Supervised membrane swimming: small G-protein lifeguards regulate PIPK signalling and monitor intracellular PtdIns(4,5)P2 pools

Regulation of PIPK (phosphatidylinositol phosphate kinase) and PtdIns(4,5)P2 signalling by small G-proteins and their effectors is key to many biological functions. Through selective recruitment and activation of different PIPK isoforms, small G-proteins such as Rho, Rac and Cdc42 modulate actin dynamics and cytoskeleton-dependent cellular events in response to extracellular signalling. These activities affect a number of processes, including endocytosis, bacterial penetration into host cells and cytolytic granule-mediated targeted cell killing. Small G-proteins and their modulators are also regulated by phosphoinositides through translocation and conformational changes. Arf family small G-proteins act at multiple sites as regulators of membrane trafficking and actin cytoskeletal remodelling, and regulate a feedback loop comprising phospholipase D, phosphatidic acid, PIPKs and PtdIns(4,5)P2, contributing to enhancement of PtdIns(4,5)P2-mediated cellular events and receptor signalling. Na+, Kir (inwardly rectifying K+), Ca2+ and TRP (transient receptor potential) ion channels are regulated by small G-proteins and membrane pools of PtdIns(4,5)P2. Yeast phosphatidylinositol 4-phosphate 5-kinases Mss4 and Its3 are involved in resistance against disturbance of sphingolipid biosynthesis and maintenance of cell integrity through the synthesis of PtdIns(4,5)P2 and downstream signalling through the Rom2/Rho2 and Rgf1/Rho pathways. Here, we review models for regulated intracellular targeting of PIPKs by small G-proteins and other modulators in response to extracellular signalling. We also describe the spatial and temporal cross-regulation of PIPKs and small G-proteins that is critical for a number of cellular functions.     

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific.Phosphoinositides (PIs) are minor lipids found in membrane fractions but implicated in a wide variety of physiological regulations in eukaryotes (Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006). Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] is a major PI in animal plasma membranes, affecting the localization and activity of various kinds of proteins carrying phosphatidylinositol-binding domains, which in turn affect the regulation of cytoskeletal organization, vesicle trafficking, cell proliferation, and cell growth during development and stress responses (Doughman et al., 2003; Downes et al., 2005; Di Paolo and De Camilli, 2006; Zonia and Munnik, 2006; Heck et al., 2007). In addition, PtdIns(4,5)P₂ is also a well-known substrate of phospholipase C, producing second messengers such as diacylglycerol, phosphatidic acid (PA), and inositol-1,4,5-trisphosphate, which are involved in the activation of intracellular signal transduction pathways (Zonia and Munnik, 2006). Transient accumulation of PtdIns(4,5)P₂ has also been observed under various kinds of environmental stress (Pical et al., 1999; DeWald et al., 2001), suggesting an important role of this lipid in the regulation of stress signal transduction pathways also in plants. These findings indicate that PtdIns(4,5)P₂ is multifunctional and involved in a variety of cellular processes. Therefore, elucidation of the mechanisms controlling the cellular levels of PtdIns(4,5)P₂ is important in understanding the significance of PI signaling in eukaryotes.PtdIns(4,5)P₂ is synthesized by phosphatidylinositol phosphate kinases (PIPKs; Anderson et al., 1999; Doughman et al., 2003; Heck et al., 2007). Physiological roles of several plant PIPKs have been reported. In Arabidopsis (Arabidopsis thaliana), AtPIP5K3 is an essential regulator of tip growth of root hairs (Kusano et al., 2008; Stenzel et al., 2008), while AtPIPK4 and AtPIPK5 are essential for pollen germination and pollen tube elongation (Ischebeck et al., 2008; Sousa et al., 2008). In addition, AtPIP5K9 was shown to interact with the cytosolic invertase CINV1 to regulate sugar-mediated root cell elongation negatively (Lou et al., 2007). Rice (Oryza sativa) OsPIPK1 is proposed to be involved in shoot growth and floral initiation through the regulation of floral induction genes (Ma et al., 2004). In animals, membrane-associated type I PIPK mainly phosphorylates the D-5 hydroxyl group of PtdIns4P to produce PtdIns(4,5)P₂ but also produces PtdIns(3,4)P₂ and PtdIns(3,5)P₂ from PtdIns3P with 5- and 4-kinase activity (Anderson et al., 1999; Heck et al., 2007), whereas type II PIPK prefers the D-4 position of PtdIns5P, producing PtdIns(4,5)P₂ in the nucleus and at the endoplasmic reticulum (Clarke et al., 2007). Thus, in animals, type I and II PIPKs are involved in the generation of PtdIns(4,5)P₂ via different pathways. Molecular biological analysis of plant PIPKs was initiated with AtPIP5K1 from Arabidopsis (Mikami et al., 1998), which phosphorylates PtdIns3P, PtdIns4P, and PtdIns(4,5)P₂ to produce PtdIns(3,4)P₂, PtdIns(4,5)P₂, and PtdIns (3,4,5)P₃, respectively, with D-4- and D-5-kinase activity (Elge et al., 2001; Westergren et al., 2001; Im et al., 2007). Similar enzymatic activity was also reported for other PIPKs from Arabidopsis (Ischebeck et al., 2008; Kusano et al., 2008; Stenzel et al., 2008). In addition, a PIPK from the moss Physcomitrella patens (designated as PpPIPK1) preferred PtdIns4P, PtdIns3P, and PtdIns(3,4)P₂ as substrates, but not PtdIns5P, producing PtdIns(4,5)P₂, PtdIns(3,4)P₂, and PtdIns(3,4,5)P₃, respectively (Saavedra et al., 2009). These findings indicate that the substrate specificity of plant PIPKs is essentially the same as that of type I PIPKs. However, AtPIP5K1 has yet to be classified as either type I or type II based on sequence comparisons of the catalytic domain (CD; Mikami et al., 1998). This was confirmed by a genome-wide analysis of PIPK genes in Arabidopsis in which all 11 PIPKs were classified as type I/II based on sequence comparisons of the CDs, which were further subdivided into subtypes A and B (Mueller-Roeber and Pical, 2002). Therefore, it is suggested that typical type I and II PIPKs are absent in plants, although further confirmation is needed.The conserved PIPK CD contains a short highly conserved region near its C-terminal end, designated the activation loop, which acts as the substrate-binding site and is responsible for the differences in substrate specificity and subcellular localization between animal type I and type II PIPKs (Kunz et al., 2000, 2002). Substrate specificities of animal type I and type II PIPKs, for example, are determined by a respective Glu and Ala at the corresponding positions in the activation loop. Moreover, it has been established that substitution of Glu to Ala results in a swap of substrate specificity and subcellular localization between the two types (Kunz et al., 2000, 2002). In contrast to animal PIPKs, a substitution in the activation loop of PpPIPK1 from Glu to Ala resulted in a nearly complete loss of type I/II activity; however, such a mutation did not fully convert the substrate specificity, although an enhancement of type II versus type I activity was observed (Saavedra et al., 2009). Since the corresponding amino acid residue is Glu in all plant PIPKs so far reported, it is suggested that there also is a plant-specific mode of substrate specificity regulation in plant type I/II PIPKs. However, enzymatic activity appears to be modified in similar ways between plant type I/II and animal type I PIPKs; that is, phosphorylation- and PA-dependent activation of PIPKs has been observed in both animals and plants (Moritz et al., 1992; Jenkins et al., 1994; Pical et al., 1999; Westergren et al., 2001; Perera et al., 2005; Saavedra et al., 2009).The regulation of plasma membrane localization of mammalian type I PIPKs remains confusing. In addition to the involvement of a Glu residue as mentioned above, the substitution of two Lys residues in the activation loop to Asn residues changes the subcellular localization from the plasma membrane to the cytosol (Kunz et al., 2000, 2002). However, Arioka et al. (2004) also showed that the plasma membrane localization of type I PIPKs is regulated by another basic amino acid pair localized downstream of the activation loop in the CD, which is not found in type II PIPKs. Interestingly, the mechanism behind plasma membrane localization of plant PIPKs seems to differ significantly from the animal one. The obvious structural feature of plant PIPKs is the presence of a repetition of membrane occupation and recognition nexus (MORN) motifs at the N-terminal half, which is conserved across the B subfamily of plant type I/II PIPKs (Mueller-Roeber and Pical, 2002). The MORN motif was first identified in mammalian junctophilin, an endoplasmic reticulum-membrane-bound component of the junctional complex between the plasma membrane and the endoplasmic reticulum (Takeshima et al., 2000). Since MORN motifs are not found in PIPKs from nonplant organisms, a plant-specific mode of PIPK activation is speculated. Indeed, a regulatory role of the MORN domain was reported in the enzymatic activation of AtPIP5K1 (Im et al., 2007) and in root hair formation, but not in enzymatic activation, of AtPIP5K3 (Stenzel et al., 2008). Moreover, the MORN domain may play a role in the plasma membrane localization of OsPIPK1 from rice and AtPIP5K1 and AtPIP5K3 from Arabidopsis (Ma et al., 2006; Im et al., 2007; Kusano et al., 2008). However, stable transformation of tobacco (Nicotiana tabacum) cells to express an AtPIP5K1 MORN domain-GFP fusion did not allow visualization of the plasma membrane localization of this protein (Im et al., 2007). Thus, it is not clear if the MORN domain functions as a plasma membrane-targeting module.Given the sequence conservation of the CD among eukaryotic PIPKs (Saavedra et al., 2009), we hypothesize that the CD is responsible for the plasma membrane localization of plant PIPKs. Thus, to gain further insight into the mechanisms regulating this issue, we dissected PpPIPK1 to determine the molecular determinants of plasma membrane localization. Here, we show that the MORN domain is not involved in the plasma membrane localization of PpPIPK1 and AtPIP5K1 in P. patens protoplasts and onion (Allium cepa) epidermal cells. We further demonstrate that two basic amino acids, but not Glu, conserved in the activation loop of the CD are required for plasma membrane localization. These findings demonstrate that the activation mode of type I/II PIPKs is plant specific and differs from that of the membrane-localized animal type I PIPKs.     

Coordinated activation of the nuclear ubiquitin ligase Cul3-SPOP by the generation of phosphatidylinositol 5-phosphate

Phosphoinositide signaling pathways regulate numerous processes in eukaryotic cells, including migration, proliferation, and survival. The regulatory lipid phosphatidylinositol 4,5-bisphosphate is synthesized by two distinct classes of phosphatidylinositol phosphate kinases (PIPKs), the type I and II PIPKs. Although numerous physiological functions have been identified for type I PIPKs, little is known about the functions and regulation of type II PIPK. Using a yeast two-hybrid screen, we identified an interaction between the type IIbeta PIPK isoform (PIPKIIbeta) and SPOP (speckle-type POZ domain protein), a nuclear speckle-associated protein that recruits substrates to Cul3-based ubiquitin ligases. PIPKIIbeta and SPOP interact and co-localize at nuclear speckles in mammalian cells, and SPOP mediates the ubiquitylation of PIPKIIbeta by Cul3-based ubiquitin ligases. Additionally, stimulation of the p38 MAPK pathway enhances the ubiquitin ligase activity of Cul3-SPOP toward multiple substrate proteins. Finally, a kinase-dead PIPKIIbeta mutant enhanced ubiquitylation of Cul3-SPOP substrates. The kinase-dead PIPKIIbeta mutant increases the cellular content of its substrate lipid phosphatidylinositol 5-phosphate (PI5P), suggesting that PI5P may stimulate Cul3-SPOP activity through a p38-dependent signaling pathway. Expression of phosphatidylinositol-4,5-bisphosphate 4-phosphatases that generate PI5P dramatically stimulated Cul3-SPOP activity and was blocked by the p38 inhibitor SB203580. Taken together, these data define a novel mechanism whereby the phosphoinositide PI5P leads to stimulation of Cul3-SPOP ubiquitin ligase activity and also implicate PIPKIIbeta as a key regulator of this signaling pathway through its association with the Cul3-SPOP complex.     

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.     

PIPKs are essential for rhizoid elongation and caulonemal cell development in the moss Physcomitrella patens

PtdIns‐4,5‐bisphosphate is a lipid messenger of eukaryotic cells that plays a critical role in processes such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels and nuclear signalling pathways. The enzymes responsible for the synthesis of PtdIns(4,5)P₂ are phosphatidylinositol phosphate kinases (PIPKs). The moss Physcomitrella patens contains two PIPKs, PpPIPK1 and PpPIPK2. To study their physiological role, both genes were disrupted by targeted homologous recombination and as a result mutant plants with lower PtdIns(4,5)P₂ levels were obtained. A strong phenotype for pipk1, but not for pipk2 single knockout lines, was obtained. The pipk1 knockout lines were impaired in rhizoid and caulonemal cell elongation, whereas pipk1‐2 double knockout lines showed dramatic defects in protonemal and gametophore morphology manifested by the absence of rapidly elongating caulonemal cells in the protonemal tissue, leafy gametophores with very short rhizoids, and loss of sporophyte production. pipk1 complemented by overexpression of PpPIPK1 fully restored the wild‐type phenotype whereas overexpression of the inactive PpPIPK1E885A did not. Overexpression of PpPIPK2 in the pipk1‐2 double knockout did not restore the wild‐type phenotype demonstrating that PpPIPK1 and PpPIPK2 are not functionally redundant. In vivo imaging of the cytoskeleton network revealed that the shortened caulonemal cells in the pipk1 mutants was the result of the absence of the apicobasal gradient of cortical F‐actin cables normally observed in wild‐type caulonemal cells. Our data indicate that both PpPIPKs play a crucial role in the development of the moss P. patens, and particularly in the regulation of tip growth.     

小立碗藓萜类物质的合成酶基因分析及其UPLC-QTOF检测

根据基因组信息和KEGG数据库分析小立碗藓基因组中合成萜类物质的基因,比较小立碗藓与酵母和拟南芥合成萜类物质基因的氨基酸序列同源性同时利用UPLC-QTOF分析小立碗藓中物质组成,来分析小立碗藓基因组中萜类物质合成的基因及小立碗藓中存在的萜类物质。与酵母相比,小立碗藓两条萜类次生代谢途径完整,途径中的基因及氨基酸丰富性更高,提示可以合成更丰富的前体物质如FPP,GPP等;小立碗藓与拟南芥的序列相似性较高,萜类背景简单。UPLC-QTOF分析检测到小立碗藓中次生代谢物质主要是芳香族化合物及各类生物碱,一种萜类物质ent-16beta-Methoxy-19-kauranoic acid。小立碗藓中本身具有合成萜类前体物质和二萜的基因,检测到少量萜类物质,适合作为萜类活性物质异源合成的底盘细胞。     

Phosphoinositide synthesis and degradation in isolated rat liver peroxisomes

Analyzing peroxisomal phosphoinositide (PId(#)) synthesis in highly purified rat liver peroxisomes we found synthesis of phosphatidylinositol 4-phosphate (PtdIns4P), PtdIns(4,5)P(2) and PtdIns(3,5)P(2). PtdIns3P was hardly detected in vitro, however, was observed in vivo after [(32)P]-phosphate labeling of primary rat hepatocytes. In comparison with other subcellular organelles peroxisomes revealed a unique PId pattern suggesting peroxisomal specificity of the observed synthesis. Use of phosphatase inhibitors enhanced the amount of PtdIns4P. The results obtained provide evidence that isolated rat liver peroxisomes synthesize PIds and suggest the association of PId 4-kinase and PId 5-kinase and PId 4-phosphatase activities with the peroxisomal membrane.     

Movin' on up: the role of PtdIns(4,5)P(2) in cell migration

Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.     

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).     

Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3). The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Mutations in the Arabidopsis phosphoinositide phosphatase gene SAC9 lead to overaccumulation of PtdIns(4,5)P2 and constitutive expression of the stress-response pathway

Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P(2) is one of the best characterized PIs; studies in which PtdIns(4,5)P(2) localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P(2) and its derivative Ins(1,4,5)P(3) accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P(2) signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P(3). We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P(2) and Ins(1,4,5)P(3) as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.     

苔藓植物蓝光/紫外光-A 信号传导的研究

种子植物含有5个已分离的光受体和至少1个未鉴定的蓝光/紫外光-A受体。隐花色素(CRY1、CRY2和CRY3) 调节植物的生长发育,而向光蛋白(PHOT1和PHOT2) 调节植物对光的营养反应。黄素可以吸收蓝光和紫外光-A,是生色团。对这些光受体的结构和作用模式已了解很多。苔藓植物小立碗藓中含有2个已分离的隐花色素(CRY1a和CRY1b),负责调节侧枝形成和生长素代谢;有4个向光蛋白(PHOTA1,PHOTA2,PHOTB1,PHOTB2) 调节叶绿体的运动。苔藓细胞内蓝光/紫外光-A刺激引发的信号转导有Ca2+参与。     

Photoperiod-regulated expression of the PpCOL1 gene encoding a homolog of CO/COL proteins in the moss Physcomitrella patens

The CONSTANS (CO) protein is a critical regulator of the photoperiodic control of flowering in Arabidopsis thaliana and Oryza sativa. We isolated a cDNA PpCOL1 encoding a homolog of the CO/CO-LIKE (COL) family proteins from a cryptogam Physcomitrella patens. The predicted PpCOL1 protein has N-terminal zinc finger and C-terminal CCT domains, which are conserved in the angiosperm CO/COL proteins. Structurally, PpCOL1 is the most closely related to the Group Ia or Ic proteins, which include AtCO and AtCOL1/2, among diverged members of the family. A transient expression assay using GFP showed that the CCT domain of PpCOL1 contains a nuclear-localizing signal. Northern blotting analyses revealed that the PpCOL1 expression is controlled by the circadian clock, and moreover, it is photoperiodically regulated at a gametophore stage when the rate of sporophyte formation is affected by day length. These observations indicate a possible involvement of PpCOL1 as a nuclear factor in the photoperiodic regulation of reproduction of Physcomitrella.     

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.