Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDβ1 on elicitation with xylanase

Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling. In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase. The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold). In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment. When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h. Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor. When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation. Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function. The possibility that PLDbeta1 is a signalling enzyme is discussed.     

脱落酸调节植物抵御水分胁迫的机制研究

干旱、盐渍、低温等均可导致植物可利用水分的亏缺,表现为水分胁迫。植物感受到水分胁迫,诱导脱落酸（abscisic acid,ABA）生物合成。ABA可通过促使气孔关闭或抑制气孔开放,使作物尽可能地降低蒸腾失水,以抵御水分胁迫。该文就植物激素ABA及其下游信号过氧化氢（hydrogenperoxide,H2O2）、一氧化氮（nitric oxide,NO）以及Ca2＋等在植物气孔运动调节方面的研究进展进行概述,以构建水分胁迫下ABA调节植物气孔运动的可能模式。     

Differential expression of two related,low-temperature-induced genes in Arabidopsis thaliana (L.) Heynh

Plant cold acclimation is correlated to expression of low-temperature-induced (lti) genes. By using a previously characterized lti cDNA clone as a probe we isolated a genomic fragment that carried two closely located lti genes of Arabidopsis thaliana. The genes were structurally related with the coding regions interrupted by three similarly located short introns and were transcribed in the same direction. The nucleotide sequences of the two genes, lti78 and lti65, predict novel hydrophilic polypeptides with molecular weights of 77856 and 64510, respectively, lti78 corresponding to the cDNA probe. Of the 710 amino acids of LTI78 and 600 amino acids of LTI65, 346 amino acids were identical between the polypeptides, which suggests that the genes may have a common origin.Both lti78 and lti65 were induced by low temperature, exogenous abscisic acid (ABA) and drought, but the responsiveness of the genes to these stimuli was markedly different. Both the levels and the temporal pattern of expression differed between the genes. Expression of lti78 was mainly responsive to low temperature, that of lti65 to drought and ABA. In contrast to the induction of lti78, which follows separate signal pathways during low-temperature, ABA and drought treatment, the drought induction of lti65 is ABA-dependent and the low-temperature induction appears to be coupled to the ABA biosynthetic pathway. This differential expression of two related genes may indicate that they have some-what different roles in the stress response.     

The transphosphatidylation activity of phospholipase D

Transphosphatidylation activity is a characteristic and remarkable property of phospholipase D (PLD) and has been studied in plants and mammalian tissues. This reaction is often used to confirm the properties and/or abnormalities of PLD activity. The mechanism for activating PLD transphosphatidylation seems multiple. Although significant changes of transphosphatidylation activity have been found in some pathological animal models, the biological significance of PLD transphosphatidylation remains largely unknown.     

A hydraulic signal in root-to-shoot signalling of water shortage

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.     

Substrate preference of stress-activated phospholipase D in Chlamydomonas and its contribution to PA formation

In response to various environmental stress conditions, plants rapidly form the intracellular lipid second messenger phosphatidic acid (PA). It can be generated by two independent signalling pathways via phospholipase D (PLD) and via phospholipase C (PLC) in combination with diacylglycerol kinase (DGK). In the green alga Chlamydomonas, the phospholipid substrates for these pathways are characterized by specific fatty acid compositions. This allowed us to establish: (i) PLD's in vivo substrate preference; and (ii) PLD's contribution to PA formation during stress signalling. Accordingly, G-protein activation (1 micro m mastoparan), hyperosmotic stress (150 mm NaCl) and membrane depolarization (50 mm KCl) were used to stimulate PLD, as monitored by the accumulation in 5 min of its unique transphosphatidylation product phosphatidylbutanol (PBut). In each case, PBut's fatty acid composition specifically matched that of phosphatidylethanolamine (PE), identifying this lipid as PLD's favoured substrate. This conclusion was substantiated by analysing the molecular species by electrospray ionization-mass spectrometry (ESI-MS/MS), which revealed that PE and NaCl-induced PBut share a unique (18 : 1)2-structure. The fatty acid composition of PA was much more complex, reflecting the different contributions from the PLC/DGK and PLD pathways. During KCl-induced stress, the PA rise was largely accounted for by PLD activity. In contrast, PLD's contribution to hyperosmotic stress-induced PA was less, being approximately 63% of the total increase. This was because the PLC/DGK pathway was activated as well, resulting in phosphoinositide-specific fatty acids and molecular species in PA.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.     

Increased expression of phospholipase Dα1 in guard cells decreases water loss with improved seed production under drought in Brassica napus

The activation of phospholipase Dα1 (PLDα1) produces lipid messenger phosphatidic acid and promotes stomatal closure in Arabidopsis. To explore the use of the PLDα1‐mediated signalling towards decreasing water loss in crop plants, we introduced Arabidopsis PLDα1 under the control of a guard cell–specific promoter AtKatI_pro into two canola (Brassica napus) cultivars. Multiple AtKatI_pro::PLDα1 lines in each cultivar displayed decreased water loss and improved biomass accumulation under hyperosmotic stress conditions, including drought and high salinity. Moreover, AtKatI_pro::PLDα1 plants produced more seeds than did WT plants in fields under drought. The results indicate that the guard cell–specific expression of PLDα1 has the potential to improve crop yield by enhancing drought tolerance.     

KCl activates phospholipase D at two different concentration ranges: distinguishing between hyperosmotic stress and membrane depolarization

Hyperosmotic stress induces the rapid formation of phosphatidic acid (PA) in Chlamydomonas moewusii via the activation of two signalling pathways: phospholipase D (PLD) and phospholipase C (PLC), the latter in combination with diacylglycerol kinase (DGK) (Munnik et al., 2000). A concomitant increase in cell Ca(2+) becomes manifest as deflagellation. When KCl was used as osmoticum we found that two concentration ranges activated deflagellation: one between 50 and 100 mm and another above 200 mm. Deflagellation in low KCl concentrations was complete within 30 sec whereas in high concentrations it took 5 min. PLC was not activated, as it was by high KCl concentrations that cause hyperosmotic stress. Moreover PLD was activated more strongly by low than by high KCl concentrations. Potassium was the most potent monovalent cation based on the induction of deflagellation and the formation of PA and PBut. During treatment, the external medium acidified, indicating an increase in H(+)-ATPase activity in order to re-establish the membrane potential. Activation of PLD and deflagellation at low KCl concentrations were abrogated by treatment with La(3+), Gd(3+) and EGTA, indicating the dependency on extracellular Ca(2+). This suggests that low concentrations of KCl depolarize the plasma membrane, resulting in the activation of H(+)-ATPases and opening voltage-dependent Ca(2+) +/- channels, observed as deflagellation and an increase in PLD activity.     

Down-regulation by elicitors of phosphatidylcholine-hydrolyzing phospholipase C and up-regulation of phospholipase A in plant cells

Phosphatidylcholine, labeled by two fluorescent fatty acids, was fed to cultured plant cells (Petrosilenum crispum, L.; VBI-0, Nicotiana benthiana, L.) and fluorescent diacylglycerol (DAG) was the major metabolite. When a glycoprotein elicitor, derived from Phytophthora sojae, was applied to the parsley cells and the small protein cryptogein from Phytophthora cryptogea was applied to the tobacco cells, these signal substances strongly and rapidly decreased the pool of fluorescent diacylglycerol and weakly increased the pool of free fluorescent fatty acid and of fluorescent lysophosphatidylcholine. The cells responded in a very similar way to the application of mastoparan, a wasp venom peptide. As phosphatidic acid was only a very minor fluorescent metabolite DAG is hypothesized to arise by the action of a phosphatidylcholine-hydrolyzing phospholipase C which was down-regulated by elicitors. Up-regulation of a phospholipase A by elicitors is also suggested by these results. This is the first evidence for phosphatidylcholine-hydrolyzing phospholipase C in plant signal transduction.     

Coordinated Regulation of Radioadaptive Response by Protein Kinase C and p38 Mitogen-Activated Protein Kinase

Eukaryotic cells are known to have an inducible or adaptive response that enhances radioresistance after a low priming dose of radiation. This radioadaptive response seems to present a novel cellular defense mechanism. However, its molecular processing and signaling mechanisms are largely unknown. Here, we studied the role of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) in the expression of radioadaptive response in cultured mouse cells. Protein immunoblot analysis using isoform-specific antibodies showed an immediate activation of PKC-alpha upon X-irradiation as indicated by a translocation from cytosol to membrane. A low priming dose caused a prolonged translocation, while a nonadaptive high dose dramatically downregulated the total PKC level. Low-dose X-rays also activated the p38 MAPK. The activation of p38 MAPK and resistance to chromosome aberration formation were blocked by SB203580, an inhibitor of p38 MAPK, and Calphostin C, an inhibitor of PKC. Furthermore, it was demonstrated that p38 MAPK was physically associated with delta1 isoform of phospholipase C (PLC-delta1), which hydrolyzed phosphatidylinositol bisphosphate into diacylglycerol, an activator of PKC, and that SB203580 also blocked the activation of PKC-alpha. These results indicate the presence of a novel mechanism for coordinated regulation of adaptive response to low-dose X-rays by a nexus of PKC-alpha/p38 MAPK/PLC-delta1 circuitry feedback signaling pathway with its breakage operated by downregulation of labile PKC-alpha at high doses or excess stimuli.     

Molecular and biochemical properties and physiological roles of plant phospholipase D

Recent advances have thrust the study of plant phospholipase D (PLD) into the molecular era. This review will highlight some of the recent progress made in elucidating the molecular and biochemical nature of plant PLDs as well as their roles in plant physiology.     

In vitro secretion of digestive phospholipase A2 by midguts isolated from tobacco hornworms,Manduca sexta

We report on secretion of phospholipase A₂ (PLA₂) by in vitro preparations of midguts isolated from tobacco hornworms, Manduca sexta. This enzyme is responsible for hydrolysis of fatty acids from the sn‐2 position of phospholipids, a necessary step in fatty acid absorption. The in vitro midgut preparations are competent to secrete PLA₂ into incubation buffer. Secretion began within the first 30 min of incubation and increased to a maximum at 8 h. We selected 2 h incubations because substantial loss of tissue integrity was observed after 8 h incubations. Using 2 h incubations, we recorded increased secretion of digestive PLA₂ from midguts incubated in buffer amended with diet or with yeast as a component of the diet. We also recorded small increases in secretion of PLA₂ from midguts incubated in buffer amended with a specific phospholipid, phosphatidylcholine. Midguts incubated in buffer amended with increased concentrations of phospholipid did not yield higher levels of PLA₂ activity. Lepidopteran midguts can be divided into three regions, and we recorded the highest secretion of PLA₂ from the middle region and lowest secretion from the anterior region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA₂, we suggest that secretion of digestive enzymes in tobacco hornworms is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Arch. Insect Biochem. Physiol. 42:179–187, 1999 .© 1999 Wiley‐Liss, Inc.     

Hyperosmotic stress rapidly generates lyso-phosphatidic acid in Chlamydomonas

Plant cells are continuously exposed to environmental stresses such as hyper-osmolarity, and have to respond in order to survive. When 32P-labelled Chlamydomonas moewusii cells were challenged with NaCl, the formation of a new radiolabelled phospholipid was stimulated, which was barely detectable before stimulation. The phospholipid was identified as lyso-phosphatidic acid (LPA), and was the only lyso-phospholipid to be accumulated. The increase in LPA was dose- and time-dependent. When other osmotically active compounds were used, the formation of LPA was also induced with similar kinetics, although salts were better inducers than non-salts. At least part of the LPA was generated by phospholipase A2 (PLA2) hydrolysing phosphatidic acid (PA). This claim is based on PA formation preceding LPA production, and PLA2 inhibitors decreasing the accumulation of LPA and promoting the conversion of PA to diacylglycerol pyrophosphate. The latter is another metabolic derivative of PA that is implicated in cell signalling. The involvement of multiple lipid-signalling pathways in hyperosmotic stress responses is discussed.     

赤霉素生物合成与信号传递对植物株高的调控

植物株高是影响作物产量和品质的重要农艺性状。赤霉素(Gibberellins,GAs)是调控植物株高的重要激素,GA相关株高基因的克隆与分子机制研究对于合理调控作物生长发育和农业生产具有极其重要的利用价值,在水稻、小麦等粮食作物育种中得到了广泛应用。为了促进GA在果树、花卉等园艺作物育种中的有效利用,文中在分子生物学水平上介绍GA生物合成和GA信号传递途径对植物株高的调控。     

Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses

To uncover new pathways involved in low-temperature signal transduction, we screened for mutants altered in cold-induced expression of RCI2A, an Arabidopsis gene that is not a member of the CBF/DREB1 regulon and is induced not only by low temperature but also by abscisic acid (ABA), dehydration (DH) and NaCl. This was accomplished by generating a line of Arabidopsis carrying a transgene consisting of the RCI2A promoter fused to the firefly luciferase coding sequence. A number of mutants showing low or high RCI2A expression in response to low temperature were identified. These mutants also displayed deregulated RCI2A expression in response to ABA, DH or NaCl. Interestingly, however, they were not altered in stress-induced expression of RD29A, a CBF/DREB1-target gene, suggesting that the mutations affect signaling intermediates of CBF/DREB1-independent regulatory pathways. Several mutants showed alterations in their tolerance to freezing, DH or salt stress, as well as in their ABA sensitivity, which indicates that the signaling intermediates defined by the corresponding mutations play an important role in Arabidopsis tolerance to abiotic stresses. Based on the mutants identified, we discuss the involvement of CBF/DREB1-independent pathways in modulating stress signaling.