Non-specific phospholipases C (NPCs) were discovered as a novel type of plant phospholipid-cleaving enzyme homologous to bacterial phosphatidylcholine-specific phospholipases C and responsible for lipid conversion during phosphate-limiting conditions. The six-gene family was established in Arabidopsis, and growing evidence suggests the involvement of two articles NPCs in biotic and abiotic stress responses as well as phytohormone actions. In addition, the diacylglycerol produced via NPCs is postulated to participate in membrane remodelling, general lipid metabolism and cross-talk with other phospholipid signalling systems in plants. This review summarises information concerning this new plant protein family and focusses on its sequence analysis, biochemical properties, cellular and tissue distribution and physiological functions. Possible modes of action are also discussed.

Summary Membrane lipids and cytoskeleton dynamics are intimately inter‐connected in the eukaryotic cell; however, only recently have the molecular mechanisms operating at this interface in plant cells been addressed experimentally. Phospholipase D (PLD) and its product phosphatidic acid (PA) were discovered to be important regulators in the membrane–cytoskeleton interface in eukaryotes. Here we report the mechanistic details of plant PLD–actin interactions. Inhibition of PLD by n‐butanol compromises pollen tube actin, and PA rescues the detrimental effect of n‐butanol on F‐actin, showing clearly the importance of the PLD–PA interaction for pollen tube F‐actin dynamics. From various candidate tobacco PLDs isoforms, we identified NtPLDβ1 as a regulatory partner of actin, by both activity and in vitro interaction assays. Similarly to published data, the activity of tobacco PIP2‐dependent PLD (PLDβ) is specifically enhanced by F‐actin and inhibited by G‐actin. We then identified the NtPLDβ1 domain responsible for actin interactions. Using sequence‐ and structure‐based analysis, together with site‐directed mutagenesis, we identified Asn323 and Thr382 of NtPLDβ1 as the crucial amino acids in the actin‐interacting fold. The effect of antisense‐mediated suppression of NtPLDβ1 or NtPLDδ on pollen tube F‐actin dynamics shows that NtPLDβ1 is the active partner in PLD–actin interplay. The positive feedback loop created by activation of PLDβ by F‐actin and of F‐actin by PA provides an important mechanism to locally increase membrane–F‐actin dynamics in the cortex of plant cells.

Phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphocholine and diacylglycerol (DAG). PC-PLC has a long tradition in animal signal transduction to generate DAG as a second messenger besides the classical phosphatidylinositol splitting phospholipase C (PI-PLC). Based on amino acid sequence similarity to bacterial PC-PLC, six putative PC-PLC genes (NPC1 to NPC6) were identified in the Arabidopsis genome. RT-PCR analysis revealed overlapping expression pattern of NPC genes in root, stem, leaf, flower, and silique. In auxin-treated P(NPC3):GUS and P(NPC4):GUS seedlings, strong increase of GUS activity was visible in roots, leaves, and shoots and, to a weaker extent, in brassinolide-treated (BL) seedlings. P(NPC4):GUS seedlings also responded to cytokinin with increased GUS activity in young leaves. Compared to wild-type, T-DNA insertional knockouts npc3 and npc4 showed shorter primary roots and lower lateral root density at low BL concentrations but increased lateral root densities in response to exogenous 0.05-1.0 μM BL. BL-induced expression of TCH4 and LRX2, which are involved in cell expansion, was impaired but not impaired in repression of CPD, a BL biosynthesis gene, in BL-treated npc3 and npc4. These observations suggest NPC3 and NPC4 are important in BL-mediated signaling in root growth. When treated with 0.1 μM BL, DAG accumulation was observed in tobacco BY-2 cell cultures labeled with fluorescent PC as early as 15 min after application. We hypothesize that at least one PC-PLC is a plant signaling enzyme in BL signal transduction and, as shown earlier, in elicitor signal transduction.

The receptor for D-myo-inositol 1,4,5-trisphosphate (InsP3-R) has been well documented in animal cells. It constitutes an important component of the intracellular calcium signalling system. Today the corresponding genes in many species have been sequenced and the antibodies against some of the InsP3-Rs are available. In contrast, very little is known about its plant counterpart. Only a few published works have dealt directly with this topic. This review summarizes the available relevant data and determines some properties of putative plant receptor(s) including the in silico search for its gene in plant genomes, in vivo evidence, its electrophysiology, the parameters of InsP3-induced calcium release and InsP3 binding, immunological cross-reactivity, and subcellular localization. Future progress in this area seems to be inevitable as, despite the efforts, its gene in plants has not been identified yet.

SUMMARYRemarkable progress in various techniques of in vivo fluorescence microscopy has brought an urgent need for reliable markers for tracking cellular structures and processes. The goal of this manuscript is to describe unexplored effects of the FM (Fei Mao) styryl dyes, which are widely used probes that label processes of endocytosis and vesicle trafficking in eukaryotic cells. Although there are few reports on the effect of styryl dyes on membrane fluidity and the activity of mammalian receptors, FM dyes have been considered as reliable tools for tracking of plant endocytosis. Using plasma membrane-localized transporters for the plant hormone auxin in tobacco BY-2 and Arabidopsis thaliana cell suspensions, we show that routinely used concentrations of FM 4-64 and FM 5-95 trigger transient re-localization of these proteins, and FM 1-43 affects their activity. The active process of re-localization is blocked neither by inhibitors of endocytosis nor by cytoskeletal drugs. It does not occur in A. thaliana roots and depends on the degree of hydrophobicity (lipophilicity) of a particular FM dye. Our results emphasize the need for circumspection during in vivo studies of membrane proteins performed using simultaneous labelling with FM dyes.