Stress- and phospholipid signalling responses in Arabidopsis PLC4-KO and -overexpression lines under salt- and osmotic stress

Several drought and salt tolerant phenotypes have been reported when overexpressing (OE) phospholipase C (PLC) genes across plant species. In contrast, a negative role for Arabidopsis PLC4 in salinity stress was recently proposed, showing that roots of PLC4-OE seedlings were more sensitive to NaCl while plc4 knock-out (KO) mutants were more tolerant. To investigate this apparent contradiction, and to analyse the phospholipid signalling responses associated with salinity stress, we performed root growth- and phospholipid analyses on plc4-KO and PLC4-OE seedlings subjected to salinity (NaCl) or osmotic (sorbitol) stress and compared these with wild type (WT). Only very minor differences between PLC4 mutants and WT were observed, which even disappeared after normalization of the data, while in soil, PLC4-OE plants were clearly more drought tolerant than WT plants, as was found earlier when overexpressing Arabidopsis PLC2, -3, -5, -7 or -9. We conclude that PLC4 plays no opposite role in salt-or osmotic stress and rather behaves like the other Arabidopsis PLCs

Acclimation to salt modifies the activation of several osmotic stress-activated lipid signalling pathways in <em>Chlamydomonas</em>

Osmotic stress rapidly activates several phospholipid signalling pathways in the unicellular alga Chlamydomonas. In this report, we have studied the effects of salt-acclimation on growth and phospholipid signalling. Growing cells on media containing 100 mM NaCl increased their salt-tolerance but did not affect the overall phospholipid content, except that levels of phosphatidylinositol phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] were reduced by one-third. When these NaCl-acclimated cells were treated with increasing concentrations of salt, the same lipid signalling pathways as in non-acclimated cells were activated. This was witnessed as increases in phosphatidic acid (PA), lyso-phosphatidic acid (L-PA), diacylglycerol pyrophosphate (DGPP), PI(4,5)P2 and its isomer PI(3,5)P2. However, all dose-dependent responses were shifted to higher osmotic-stress levels, and the responses were lower than in non-acclimated cells. When NaCl-acclimated cells were treated with other osmotica, such as KCl and sucrose, the same effects were found, illustrating that they were due to hyperosmotic rather than hyperionic acclimation. The results indicate that acclimation to moderate salt stress modifies stress perception and the activation of several downstream pathways

Multiple PLDs Required for High Salinity and Water Deficit Tolerance in Plants

High salinity and drought have received much attention because they severely affect crop production worldwide. Analysis and comprehension of the plant's response to excessive salt and dehydration will aid in the development of stress-tolerant crop varieties. Signal transduction lies at the basis of the response to these stresses, and numerous signaling pathways have been implicated. Here, we provide further evidence for the involvement of phospholipase D (PLD) in the plant's response to high salinity and dehydration. A tomato (Lycopersicon esculentum) α-class PLD, LePLDα1, is transcriptionally up-regulated and activated in cell suspension cultures treated with salt. Gene silencing revealed that this PLD is indeed involved in the salt-induced phosphatidic acid production, but not exclusively. Genetically modified tomato plants with reduced LePLDα1 protein levels did not reveal altered salt tolerance. In Arabidopsis (Arabidopsis thaliana), both AtPLDα1 and AtPLDδ were found to be activated in response to salt stress. Moreover, pldα1 and pldδ single and double knock-out mutants exhibited enhanced sensitivity to high salinity stress in a plate assay. Furthermore, we show that both PLDs are activated upon dehydration and the knock-out mutants are hypersensitive to hyperosmotic stress, displaying strongly reduced growth

Phospholipid Signaling Responses in Salt-Stressed Rice Leaves

Salinity is one of the major environmental factors limiting growth and productivity of rice plants. In this study, the effect of salt stress on phospholipid signaling responses in rice leaves was investigated. Leaf cuts were radiolabeled with 32P-orthophosphate and the lipids extracted and analyzed by thin-layer chromatography, autoradiography and phosphoimaging. Phospholipids were identified by co-migration of known standards. Results showed that 32Pi was rapidly incorporated into the minor lipids, phos-phatidylinositol bisphosphate (PIP2) and phosphatidic acid (PA) and, interestingly, also into the structural lipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), which normally label relatively slowly, like phosphatidylcholine (PC) and phosphatidylinositol (PI). Only very small amounts of PIP2 were found. However, in response to salt stress (NaCl), PIP2 levels rapidly (<30 min) increased up to 4-fold, in a time- and dose-dependent manner. PA and its phosphorylated product, diacylglyc-erolpyrophosphate (DGPP), also increased upon NaCl stress, while cardiolipin (CL) levels decreased. All other phospholipid levels remained unchanged. PA signaling can be generated via the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK) or directly via phospholipase D (PLD). The latter can be measured in vivo, using a transphosphatidylation assay. Interestingly, these measurements revealed that salt stress inhibited PLD activity, indicating that the salt stress-induced PA response was not due to PLD activity. Comparison of the 32P-lipid responses in salt-tolerant and salt-sensitive cultivars revealed no significant differences. Together these results show that salt stress rapidly activates several lipid responses in rice leaves but that these responses do not explain the difference in salt tolerance between sensitive and tolerant cultivars

Rapid phosphatidic acid accumulation in response to low temperature stress in Arabidopsis is generated through diacylglycerol kinase.

Phosphatidic acid (PtdOH) is emerging as an important signaling lipid in abiotic stress responses in plants. The effect of cold stress was monitored using (32)P-labeled seedlings and leaf discs of Arabidopsis thaliana. Low, non-freezing temperatures were found to trigger a very rapid (32)P-PtdOH increase, peaking within 2 and 5 min, respectively. In principle, PtdOH can be generated through three different pathways, i.e., (1) via de novo phospholipid biosynthesis (through acylation of lyso-PtdOH), (2) via phospholipase D hydrolysis of structural phospholipids, or (3) via phosphorylation of diacylglycerol (DAG) by DAG kinase (DGK). Using a differential (32)P-labeling protocol and a PLD-transphosphatidylation assay, evidence is provided that the rapid (32)P-PtdOH response was primarily generated through DGK. A simultaneous decrease in the levels of (32)P-PtdInsP, correlating in time, temperature dependency, and magnitude with the increase in (32)P-PtdOH, suggested that a PtdInsP-hydrolyzing PLC generated the DAG in this reaction. Testing T-DNA insertion lines available for the seven DGK genes, revealed no clear changes in (32)P-PtdOH responses, suggesting functional redundancy. Similarly, known cold-stress mutants were analyzed to investigate whether the PtdOH response acted downstream of the respective gene products. The hos1, los1, and fry1 mutants were found to exhibit normal PtdOH responses. Slight changes were found for ice1, snow1, and the overexpression line Super-ICE1, however, this was not cold-specific and likely due to pleiotropic effects. A tentative model illustrating direct cold effects on phospholipid metabolism is presented

Extracellular spermine triggers a rapid intracellular phosphatidic acid response in arabidopsis, involving PLDδ activation and stimulating ion flux

Polyamines, such as putrescine (Put), spermidine (Spd), and spermine (Spm), are low-molecular-weight polycationic molecules found in all living organisms. Despite the fact that they have been implicated in various important developmental and adaptative processes, their mode of action is still largely unclear. Here, we report that Put, Spd, and Spm trigger a rapid increase in the signaling lipid, phosphatidic acid (PA) in Arabidopsis seedlings but also mature leaves. Using time-course and dose-response experiments, Spm was found to be the most effective; promoting PA responses at physiological (low μM) concentrations. In seedlings, the increase of PA occurred mainly in the root and partly involved the plasma membrane polyamine-uptake transporter (PUT), RMV1. Using a differential 32Pi-labeling strategy combined with transphosphatidylation assays and T-DNA insertion mutants, we found that phospholipase D (PLD), and in particular PLDδ was the main contributor of the increase in PA. Measuring non-invasive ion fluxes (MIFE) across the root plasma membrane of wild type and pldδ-mutant seedlings, revealed that the formation of PA is linked to a gradual- and transient efflux of K+. Potential mechanisms of how PLDδ and the increase of PA are involved in polyamine function is discussed