proteins between the shank and the tip. Although AFs play a key role in PM internalization in the shank, the role of MTs in exo-endocytosis needs to be characterized. The present results show that integrity of the MT cytoskeleton is necessary to control exo-endocytosis events in the tip. MT polymerization plays a role in promoting PM invagination in the apex of tobacco pollen tubes since nocodazole affected PM internalization in the tip and subsequent migration of endocytic vesicles from the apex for degradation. MT depolymerization in the apex and shank was associated with misallocation of a significantly greater amount of internalized PM to the Golgi apparatus and its early recycling to the secretory pathway. Fluorescence Recovery After Photobleaching (FRAP) experiments also showed that MT depolymerization in the tip region influenced the rate of exocytosis in the central domain of the apical PM.
文摘Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.
基金supported by National Key Project of China (No. 2001BA302B)
文摘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.
基金the National Natural Science Foundation of China (Grants Nos. 30070454 and 30471045)
文摘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.
基金supported by the FAPERJ,CAPES and CNPq of Brazil.
文摘The success of sexual plant reproduction is directly influenced by specific interactions between the pollen and pistil.Light,fluorescence and scanning electron microscopy techniques were used to evaluate the steps of pollination in sour passion fruit plants(Passiflora edulis Sims).In the compatible interaction,pollen tubes grow through stigma projections towards the ovary.The pollen grain surface was found to be spheroidal and to consist of heteroreticulate exine with six colpi.Furthermore,analysis in vivo of pollenpistil interactions indicated that stigmas of flowers 24 hours before anthesis are unable to discriminate compatible(genetically unrelated)and incompatible(genetically related)pollen grains.Taken together,these results provide insight into the cellular mechanisms underlying pollination in passion fruit plants.
基金This work was supported by the grant from Agricultural Ministry of China(No.2016ZX08009-003-002).
文摘SWEET transporters play a pivotal role in sugar transport in plants.However,their functions in pollen tube growth,especially in coniferous species remain unknown.Here,we used RT-qPCR to reveal that a SWEET1 gene was specifically expressed in pollen and pollen tubes of Picea wilsonii.A pollen germination assay showed that PwSWEET1 was induced by H3BO3 but not by Ca^2+.In a sugar specificity experiment,sucrose(Suc)and glucose(Glc)were effective sugars for pollen germination and pollen tube growth.PwSWEET1 expression was induced most by Suc and Glc.Heterologous expression of PwSWEET1 in yeast showed that PwSWEET1 can restore the glucose absorption in yeast strain EBY.VW4000,which has a hexose absorption defect,and the absorption of glucose is pH-independent.This evidence supports the involvment of PwSWEET1 in boron-dependent glucose transport in pollen germination and pollen tube growth of Picea wilsonii.
文摘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
文摘We examined the influence of storage time on germinability and tube growth of freeze stored pollens collected from 25 wild male plants in Actinidia eriantha variety. Pollens were stored in freezer at - 20°C for six months and one year periods to determine changing at germinability in time. In vitro germination was conducted in certain cultural medium defined for Actinidia genus. The results showed that the germination percentages and tube lengths of genotypes decreased at the end of storage period. MH22, MH45, MH47, MH56, MH67, MH70, MH71, MH72, MH74, MH55 and MH61 genotypes were evaluated as vigor genotypes, because they maintained their viability and germination capability displaying statistically insignificant decreasing although their tube lengths significantly decreased except MH67. This investigation provided to determine some robust wild male germplasm resources in A. eriantha in point of durability of pollens against long term conservation for using at future pollination and breeding programs.
基金This work was supported by the Grant 31872050 from the National Natural Science Foundation of Chinathe Agricultural Seed Engineering of Shandong Province(Grant No.2020LZGC008)+1 种基金the Agricultural Science and Technology Innovation Project for Shandong Academy of Agricultural Sciences(Grant No.CXGC2022A13)the Key R&D Program of Linyi City(Grant No.2020ZX010).
文摘It is generally agreed that many Vitis vinifera L.cultivars are self-fertile,where self-pollination often occurs before capfall in a process called cleistogamy.Therefore,it is difficult to identify the right time to remove stamens before self-pollination during the cross-breeding of grape.For this paper,we observed the process of grape flowering and measured the pollen viability and stigma receptivity of grape flowers of‘Shine Muscat’in order to identify the starting time of self-pollination before capfall and to provide useful information for improving the efficiency of cross-breeding.The results demonstrate that the anther is not cracked during the visible clusters and separated clusters stages.Meanwhile,in the separated floral buds,flowering begins,and full bloom stages,the pollen viability is 60.7%,73.2%and 80.3%,respectively;however,at the berry set stage,pollen viability drops to zero.The top of the mature stigma is composed of a layer of nearly cylindrical papillary cells,and the stigma receptivity for pollen changes with the development of flowers:in particular,no reaction was observed in the visible clusters stage;weak positive reaction at the separated clusters stage;strong positive reaction at the separated floral buds,flowering begins,and full bloom stages;and no reaction at the berry set stage.In the separated floral buds stage,pollen tubes were seen germinating in the style.In the flowering begins stage,more pollen tubes were observed at the entry of the ovary.During the full bloom stage,most pollen tubes elongated into the ovary base and some entered the pearl hole.At the berry set stage,newborn endosperm nucleus could be seen in the ovule.From the above,we can conclude that the initiation time of closed fertilization for‘Shine Muscat’grape can be judged as the separated floral buds stage,and it is best to discard the stamen before the separated floral buds stage when conducting cross-breeding.
基金supported by the National Natural Science Foundation of China (32170330, 32170331 and 31970195)Hainan Yazhou Bay Seed Lab (B23YQ1510)the Fundamental Research Funds for the Central Universities (lzujbky-2022-ey06)。
文摘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.
基金supported by the National Natural Science Foundation of China (31772276)the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, Chinathe China Postdoctoral Science Foundation (2017M620213)。
文摘Mildew resistance locus O(MLO) is a plant-specific gene family that plays an important role in the growth and development of plants and their interactions with the environment. However, the available information on this gene family in pear is limited. Here, 24 PbrMLO genes were identified and divided into five subfamilies(I, II, III, IV and V). Wholegenome duplication(WGD) and dispersed duplication contributed to the expansion of the PbrMLO family. In addition, gene expression analysis revealed that PbrMLO genes were distributed in various pear tissues, suggesting their diverse functions. We selected PbrMLO23 for further functional analysis. Expression profile analysis by qRT-PCR showed that PbrMLO23 was highly expressed in pollen. Subcellular localization analysis showed that PbrMLO23 was located on the plasma membrane. When the expression level of PbrMLO23 was knocked down by using antisense oligonucleotides, pollen tube lengths increased, indicating that PbrMLO23 plays a functional role in inhibiting pollen tube growth. In summary, these results provide evolutionary insight into PbrMLO and its functional characteristics and lay a foundation for further analysis of the functions of PbrMLO members in pear.
基金Supported by Natural Science Foundation of Beijing(6122004)Project of Science and Technology Innovation of Beijing(PXM2013-014207-000051,PXM2014-014207-000018)+2 种基金Science and Technology Promotion Project of Beijing(PXM2013-014207-000079,PXM2014-014207-000081)Project of Beijing Laboratory of Urban and Rural Ecological Environment(PXM2015-014207-000014)Project of Construction of Innovative Teams and TeacherCareer Development for Universities and Colleges under Beijing Municipality(IDHT20150503)
文摘The pollination methods and embryo rescue techniques of hybridization between I.ilium Oriental hybrids were studied in this paper. The results showed that : ( 1 ) in the combination of ' Justina' x ' Shala', pollen tubes were observed to grow downward in styles and target to ovules in ovaries when normal sti^na 13o1- lination method was used; pollen tubes were found to grow directionless to a different degree at stylar cuts and in ovaries when cut-style pollination method was used; the longer the styles were left, the more pollen tubes toward to ovules were observed and more plump seeds were produced; the maximum plump seeds were obtained via normal stigma pollination. (2) Hybridization experiments were performed for the 56 combinations of Lilium Oriental hybrids with normal stigma pollina- tion, and one combination generated no plump ovule and three combinations generated fewer than 10 plump ovules while the other 52 combinations generated plenty of plump ovules, which preliminarily indicated that normal stigma pollination method could be used as an universal pollination technique for hybridization between Lilium Oriental hybrids. The experiment results of different embryo rescue techniques showed that using cut-ovule inoculation about 70 d after pollination had a fas- ter germination speed and a higher germination rate of hybrid embryo than normal ovule inoculation, and embryo rescue efficiency of the former method was obviously higher than "embryo isolated inoculation" on 40 d after pollination. The research suggested that "cut-ovule inoculation" about 70 d after pollination could be regarded as an universal embryo rescue technique for hybridization between Lilium Oriental hybrids.
基金supported by the Fundamental Research Funds for the Central Universities(2572021DT01)the National Natural Science Foundation of China(31770637)+1 种基金the Innovation Project of State Key Laboratory of Tree Genetics and Breeding(2014A01)the Heilongjiang Touyan Innovation Team Program(Tree Genetics and Breeding Innovation Team)。
文摘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.