Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcll is male sterile as a result of the failure ofpollen germination. We show that the bcll mutant allele cannot be transmitted by male gametophytes and no homozygous bcll mutants were obtained. Analysis of pollen developmental stages indicates that the bcll mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting （VPS） in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.     

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.     

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.     

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.     

Plant villins:Versatile actin regulatory proteins

Regulation of actin dynamics is a central theme in cell biology that is important for different aspects of cell physiology.Villin, a member of the villin/gelsolin/fragmin superfamily of proteins, is an important regulator of actin. Villins contain six gelsolin homology domains(G1–G6) and an extra headpiece domain. In contrast to their mammalian counterparts, plant villins are expressed widely, implying that plant villins play a more general role in regulating actin dynamics. Some plant villins have a de fined role in modifying actin dynamics in the pollen Invitube; most of their in vivo activities remain to be ascertained.Recently, our understanding of the functions and mechanisms of action for plant villins has progressed rapidly, primarily due to the advent of Arabidopsis thaliana genetic approaches and imaging capabilities that can visualize actin dynamics at the single filament level in vitro and in living plant cells. In this review,we focus on discussing the biochemical activities and modes of regulation of plant villins. Here, we present current understanding of the functions of plant villins. Finally, we highlight some of the key unanswered questions regarding the functions and regulation of plant villins for future research.     

Dimethyl Sulfoxide Is Feasible for Plant Tubulin Assembly In vitro： A Comprehensive Analysis

It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro. However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro. In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2 mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 ℃. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 ℃, or following the addition to or the removal of Ca^2 from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-) carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.     

Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice

Alpha-picolinic acid (PA), a metabolite of tryptophan and an inducer of apoptosis in the animal cell, has been reported to be a toxin produced by some of plant fungal pathogens and used in screening for disease resistant mutants. Here, we report that PA is an efficient apoptosis agent triggering cell death of hypersensitive-like response in planta. Confirmed by Fluorescence Activated Cell Sorter (FACS), rice suspension cells and leaves exhibited programmed cell death induced by PA. The PA-induced cell death was associated with the accumulation of reactive oxygen species that could be blocked by diphenylene iodonium chloride, indicating that the generation of reactive oxygen species was NADPHoxidase dependent. We also demonstrated the induction of rice defense-related genes and subsequent resistant enhancement by PA against the rice blast fungus Magnaporthe grisea. Hence, it was concluded that the PA-stimulated defense response likely involves the onset of the hypersensitive response in rice, which also provides a simple eliciting tool for studying apoptosis in the plant cell.     

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.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.     

Glucose transporter 4 can be inserted in the membrane without exposing its catalytic site for photolabeling from the medium

Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100nmol/L insulin and 10μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P3.     

SDG714 Regulates Specific Gene Expression and Consequently Affects Plant Growth via H3K9 Dimethylation

Histone lysine methylation is known to be involved in the epigenetic regulation of gene expression in all eukaryotes including plants. Here we show that the rice SDG714 is primarily responsible for dimethylation but not trimethylation on histone H3K9 in vivo. Overexpression of YFP-SDG714 in Arabidopsis significantly inhibits plant growth and this inhibition is associated with an enhanced level of H3K9 dimethylation. Our microarray results show that many genes essential for the plant growth and development were downregulated in transgenic Arabidopsis plants overexpressing YFP-SDG714. By chromatin immunoprecipitation analysis, we show that YFP-SDG714 is targeted to specific chromatin regions and dimethylate the H3K9, which is linked with heterochromatinization and the downregulation of genes. Most interestingly, when YFP-SDG714 production is stopped, the inhibited plants can partially restore their growth, suggesting that the perturbation of gene expression caused by YFP-SDG714 is revertible. Taken together, our results point to an important role of SDG714 in H3K9 dimethylation, suppression of gene expression and plant growth, and provide a potential method to regulate gene expression and plant development by an on-off switch of SDG714 expression.     

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.     

Clues to the signals for chloroplast photo-relocation from the lifetimes of accumulation and avoidance responses

Chloroplast photo-relocation movement is crucial for plant survival; however, the mechanism of this phenomenon is still poorly understood. Especially, the signal that goes from photoreceptor to chloroplast is unknown, although the photoreceptors(phototropin 1 and 2) have been identified and an actin structure(chloroplast actin filaments) has been characterized that is specific for chloroplast movement. Here,in gametophytes of the fern Adiantum capillus-veneris,gametophores of the moss Physcomiterella patens, and leaves of the seed plant Arabidopsis thaliana, we sought to characterize the signaling system by measuring the lifetime of the induced response. Chloroplast movements were induced by microbeam irradiation with high-intensity blue light and recorded. The lifetime of the avoidance state was measured as a lag time between switching off the beam and the loss of avoidance behavior, and that of the accumulation state was measured as the duration of accumulation behavior following the extinction of the beam. The lifetime for the avoidance response state is approximately 3–4 min and that for the accumulation response is 19–28 min. These data suggest that the two responses are based on distinct signals.     

ATP-binding cassette transporter enhances tolerance to DDT in Tetrahymena

The reuse of dichlorodiphenyltrichloroethane(DDT) as an indoor residual spray was permitted by the World Health Organization in 2007, and approximately 14 countries still use DDT to control disease vectors. The extensive exposure of insects to DDT has resulted in the emergence of DDT resistance, especially in mosquitoes, and the mechanism for this resistance in mosquitoes has been widely reported. Spraying can also introduce DDT directly into surface water, and DDT can subsequently accumulate in microorganisms, but the mechanism for the resistance to DDT degradation in microorganisms is unclear. Using whole-genome microarray analysis, we detected an abcb15 gene that was up-regulated in a specific manner by DDT treatment in T. thermophile. The deduced ABCB15 peptide sequence had two transmembrane domains(TMDs) and two nucleotide-binding domains(NBDs) to form the structure TMD-NBD-TMD-NBD, and each NBD contained three conserved motifs: Walker-A, C-loop, and Walker-B, which indicated the T. thermophila abcb15 was a typical ABC transporter gene. The expression of ABCB15 fused with a C-terminal green fluorescent protein was found to be on the periphery of the cell, suggesting that ABCB15 was a membrane pump protein. In addition, cells with abcb15 partially knocked down(abcb15-KD) grew slower than wild-type cells in the presence of 256 mg L-1 DDT, indicating the tolerance of abcb15-KD strain to DDT exposure was decreased. Thus, we suggest that in Tetrahymena, the membrane pump protein encoded by ABCT gene abcb15 can enhance the tolerance to DDT and protect cells from this exogenous toxin by efficiently pumping it to the extracellular space.