Arabidopsis shaker pollen inward K~+ channel SPIK functions in SnRK1 complex-regulated pollen hydration on the stigma

Pollen hydration is a critical step that determines pollen germination on the stigma. KINβγ is a plantspecific subunit of the SNF1-related protein kinase 1 complex(SnRK1 complex). In pollen of the Arabidopsis kinbg mutant, the levels of reactive oxygen species were decreased which lead to compromised hydration of the mutant pollen on the stigma. In this study, we analyzed gene expression in kinβγ mutant pollen by RNA-seq and found the expression of inward shaker K~+ channel SPIK was down-regulated in the kinβγ pollen. Furthermore, we showed that the pollen hydration of the Arabidopsis spik mutant was defective on the wild-type stigma, although the mutant pollen demonstrated normal hydration in vitro.Additionally, the defective hydration of spik mutant pollen could not be rescued by the wild-type pollen on the stigma,indicating that the spik mutation deprived the capability of pollen absorption on the stigma. Our results suggest that the Arabidopsis SnRK1 complex regulates SPIK expression,which functions in determining pollen hydration on the stigma.     

Pollen and Resource Limitation in Veratrum nigrum L. （Liliaceae）, an Andromonoecious Herb

Pollen limitation and resource limitation were invoked to account for the pattern that flowering plants produce more flowers and ovules than fruits and seeds. This study aimed to determine their relative importance in Veratrum nigrum, a self-compatible, perennial, andromonoecious herb. In order to determine whether female production was limited by pollen grains on stigmas or by available resources, we performed supplemental hand pollination in three populations, male-flower-bud removal in three other populations, and emasculation of hermaphroditic flowers in still another population, resulting in a total of seven populations experimentally manipulated. Across the three populations, supplemental hand pollination did not significantly increase fruit set, seed number per fruit, and total seed production per individual, nor did emasculation of hermaphroditic flowers. Taken together, our results suggest that pollen grains deposited on stigmas were abundant enough to fertilize all the ovules. Male-flower-bud removal significantly increased the mean size of hermaphroditic flowers in all three populations. Female reproductive success was increased in one population, but not in the other two populations possibly due to heavy flower/seed predation. We concluded that the female reproductive success of V. nigrum was not limited by pollen grains but by available resources, which is consistent with Bateman＇s principle. Furthermore, the female reproduction increase of male-flower-bud removal individuals might suggest a trade-off between male and female sexual functions.     

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

OsGCD1,a novel player in rice intine construction

<正>In angiosperms,pollen development is an intricate indispensable process for successful sexual reproduction,which involves accurately and tightly controlled regulations and requires numerous genes expressed in both sporophytic and gametophytic tissues of the anther(McCormick,2004).Understanding the molecular mechanism underlying pollen development is of great significance for the construction of male sterile lines and crop breeding.However,many critical issues concerning pollen development still need to be elucidated.Recently,targeted editing of pollen development-related genes mediated by CRISPR/Cas9 system has provided powerful tools to facilitate the relevant studies in crops to figure out these issues(Li et al., 2016,2017).     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.     

An Image Skeletonization-Based Tool for Pollen Tube Morphology Analysis and Phenotyping

The mechanism underlying pollen tube growth involves diverse genes and molecular pathways. Alterations in the regulatory genes or pathways cause phenotypic changes reflected by cellular morphology, which can be captured using fluorescence microscopy. Determining and classifying pollen tube morphological phenotypes in such microscopic images is key to our understanding the involvement of genes and pathways. In this context, we propose a computational method to extract quantitative morphological features, and demonstrate that these features reflect morphological differences relevant to distinguish different defects of pollen tube growth. The corresponding software tool furthermore includes a novel semi-automated image segmentation approach, allowing to highly accurately identify the boundary of a pollen tube in a microscopic image.     

Assessing protein-DNA interactions: Pros and cons of classic and emerging techniques

正Investigation of protein-DNA interactions provides important information for understanding gene function and regulation,but identification and validation of specific interactions remain major challenges in the post-genomics era.Therefore,effective and economical methods to assess protein-DNA interactions are highly sought-after by molecular biologists.Choosing the appropriate method to examine a specific protein-DNA interaction also remains a crucial challenge     

Sexual Plant Reproduction: a Fertile and Flourishing Field

Since the very beginning of plant science, sexual plant reproduction (SPR) has proved an attractive and enduring topic for generations of botanists. With the rapid development of modern technology, a significant acceleration has occurred in our understanding on the developmental mechanisms of plant reproductive processes, particularly the evolution of double fertilisation, signalling in pollen tube orientation, molecular characterisation of plant gametes, maternal to zygotic transitions and parental gene involvement in early embryogenesis. This is reflected not only by several recent high-ranking research papers, but also by the frequent conferences and workshops on these topics. These include the 2008 XXth International Congress on SPR in Bras´齦ia and “Frontiers in SPR III” in Tucson, as well as “Cell-Cell Communication in Plant Reproduction” held in 2009 in Bath. The continuing efforts from around the world indicate that SPR is still a fertile and flourishing field, with great expectations for the coming decade. Investigations on SPR have been enhanced recently by their underlying relationship to agriculture. With the development of molecular crop breeding comes an increasing requirement for knowledge on molecular mechanisms of SPR, both general regulatory processes, and in particular, signalling for cell-cell communication in several critical steps. To reveal mechanisms behind some well-known phenomena in modern agriculture, such as compatibility of crop crosses and fertility determination of remote hybrids and heterosis, one must first precisely understand the basic developmental processes and molecular mechanisms involved in SPR. Thus, it is not surprising that the field has attracted much attention in recent years. In fact, advances in the study of SPR throw new light on investigations for solving many existing problems in agriculture. This special issue presents current research on multiple aspects of SPR, both pre- and post-fertilisation. Hence, this special issue of the Journal of Integrative Plant Biology features a diverse collection of papers. One paper, “The earliest normal flower in Liaoning Province, China” (see pages 800–811), presents evidence of component parts and morphology of early angiosperm flowers. Four papers, “A genome-wide functional characterisation of Arabidopsis regulatory calcium sensors in pollen tubes” (see pages 751–761), “GNOM-LIKE 2, encoding an ARF-GEF protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis” (see pages 762–773), “Regulation of actin dynamics in the pollen tubes:control of actin polymer level” (see pages 740–750) and “Pollen tube growth: a delicate equilibrium between secretory and endocytic pathways” (see pages 727–739), deal with molecular mechanisms of pollen tube growth and orientation, a hot field in SPR. Amongst these papers, Prof. Moscatelli offers a deep insight into dynamic control of vesicle trafficking in pollen tubes. In the signalling cascade of vesicle trafficking, calcium and actin dynamics play essential roles in the balance between secretory and endocytic pathways. GNOM-LIKE 2, as an ARF-GEF- and BFA-sensitive protein,may indirectly influence calcium and actin dynamics during pollen germination and pollen tube growth. “Premature tapetum degeneration: a major cause of abortive pollen development in photoperiod sensitive genic male sterility in rice” (see pages 774–781) presents new evidence for the interaction between tapetum and pollen development. “Comparative detection of calcium fluctuations in single female sexual cells of tobacco to distinguish calcium signals triggered by in vitro fertilisation” (see pages 782–791) reports observations on calcium dynamics during sperm-central cell fusion, and “Oil body biogenesis during Brassica napus embryogenesis” (see pages 792–799) focuses on post-fertilisation developmental events, mainly on oil body generation and accumulation during embryogenesis in both wild type and mutants. A review paper, “Analyses of sexual reproductive success in transgenic and/or mutant plants” (see pages 719–726), gives a critical evaluation of current techniques used for investigating genes that affect pistil development, and provides useful information and technical guidance to readers in this field. These papers coherently address the common theme: development and its mechanisms in plant reproductive processes. In this way, we hope to create an atmosphere in which extensive discussion can stimulate and lead to interactions between scientists with different perspectives in order to inspire future directions. We sincerely hope that this special issue of JIPB will provide a platform to highlight current advances and novel findings in the study of SPR. Finally, we express our appreciation to JIPB for supporting this special issue and to all authors for their great contributions. Mengxiang Sun, Professor The Editor for this Special Issue College of Life Sciences, Wuhan University, China     

Cysteine-rich peptides: signals for pollen tube guidance,species isolation and beyond

<正>In flowering plants, the two sperm cells are passive cargo transported into the ovule by pollen tubes (Zhang et al.,2017). Therefore, the precise guidance of pollen tubes is critical for successful double fertilization (Dresselhaus and Franklin-Tong, 2013). A series of male-female interactions are required to guarantee the spatiotemporal regulation of pollen tube guidance because during growth, pollen tubes     

Pollen Tube Growth： a Delicate Equilibrium Between Secretory and Endocytic Pathways

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles （SVs） in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane （PM）, defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.     

Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible Iines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

Novel Adaptors of Amyloid Precursor Protein Intracellular Domain and Their Functional Implications

Amyloid precursor protein intracellular domain(AICD) is one of the potential candidates in deciphering the complexity of Alzheimer’s disease.It plays important roles in determining cell fate and neurodegeneration through its interactions with several adaptors.The presence or absence of phosphorylation at specific sites determines the choice of partners.In this study,we identified 20 novel AICDinteracting proteins by in vitro pull down experiments followed by 2D gel electrophoresis and MALDI-MS analysis.The identified proteins can be grouped into different functional classes including molecular chaperones,structural proteins,signaling and transport molecules,adaptors,motor proteins and apoptosis determinants.Interactions of nine proteins were further validated either by colocalization using confocal imaging or by co-immunoprecipitation followed by immunoblotting.The cellular functions of most of the proteins can be correlated with AD.Hence,illustration of their interactions with AICD may shed some light on the disease pathophysiology.     

Molecular Soil Biology (MSB)

正Molecular Soil Biology(ISSN 1925-2005),indexed by international database Pro Quest,is an open access,peer reviewed online journal publishing the original research articles,short communication of novel findings and reviews in all the aspects of life in the soil.The journal publishes in describing and explaining biological processes in soil in terms of soil micro-structure,soil micro-ecosystems,soil microbiology and molecular interactions among soil,microbes and plants,environmental stress     

The structure of Rap1 in complex with RIAM reveals specificity determinants and recruitment mechanism

The small GTPase Rap1 induces integrin activation via an inside-out signaling pathway mediated by the Rapl-interacting adaptor mol- ecule （RIAM）. Blocking this pathway may suppress tumor metastasis and other diseases that are related to hyperactive integrins. However, the molecular basis for the specific recognition of RIAM by Rap1 remains largely unknown. Herein we present the crystal structure of an active, GTP-bound GTPase domain of Rap1 in complex with the Ras association （RA）-pleckstrin homology （PH） structural module of RIAM at 1.65 A. The structure reveals that the recognition of RIAM by Rap1 is governed by side-chain interactions. Several side chains are critical in determining specificity of this recognition, particularly the Lys31 residue in Rap1 that is oppositely charged compared with the Glu31/Asp31 residue in other Ras GTPases. Lys31 forms a salt bridge with RIAM residue Glu212, making it the key specificity determinant of the interaction. We also show that disruption of these interactions results in reduction of Rapl：RIAM association, leadingto a loss of co-clustering and cell adhesion. Our findings elucidate the molecular mechanism by which RIAM med- iates Rapl-induced integrin activation. The crystal structure also offers new insight into the structural basis for the specific recruitment of RA-PH module-containing effector proteins by their smaU GTPase partners.     

GPS in pollen tubes: Found!

正Sexual reproduction of flowering plants depends on the delivery of two immotile sperm cells to the female gametophyte(FG),i.e.the embryo sac,through the growth of a pollen tube,a long cylindrical cellular extension from a pollen grain.The journey of pollen tubes toward the embryo sacs is led by female cues,which guide the path of the pollen tubes.Recognition between the male-female gametophytes in some cases precedes successful fertilization     

芫花的花粉形态观察及生活力和萌发率分析

Pollen morphology of Daphne genkwa Sieb.et Zucc.was observed by scanning electron microscope(SEM),and its viability and germination rate were measured by TTC and sucrose in vitro culture methods.The results show that pollen is stenopalynous type with a diameter of 15.6-21.6 μm.Per pollen has 10-16 apertures which is irregular circular with a diameter of 1.4-2.0 μm.Surface ornamentation of pollen is rough reticulate pattern which is circular polygon(tetragon-heptagon,mostly pentagon-hexagon).Pollen viability is 51% by TTC.Sucrose of different concentrations has a significant effect on pollen germination rate during pollen culture.And pollen germination rate is the highest with a percentage of 27.0% in medium containing 50 g·L-1 sucrose,while pollen could not germinate in medium containing sucrose over 250 g·L-1.Otherwise,there is the phenomenon of "multi-aperture germination" during pollen germinating.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 （gnl2-1） and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Identification, Expression and Functional Analysis of a Receptor-like Cytoplasmic Kinase, OsRLCK1, in Rice

Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCK1, which belongs to the receptor-like cytoplasmic kinase Ⅷ subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCK1 is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCK1 recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCK1 is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.