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.

A phosphoinositide 5-phosphatase from Solanum tuberosum is activated by PAMP-treatment and may antagonize phosphatidylinositol 4,5-bisphosphate at Phytophthora infestans infection sites.

Potato (Solanum tuberosum) plants susceptible to late blight disease caused by the oomycete Phytophthora infestans display enhanced resistance upon infiltration with the pathogen-associated molecular pattern (PAMP), Pep-13. Here, we characterize a potato gene similar to Arabidopsis 5-phosphatases which was identified in transcript arrays performed to identify Pep-13 regulated genes, and termed StIPP. Recombinant StIPP protein specifically dephosphorylated the D5-position of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ) in vitro. Other phosphoinositides or soluble inositolpolyphosphates were not converted. When transiently expressed in tobacco (Nicotiana tabacum) pollen tubes, a StIPP-YFP fusion localized to the subapical plasma membrane and antagonized PtdIns(4,5)P2 -dependent effects on cell morphology, indicating in vivo functionality. Phytophthora infestans-infection of N. benthamiana leaf epidermis cells resulted in relocalization of StIPP-GFP from the plasma membrane to the extra-haustorial membrane (EHM). Colocalizion with the effector protein RFP-AvrBlb2 at infection sites is consistent with a role of StIPP in the plant-oomycete interaction. Correlation analysis of fluorescence distributions of StIPP-GFP and biosensors for PtdIns(4,5)P2 or phosphatidylinositol 4-phosphate (PtdIns4P) indicate StIPP activity predominantly at the EHM. In Arabidopsis protoplasts, expression of StIPP resulted in the stabilization of the PAMP receptor, FLAGELLIN-SENSITIVE 2, indicating that StIPP may act as a PAMP-induced and localized antagonist of PtdIns(4,5)P2 -dependent processes during plant immunity.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

Coordinated Localization and Antagonistic Function of NtPLC3 and PI4P 5-Kinases in the Subapical Plasma Membrane of Tobacco Pollen Tubes.

Polar tip growth of pollen tubes is regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which localizes in a well-defined region of the subapical plasma membrane. How the PtdIns(4,5)P2 region is maintained is currently unclear. In principle, the formation of PtdIns(4,5)P2 by PI4P 5-kinases can be counteracted by phospholipase C (PLC), which hydrolyzes PtdIns(4,5)P2. Here, we show that fluorescence-tagged tobacco NtPLC3 displays a subapical plasma membrane distribution which frames that of fluorescence-tagged PI4P 5-kinases, suggesting that NtPLC3 may modulate PtdIns(4,5)P2-mediated processes in pollen tubes. The expression of a dominant negative NtPLC3 variant resulted in pollen tube tip swelling, consistent with a delimiting effect on PtdIns(4,5)P2 production. When pollen tube morphologies were assessed as a quantitative read-out for PtdIns(4,5)P2 function, NtPLC3 reverted the effects of a coexpressed PI4P 5-kinase, demonstrating that NtPLC3-mediated breakdown of PtdIns(4,5)P2 antagonizes the effects of PtdIns(4,5)P2 overproduction in vivo. When analyzed by spinning disc microscopy, fluorescence-tagged NtPLC3 displayed discontinuous membrane distribution omitting punctate areas of the membrane, suggesting that NtPLC3 is involved in the spatial restriction of plasma membrane domains also at the nanodomain scale. Together, the data indicate that NtPLC3 may contribute to the spatial restriction of PtdIns(4,5)P2 in the subapical plasma membrane of pollen tubes.

Pho85 and PI(4,5)P2 regulate different lipid metabolic pathways in response to cold.

Lipid homeostasis allows cells to adjust membrane biophysical properties in response to changes in environmental conditions. In the yeast Saccharomyces cerevisiae, a downward shift in temperature from an optimal reduces membrane fluidity, which triggers a lipid remodeling of the plasma membrane. How changes in membrane fluidity are perceived, and how the abundance and composition of different lipid classes is properly balanced, remain largely unknown. Here, we show that the levels of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], the most abundant plasma membrane phosphoinositide, drop rapidly in response to a downward shift in temperature. This change triggers a signaling cascade transmitted to cytosolic diphosphoinositol phosphate derivatives, among them 5-PP-IP4 and 1-IP7, that exert regulatory functions on genes involved in the inositol and phospholipids (PLs) metabolism, and inhibit the activity of the protein kinase Pho85. Consistent with this, cold exposure triggers a specific program of neutral lipids and PLs changes. Furthermore, we identified Pho85 as playing a key role in controlling the synthesis of long-chain bases (LCBs) via the Ypk1-Orm2 regulatory circuit. We conclude that Pho85 orchestrates a coordinated response of lipid metabolic pathways that ensure yeast thermal adaptation.

A PAMP-triggered MAPK cascade inhibits phosphatidylinositol 4,5-bisphosphate production by PIP5K6 in Arabidopsis thaliana.

The phosphoinositide kinase PIP5K6 has recently been identified as a target for the mitogen-activated protein kinase (MAPK) MPK6. Phosphorylation of PIP5K6 inhibited the production of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2 ), impacting membrane trafficking and cell expansion in pollen tubes. Here, we analyzed whether MPK6 regulated PIP5K6 in vegetative Arabidopsis cells in response to the pathogen-associated molecular pattern (PAMP) flg22. Promoter-ß-glucuronidase analyses and quantitative real-time reverse transcription polymerase chain reaction data show PIP5K6 expressed throughout Arabidopsis tissues. Upon flg22 treatment of transgenic protoplasts, the PIP5K6 protein was phosphorylated, and this modification was reduced for a PIP5K6 variant lacking MPK6-targeted residues, or in protoplasts from mpk6 mutants. Upon flg22 treatment of Arabidopsis plants, phosphoinositide levels mildly decreased and a fluorescent reporter for PtdIns(4,5)P2 displayed reduced plasma membrane association, contrasting with phosphoinositide increases reported for abiotic stress responses. Flg22 treatment and chemical induction of the upstream MAPK kinase, MKK5, decreased phosphatidylinositol 4-phosphate 5-kinase activity in mesophyll protoplasts, indicating that the flg22-activated MAPK cascade limited PtdIns(4,5)P2 production. PIP5K6 expression or PIP5K6 protein abundance changed only marginally upon flg22 treatment, consistent with post-translational control of PIP5K6 activity. PtdIns(4,5)P2 -dependent endocytosis of FM 4-64, PIN2 and the NADPH-oxidase RbohD were reduced upon flg22 treatment or MKK5 induction. Reduced RbohD-endocytosis was correlated with enhanced ROS production. We conclude that MPK6-mediated phosphorylation of PIP5K6 limits the production of a functional PtdIns(4,5)P2 pool upon PAMP perception.

MAPKs Influence Pollen Tube Growth by Controlling the Formation of Phosphatidylinositol 4,5-Bisphosphate in an Apical Plasma Membrane Domain.

An apical plasma membrane domain enriched in the regulatory phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is critical for polar tip growth of pollen tubes. How the biosynthesis of PtdIns(4,5)P2 by phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) is controlled by upstream signaling is currently unknown. The pollen-expressed PI4P 5-kinase PIP5K6 is required for clathrin-mediated endocytosis and polar tip growth in pollen tubes. Here, we identify PIP5K6 as a target of the pollen-expressed mitogen-activated protein kinase MPK6 and characterize the regulatory effects. Based on an untargeted mass spectrometry approach, phosphorylation of purified recombinant PIP5K6 by pollen tube extracts could be attributed to MPK6. Recombinant MPK6 phosphorylated residues T590 and T597 in the variable insert of the catalytic domain of PIP5K6, and this modification inhibited PIP5K6 activity in vitro. PIP5K6 interacted with MPK6 in yeast two-hybrid tests, immuno-pull-down assays, and by bimolecular fluorescence complementation at the apical plasma membrane of pollen tubes. In vivo, MPK6 expression resulted in reduced plasma membrane association of a fluorescent PtdIns(4,5)P2 reporter and decreased endocytosis without impairing membrane association of PIP5K6. Effects of PIP5K6 expression on pollen tube growth and cell morphology were attenuated by coexpression of MPK6 in a phosphosite-dependent manner. Our data indicate that MPK6 controls PtdIns(4,5)P2 production and membrane trafficking in pollen tubes, possibly contributing to directional growth.

Guilt by Association: A Phenotype-Based View of the Plant Phosphoinositide Network.

Eukaryotic membranes contain small amounts of phospholipids that have regulatory effects on the physiological functions of cells, tissues, and organs. Phosphoinositides (PIs)-the phosphorylated derivatives of phosphatidylinositol-are one example of such regulatory lipids. Although PIs were described in plants decades ago, their contribution to the regulation of physiological processes in plants is not well understood. In the past few years, evidence has emerged that PIs are essential for plant function and development. Recently reported phenotypes associated with the perturbation of different PIs suggest that some subgroups of PIs influence specific processes. Although the molecular targets of PI-dependent regulation in plants are largely unknown, the effects of perturbed PI metabolism can be used to propose regulatory modules that involve particular downstream targets of PI regulation. This review summarizes phenotypes associated with the perturbation of the plant PI network to categorize functions and suggest possible downstream targets of plant PI regulation.

Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

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)P(2)). 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)P(2). In N. crassa hyphae, PtdIns(4,5)P(2) 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)P(2) 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.

ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization.

Jasmonates are important signals in plant stress responses and plant development. An essential step in the biosynthesis of jasmonic acid (JA) is catalysed by ALLENE OXIDE CYCLASE (AOC) which establishes the naturally occurring enantiomeric structure of jasmonates. In Arabidopsis thaliana, four genes encode four functional AOC polypeptides (AOC1, AOC2, AOC3, and AOC4) raising the question of functional redundancy or diversification. Analysis of transcript accumulation revealed an organ-specific expression pattern, whereas detailed inspection of transgenic lines expressing the GUS reporter gene under the control of individual AOC promoters showed partially redundant promoter activities during development: (i) In fully developed leaves, promoter activities of AOC1, AOC2, and AOC3 appeared throughout all leaf tissue, but AOC4 promoter activity was vascular bundle-specific; (ii) only AOC3 and AOC4 showed promoter activities in roots; and (iii) partially specific promoter activities were found for AOC1 and AOC4 in flower development. In situ hybridization of flower stalks confirmed the GUS activity data. Characterization of single and double AOC loss-of-function mutants further corroborates the hypothesis of functional redundancies among individual AOCs due to a lack of phenotypes indicative of JA deficiency (e.g. male sterility). To elucidate whether redundant AOC expression might contribute to regulation on AOC activity level, protein interaction studies using bimolecular fluorescence complementation (BiFC) were performed and showed that all AOCs can interact among each other. The data suggest a putative regulatory mechanism of temporal and spatial fine-tuning in JA formation by differential expression and via possible heteromerization of the four AOCs.

Role of cis-12-oxo-phytodienoic acid in tomato embryo development.

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.

Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum.

The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.

Variable Regions of PI4P 5-Kinases Direct PtdIns(4,5)P(2) Toward Alternative Regulatory Functions in Tobacco Pollen Tubes.

The apical plasma membrane of pollen tubes contains different PI4P 5-kinases that all produce phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] but exert distinct cellular effects. In the present example, overexpression of Arabidopsis AtPIP5K5 or tobacco NtPIP5K6-1 caused growth defects previously attributed to increased pectin secretion. In contrast, overexpression of Arabidopsis AtPIP5K2 caused apical tip swelling implicated in altering actin fine structure in the pollen tube apex. AtPIP5K5, NtPIP5K6-1, and AtPIP5K2 share identical domain structures. Domains required for correct membrane association of the enzymes were identified by systematic deletion of N-terminal domains and subsequent expression of fluorescence-tagged enzyme truncations in tobacco pollen tubes. A variable linker region (Lin) contained in all PI4P 5-kinase isoforms of subfamily B, but not conserved in sequence, was recognized to be necessary for correct subcellular localization of AtPIP5K5, NtPIP5K6-1, and AtPIP5K2. Deletion of N-terminal domains including the Lin domain did not impair catalytic activity of recombinant AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 in vitro; however, the presence of the Lin domain was necessary for in vivo effects on pollen tube growth upon overexpression of truncated enzymes. Overexpression of catalytically inactive variants of AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 did not influence pollen tube growth, indicating that PtdIns(4,5)P(2) production rather than structural properties of PI4P 5-kinases was relevant for the manifestation of growth phenotypes. When Lin domains were swapped between NtPIP5K6-1 and AtPIP5K2 and the chimeric enzymes overexpressed in pollen tubes, the chimeras reciprocally gained the capabilities to invoke tip swelling or secretion phenotypes, respectively. The data indicate that the Lin domain directed the enzymes into different regulatory contexts, possibly contributing to channeling of PtdIns(4,5)P(2) at the interface of secretion and actin cytoskeleton.

Functional cooperativity of enzymes of phosphoinositide conversion according to synergistic effects on pectin secretion in tobacco pollen tubes.

The Arabidopsis phosphoinositide kinases PI4Kß1 and PIP5K5 have been implicated in the control of directional vesicle trafficking underlying polar tip growth in pollen tubes. PI4Kß1 and PIP5K5 catalyze key consecutive steps of phosphoinositide conversion, and it appears obvious that phosphatidylinositol-4-phosphate formed by PI4Kß1 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 PI4Kß1, 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 PI4Kß1 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 PI4Kß1 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 PI4Kß1 and PIP5K5 acting on PtdIns formed by PIS2 rather than PIS1. Possible ramifications for the preferential channeling of phosphoinositide intermediates between particular isoforms of PI pathway enzymes are discussed.

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)P(2)] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P(2) production at that location are uncharacterized, functions of PtdIns(4,5)P(2) 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)P(2) production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P(2) 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)P(2) via regulatory effects on membrane trafficking and/or apical pectin deposition.

Salt-stress-induced association of phosphatidylinositol 4,5-bisphosphate with clathrin-coated vesicles in plants.

Plants exposed to hyperosmotic stress undergo changes in membrane dynamics and lipid composition to maintain cellular integrity and avoid membrane leakage. Various plant species respond to hyperosmotic stress with transient increases in PtdIns(4,5)P(2); however, the physiological role of such increases is unresolved. The plasma membrane represents the outermost barrier between the symplast of plant cells and its apoplastic surroundings. In the present study, the spatio-temporal dynamics of stress-induced changes in phosphoinositides were analysed in subcellular fractions of Arabidopsis leaves to delineate possible physiological roles. Unlabelled lipids were separated by TLC and quantified by gas-chromatographic detection of associated fatty acids. Transient PtdIns(4,5)P(2) increases upon exposure to hyperosmotic stress were detected first in enriched plasmamembrane fractions, however, at later time points, PtdIns(4,5)P(2) was increased in the endomembrane fractions of the corresponding two-phase systems. When major endomembranes were enriched from rosette leaves prior to hyperosmotic stress and during stimulation for 60 min, no stress-induced increases in the levels of PtdIns(4,5)P(2) were found in fractions enriched for endoplasmic reticulum, nuclei or plastidial membranes. Instead, increased PtdIns(4,5)P(2) was found in CCVs (clathrin-coated vesicles), which proliferated several-fold in mass within 60 min of hyperosmotic stress, according to the abundance of CCV-associated proteins and lipids. Monitoring the subcellular distribution of fluorescence-tagged reporters for clathrin and PtdIns(4,5)P(2) during transient co-expression in onion epidermal cells indicates rapid stress-induced co-localization of clathrin with PtdIns(4,5)P(2) at the plasma membrane. The results indicate that PtdIns(4,5)P(2) may act in stress-induced formation of CCVs in plant cells, highlighting the evolutionary conservation of the phosphoinositide system between organismic kingdoms.

The AOC promoter of tomato is regulated by developmental and environmental stimuli.

The allene oxide cyclase (AOC) catalyzes the formation of cis-(+)-12-oxophytodienoic acid, an intermediate in jasmonate biosynthesis and is encoded by a single copy gene in tomato. The full length AOC promoter isolated by genome walk contains 3600 bp. Transgenic tomato lines carrying a 1000 bp promoter fragment and the full length promoter, respectively, in front of the beta-glucuronidase (GUS)-encoding uidA gene and several tobacco lines carrying the full length tomato AOC promoter before GUS were used to record organ- and tissue-specific promoter activities during development and in response to various stimuli. High promoter activities corresponding to immunocytochemically detected occurrence of the AOC protein were found in seeds and young seedlings and were confined to the root tip, hypocotyl and cotyledons of 3-d-old seedlings. In 10-d-old seedlings promoter activity appeared preferentially in the elongation zone. Fully developed tomato leaves were free of AOC promoter activity, but showed high activity upon wounding locally and systemically or upon treatment with JA, systemin or glucose. Tomato flowers showed high AOC promoter activities in ovules, sepals, anthers and pollen. Most of the promoter activity patterns found in tomato with the 1000 bp promoter fragment were also detected with the full length tomato AOC promoter in tobacco during development or in response to various stimuli. The data support a spatial and temporal regulation of JA biosynthesis during development and in response to environmental stimuli.

The type B phosphatidylinositol-4-phosphate 5-kinase 3 is essential for root hair formation in Arabidopsis thaliana.

Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant plants homozygous for T-DNA-disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair-specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.