Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P₂ effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P₂-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P₂. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P₂ in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P₂ in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P₂ functions to coordinate cytoskeletal dynamics and secretion.

The apical plasma membrane of pollen tubes contains nanodomains where the regulatory phospholipid PtdIns(4,5)P₂ exerts a stabilizing effect on the actin cytoskeleton.     

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P₂ from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P₂ to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of ³H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.     

Identification of PtdIns(3,4)P2 effectors in human platelets using quantitative proteomics

After decades in PtdIns(3,4,5)P₃'s shadow, PtdIns(3,4)P₂ has now emerged as a bona fide regulator of important cellular events, including endocytosis and cell migration. New understanding of PtdIns(3,4)P₂'s cellular roles has been possible via novel approaches to observe and quantify cellular PtdIns(3,4)P₂ dynamics, alongside methods to target the kinases and phosphatases governing phosphoinositide turnover. Despite this, the mechanisms by which PtdIns(3,4)P₂ orchestrates its cellular roles remain more poorly understood, most notably because, to date, few PtdIns(3,4)P₂ effectors have been identified. Here, we develop and apply an affinity-proteomics strategy to conduct a global screen for PtdIns(3,4)P₂ interactors in human platelets; a primary cell type with striking PtdIns(3,4)P₂ accumulation. Through an integrated approach, coupling affinity capture of PtdIns(3,4)P₂-binding proteins to both label-free and isobaric tag-based quantitative proteomics, we identify a diverse PtdIns(3,4)P₂ interactome. Included are long-established PtdIns(3,4)P₂-binding proteins such as PLEKHA1, PLEKHA2, AKT and DAPP1, and a host of potentially novel effectors, including MTMR5, PNKD, RASA3 and GAB3. The PtdIns(3,4)P₂ interactome shows an enrichment of pleckstrin homology (PH) domain-containing proteins, and through bioinformatics and array analyses we characterise the PH domain of MTMR5 and define its phosphoinositide selectivity. The interactome is also diverse in function, including several proteins known to support protein trafficking and cytoskeletal mobilisation. Such proteins have the ability to drive key platelet events, and to fulfil recently-defined roles for PtdIns(3,4)P₂ in a wider range of cell types. Moreover, this study will serve as a valuable resource for the future characterisation of effector-driven PtdIns(3,4)P₂ function.     

Arabidopsis FAB1/PIKfyve Proteins Are Essential for Development of Viable Pollen

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is a phospholipid that has a role in controlling membrane trafficking events in yeast and animal cells. The function of this lipid in plants is unknown, although its synthesis has been shown to be up-regulated upon osmotic stress in plant cells. PtdIns(3,5)P₂ is synthesized by the PIKfyve/Fab1 family of proteins, with two orthologs, FAB1A and FAB1B, being present in Arabidopsis (Arabidopsis thaliana). In this study, we attempt to address the role of this lipid by analyzing the phenotypes of plants mutated in FAB1A and FAB1B. It was not possible to generate plants homozygous for mutations in both genes, although single mutants were isolated. Both homozygous single mutant plant lines exhibited a leaf curl phenotype that was more marked in FAB1B mutants. Genetic transmission analysis revealed that failure to generate double mutant lines was entirely due to inviability of pollen carrying mutant alleles of both FAB1A and FAB1B. This pollen displayed severe defects in vacuolar reorganization following the first mitotic division of development. The presence of abnormally large vacuoles in pollen at the tricellular stage resulted in the collapse of the majority of grains carrying both mutant alleles. This demonstrates a crucial role for PtdIns(3,5)P₂ in modulating the dynamics of vacuolar rearrangement essential for successful pollen development. Taken together, our results are consistent with PtdIns(3,5)P₂ production being central to cellular responses to changes in osmotic conditions.Phosphoinositides make up a minor fraction of total membrane lipids in all eukaryotic organisms. Their production is spatially restricted to the cytoplasmic leaflet of specific organellar membranes and temporally regulated by phosphatidylinositol (PtdIns) kinases and phosphatases. Three of the five hydoxyl groups of PtdIns can be phosphorylated, either singly or combinatorially, to produce seven different phosphoinositides. These different phosphoinositides can recruit and/or activate proteins with specific phosphoinositide-binding domains and have been implicated in the regulation of many important cellular functions, including membrane trafficking, cell growth, and cytoskeleton remodeling (Di Paolo and De Camilli, 2006).In animal cells, phosphorylation at the 3 position of PtdIns and its phosphorylated derivatives can be carried out by three different classes of PtdIns 3-kinase (classes I–III; Cantley, 2002). Plants and yeast only have class III PtdIns 3-kinases that are orthologs of the Saccharomyces cerevisiae protein Vps34p (Mueller-Roeber and Pical, 2002). Vps34p orthologs are thought to use PtdIns as their sole lipid substrate and produce PtdIns 3-phosphate (PtdIns3P). PtdIns3P is involved in endosomal/lysosomal protein sorting in eukaryotic cells in addition to cellular signaling events (Backer, 2008).In plants, PtdIns3P is essential for normal growth and development. Arabidopsis (Arabidopsis thaliana) plants carrying a VPS34 antisense construct have severe developmental defects (Welters et al., 1994). Furthermore, using pharmacological inhibitors of PtdIns3P production and analysis of transgenic plants defective in downstream signaling from PtdIns3P, it has been shown that this lipid has a role to play in many diverse physiological processes, such as root hair growth (Lee et al., 2008a). The phenotypes observed in studies of PtdIns3P function in plants are consistent with a role in endosomal and vacuolar trafficking in plants (Kim et al., 2001; Lee et al., 2008a), as in other eukaryotes. Recently, an attempt to generate vps34 homozygous mutant plant lines was unsuccessful due to failure of the mutant vps34 allele to transmit through the male germ line (Lee et al., 2008b).Importantly, PtdIns3P is the precursor to another phosphoinositide, PtdIns 3,5-bisphosphate [PtdIns(3,5)P₂], which also has vital roles in endosomal trafficking in eukaryotes (Dove et al., 2009). Thus, it is possible that some of the effects in plants attributed to PtdIns3P in previous studies may actually be due to an inability of cells to produce PtdIns(3,5)P₂. PtdIns(3,5)P₂ is produced by the PtdIns3P 5-kinases PIKfyve and Fab1p in animal and yeast cells, respectively. PIKfyve/Fab1p proteins have an N-terminal FYVE domain necessary for binding to PtdIns3P-containing membranes, a central Cpn60_TCP1 (for HSP chaperonin T complex 1) homology domain, and a C-terminal kinase domain. In Arabidopsis, there are a number of genes encoding putative Fab1p homologs, but only two of them, FAB1A (At4g33240) and FAB1B (At3g14270), encode proteins having FYVE domains at their N termini (Mueller-Roeber and Pical, 2002). It is likely that these proteins are PtdIns3P 5-kinases in Arabidopsis. Despite the importance of PtdIns(3,5)P₂ in yeast and animals, very little is known about its function in plants. However, it has been shown that hyperosmotic stress can induce the rapid synthesis of PtdIns(3,5)P₂ in cell suspension cultures from a number of plant species (Meijer and Munnik, 2003) and in pollen tubes from tobacco (Nicotiana tabacum; Zonia and Munnik, 2004). This production is consistent with a requirement for PtdIns(3,5)P₂ in vacuolar membrane reorganization, as water moves from the vacuole to the cytosol upon cells being placed under hyperosmotic stress. In animal cells, defective PtdIns(3,5)P₂ production leads to cytoplasmic vacuolation of endosome-derived membranes (Ikonomov et al., 2001; Jefferies et al., 2008). It seems that there is a general requirement in all eukaryotes for PtdIns(3,5)P₂ production in endomembrane remodeling. This remodeling could be mediated by proteins that bind to PtdIns(3,5)P₂. A number of candidates have been identified, including yeast Svp1p (Dove et al., 2004), its mammalian homolog WIP149 (Jeffries et al., 2004), CHMP3 (Whitley et al., 2003), and Ent3p (Friant et al., 2003).In this study, we aimed to further investigate the role of PtdIns(3,5)P₂ in plant physiology and the function of PIKfyve/Fab1p orthologs in Arabidopsis by generating mutant plant lines homozygous for T-DNA insertions in both FAB1A and FAB1B. We failed to generate double homozygous fab1a/fab1b knockout plants but observed subtle phenotypes in both fab1a and fab1b single homozygous plants. The data show that pollen with a fab1a/fab1b genotype has an abnormal vacuolar phenotype and does not contribute to the next generation. Our data are consistent with the hypothesis that the male gametophytic defect observed in vps34 mutant pollen (Lee et al., 2008b) is due to an inability of this pollen to generate PtdIns(3,5)P₂ and is not a direct result of the lack of PtdIns3P.     

Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

Fab1p Is Essential for PtdIns(3)P 5-Kinase Activity and the Maintenance of Vacuolar Size and Membrane Homeostasis

The Saccharomyces cerevisiae FAB1 gene encodes a 257-kD protein that contains a cysteine-rich RING-FYVE domain at its NH₂-terminus and a kinase domain at its COOH terminus. Based on its sequence, Fab1p was initially proposed to function as a phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinase (Yamamoto et al., 1995). Additional sequence analysis of the Fab1p kinase domain, reveals that Fab1p defines a subfamily of putative PtdInsP kinases that is distinct from the kinases that synthesize PtdIns(4,5)P₂. Consistent with this, we find that unlike wild-type cells, fab1Δ, fab1^tsf, and fab1 kinase domain point mutants lack detectable levels of PtdIns(3,5)P₂, a phosphoinositide recently identified both in yeast and mammalian cells. PtdIns(4,5)P₂ synthesis, on the other hand, is only moderately affected even in fab1Δ mutants. The presence of PtdIns(3)P in fab1 mutants, combined with previous data, indicate that PtdIns(3,5)P₂ synthesis is a two step process, requiring the production of PtdIns(3)P by the Vps34p PtdIns 3-kinase and the subsequent Fab1p- dependent phosphorylation of PtdIns(3)P yielding PtdIns(3,5)P₂. Although Vps34p-mediated synthesis of PtdIns(3)P is required for the proper sorting of hydrolases from the Golgi to the vacuole, the production of PtdIns(3,5)P₂ by Fab1p does not directly affect Golgi to vacuole trafficking, suggesting that PtdIns(3,5)P₂ has a distinct function. The major phenotypes resulting from Fab1p kinase inactivation include temperature-sensitive growth, vacuolar acidification defects, and dramatic increases in vacuolar size. Based on our studies, we hypothesize that whereas Vps34p is essential for anterograde trafficking of membrane and protein cargoes to the vacuole, Fab1p may play an important compensatory role in the recycling/turnover of membranes deposited at the vacuole. Interestingly, deletion of VAC7 also results in an enlarged vacuole morphology and has no detectable PtdIns(3,5)P₂, suggesting that Vac7p functions as an upstream regulator, perhaps in a complex with Fab1p. We propose that Fab1p and Vac7p are components of a signal transduction pathway which functions to regulate the efflux or turnover of vacuolar membranes through the regulated production of PtdIns(3,5)P₂.     

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

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

The phospholipid flippase ALA3 regulates pollen tube growth and guidance in Arabidopsis

Pollen tube guidance regulates the growth direction and ovule targeting of pollen tubes in pistils, which is crucial for the completion of sexual reproduction in flowering plants. The Arabidopsis (Arabidopsis thaliana) pollen-specific receptor kinase (PRK) family members PRK3 and PRK6 are specifically tip-localized and essential for pollen tube growth and guidance. However, the mechanisms controlling the polar localization of PRKs at the pollen tube tip are unclear. The Arabidopsis P4-ATPase ALA3 helps establish the polar localization of apical phosphatidylserine (PS) in pollen tubes. Here, we discovered that loss of ALA3 function caused pollen tube defects in growth and ovule targeting and significantly affected the polar localization pattern of PRK3 and PRK6. Both PRK3 and PRK6 contain two polybasic clusters in the intracellular juxtamembrane domain, and they bound to PS in vitro. PRK3 and PRK6 with polybasic cluster mutations showed reduced or abolished binding to PS and altered polar localization patterns, and they failed to effectively complement the pollen tube-related phenotypes of prk mutants. These results suggest that ALA3 influences the precise localization of PRK3, PRK6, and other PRKs by regulating the distribution of PS, which plays a key role in regulating pollen tube growth and guidance.

AMINOPHOSPHOLIPID ATPASE3 guides pollen tubes by regulating the distribution of anionic phospholipids to affect the precise localization of certain pollen-specific receptor kinases at pollen tubes.

IN A NUTSHELL Background: In flowering plants, pollen tube guidance regulates the rapid growth and timely targeting of the pollen tube to the ovule in the pistil during sexual reproduction, when signaling between the male and female gametophytes occur. The small peptide-RLK signaling module is essential for the interaction between the male and female gametophyte. Certain members of the pollen-specific receptor kinase (PRK) family have different subcellular localization patterns in Arabidopsis pollen tubes and play critical roles in pollen tube growth and guidance. However, the molecular mechanisms that regulate and maintain the polar localization of PRKs at the pollen tube tip are still unknown. Question: We were interested in exploring how Arabidopsis P4-ATPase (aminophospholipid ATPase, ALA) precisely regulates pollen tube guidance and maintains the polar localization patterns of PRK6 and PRK3. How plant ALA family members regulate pollen tube guidance has not yet been documented. Findings: The loss of ALA3 function not only caused sluggish pollen tube growth and aberrant ovule targeting but also affected the polar localization patterns of several PRKs at the pollen tube tip. Members of the PRKs family can directly interact with anionic phospholipids such as phosphatidylserine (PS), and the capacity of PRK3/6 to bind anionic phospholipids is crucial for both their polar localization and physiological functions. ALA3 establishes and maintains the polar distribution of PS, which influences secretory vesicles-mediated polar trafficking at the pollen tube tip to affect the distribution of PRK3 and PRK6. On the other hand, PS might also directly recruit PRK3 and PRK6 to the pollen tube tip and sustain their localization. Next steps: The localization of PRKs is a complex, finely regulated process. The C-termini of PRKs may also affect their polar distribution. More research is required to reveal how the C-terminus domain precisely controls the localization of PRKs.     

The gar2 and rga alleles increase the growth of gibberellin-deficient pollen tubes in Arabidopsis

Ectopic expression in Arabidopsis of a pea (Pisum sativum) cDNA (2ox2) encoding a gibberellin (GA) 2-oxidase (PsGA2ox2), involved in the deactivation of biologically active GAs, has been used to establish a role for GAs in promoting pollen tube growth. One line, 35S:2ox2/28c, when homozygous for the transgene, exhibits a novel small fruit phenotype. The 28c transgene reduces pollen tube growth, and this results in a reduced number of fertilized seeds that are only present at the end of the silique nearest the stigma. To confirm that the 28c pollen tube phenotype is due to sense expression of the 2ox2 mRNA, a “hairpin” RNA interface silencing construct, designed to silence 2ox2 expression, has been used to restore pollen tube growth and fruit development. The interaction between 28c and other mutants with increased GA response has also been examined to provide further evidence that GAs play an important role in pollen tube growth. Based on the ability of mutant alleles to suppress the 35S:2ox2/28c phenotype, we define new roles for the gar2-1 and rga alleles in GA signaling during pollen tube elongation in addition to their previously established roles in vegetative tissues. In contrast to the constitutive GA response observed in internodes and leaves lacking RGA and GAI, the rga-2 gai-d5 mutant combination is only a partial suppressor of the 28c phenotype. Because the dominant dwarfing gai-1 allele reduces GA response in vegetative tissues, its effect on plant fertility has been examined. Although gai-1 reduces seed set, this appears to reflect defects in reproductive development other than pollen tube function. Finally, we show that the genetic background (Landsberg erecta or Columbia) modifies the 28c phenotype and that this effect is not due to the ER/er difference between these two ecotypes.     

Arabidopsis LIM proteins PLIM2a and PLIM2b regulate actin configuration during pollen tube growth

The pollen tube grows rapidly, exclusively at its tip, to deliver its sperm for fertilization. The polarized tip growth of pollen tubes is dependent on the highly dynamic actin cytoskeleton. Plant LIM proteins (named after initials of containing proteins Lin11, Isl-1, and Mec-3) have been shown to regulate actin bundling in different cells, however, their roles in pollen tube growth have remained obscure. Here, we report the function of Arabidopsis LIM proteins PLIM2a and PLIM2b in pollen tube growth. The PLIM2a mutation resulted in short and swollen Arabidopsis pollen tube with defective actin bundles. The expression of the construct green fluorescent protein (GFP)-PLIM2b led to fluorescence of the actin bundles in germinating pollen and also the long actin bundles along the growing pollen tubes in Arabidopsis, but not of the short and sparse actin bundles that characterize the tip regions of the pollen tubes. There is a partially redundant function between PLIM2a and PLIM2b in the shank actin bundle organization during Arabidopsis pollen tube growth, as PLIM2b could rescue for the defective shank actin bundles in PLIM2a mutation pollen tubes. This report suggests critical roles of PLIM2a/PLIM2b in actin configuration during Arabidopsis pollen germination and tube growth.     

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1? and sjl3?, and the temperature-sensitive mutant sac1^ts/sjl3? at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1? and sac1^ts/sjl3? mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1? strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1? strain, although autophagosomes were present in the cytoplasm. In the sac1^ts/sjl3? strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3? strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.     

Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton

Main conclusion

Pollen tube growth in styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the adverse effect of high temperatures during square development. High-temperature stress during flowering influences the square development of upland cotton (Gossypium hirsutum L.) and cotton yield. Although it is well known that square development is sensitive to high temperature, high-temperature sensitive stages of square development and the effects of high temperature on pollen tube growth in the styles are unknown. The effect of high temperature on anther development corresponding to pollen vigor is unknown during anther development. The objectives of this study were to identify the stages of square development that are sensitive to high temperatures (37/30 and 40/34 °C), to determine whether the abnormal development of squares influenced by high temperature is responsible for the variation in the in vitro germination percent of pollen grains at anthesis, to identify the effect of high temperature on pollen germination in the styles, and to determine pollen thermotolerance heterosis. Our results show that the stages from the sporogenous cell to tetrad stage (square length <6.0 mm) were the most sensitive to high temperature, and the corresponding pollen viability at anthesis was consistent with the changes in the square development stage. Pollen tube growth in the styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the effect of high temperature during square development. The thermotolerance of hybrid F₁ pollen showed heterosis, and pollen viability could be used as a criterion for screening for high-temperature tolerance cultivars. These results can be used in breeding to develop new cotton cultivars that can withstand high-temperature conditions, particularly in a future warmer climate.

     

BRET-monitoring of the dynamic changes of inositol lipid pools in living cells reveals a PKC-dependent PtdIns4P increase upon EGF and M3 receptor activation

Deciphering many roles played by inositol lipids in signal transduction and membrane function demands experimental approaches that can detect their dynamic accumulation with subcellular accuracy and exquisite sensitivity. The former criterion is met by imaging of fluorescence biosensors in living cells, whereas the latter is facilitated by biochemical measurements from populations. Here, we introduce BRET-based biosensors able to detect rapid changes in inositol lipids in cell populations with both high sensitivity and subcellular resolution in a single, convenient assay. We demonstrate robust and sensitive measurements of PtdIns4P, PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ dynamics, as well as changes in cytoplasmic Ins(1,4,5)P₃ levels. Measurements were made during either experimental activation of lipid degradation, or PI 3-kinase and phospholipase C mediated signal transduction. Our results reveal a previously unappreciated synthesis of PtdIns4P that accompanies moderate activation of phospholipase C signaling downstream of both EGF and muscarinic M3 receptor activation. This signaling-induced PtdIns4P synthesis relies on protein kinase C, and implicates a feedback mechanism in the control of inositol lipid metabolism during signal transduction.     

Phosphoinositides and the endocytic pathway

Phosphorylation of the phosphatidylinositol headgroup generates seven varieties of phosphoinositide of which PtdIns(4,5)P₂, PtdIns3P and PtdIns (3,5)P₂ have established roles on the endocytic pathway. In this review, we discuss the enzymes responsible for generation and turnover of these lipids, which are keys to determining compartmental identity and the flux of material through the endocytic system. The enzymatic generation of lipids serves as an amplification mechanism through which a wide variety of effector molecules can be recruited.     

Roles of phosphoinositides in regulation of stomatal movements

Guard cells sense various environmental and internal stimuli and, in response, modulate the stomatal aperture to a size optimal for growth and adaptation. Among the many factors involved in the fine regulation of stomata, we have focused our studies on the role of phosphoinositides. Our recent study published in the Plant Journal (52:803–16) provides evidence for an important role for phosphatidylinositol 4,5-bis-phosphate (PtdIns(4,5)P₂) in inducing stomatal opening. Light induces translocation of a PtdIns(4,5)P₂-binding protein from the cytosol to the plasma membrane and treatments that increase the intracellular PtdIns(4,5)P₂ level induce stomatal opening in the absence of light irradiation. Inhibition of anion channel activity, a negative regulator for stomatal opening, was suggested as a mechanism of PtdIns(4,5)P₂-induced stomatal opening. We also reported that phosphatidylinositol 3-phosphate (PtdIns(3)P) and phosphatidylinositol 4-phosphate (PtdIns(4)P) regulate actin dynamics in guard cells. The effects of the phosphoinositides were specific, and were not induced by other lipids with similar structures. The roles of different interacting partners are likely to be important for these lipids to produce specific changes in guard cell activity.Key words: PtdIns(4,5)P₂; PtdIns(4)P; Ins(1,4,5)P₃; anion channel; PIP kinase; phospholipase C; stomatal opening; guard cells     

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.