Plant Phosphatidylcholine-Hydrolyzing Phospholipases C NPC3 and NPC4 with Roles in Root Development and Brassinolide Signaling in Arabidopsis thaliana

Abstract

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.

Does diacylglycerol serve as a signaling molecule in plants?

“…23 In tobacco cell cultures, treatment with brassinolide raises the DAG content within 15 min through the elevation of PC/PLC activity in a concentration-dependent manner; at the same time the size of the PA pool is not significantly increased. 24 The DAG content of Dunaliella salina cells is rather high in comparison with most animal tissues, particularly in the chloroplast and plasma membrane. When confronted with osmotic shock, the plasma membrane DAG content increases markedly to a level sufficient to consider DAG to be a genuine potential secondary messenger in PLC-mediated signal transduction.…”

Biology and Biochemistry of Plant Phospholipases

“…These genes can be subdivided into two groups based on homology, with NPC1, NPC2 and NPC6 forming one group, and NPC3, NPC4 and NPC5 falling into the second (Wimalasekera et al, 2010). NPC1 and NPC2 are located on chromosome I and II, respectively, with the remaining genes found on chromosome III.…”

“…All of the Arabidopsis NPCs possess three conserved domains of unknown function; the first NPC subgroup also contains a putative secretory sequence at the N-terminus (Nakamura et al, 2005). In contrast, the three nonsecreted NPCs posses a potential zinc finger motif at their C-termini (Wimalasekera et al, 2010), but the importance of this region has not yet been demonstrated.…”

Function of polar glycerolipids in flower development in Arabidopsis thaliana

“…A similar trend was observed in NPC4, but was induced to a lesser extent. GUS-reporter expression analysis showed that NPC4 is expressed in immature anthers, which suggests a role of NPC4 in anther development [95]. Floral-organspecificity study in mature flowers showed that NPC6 is highly expressed in pistils [11].…”

“…Tissue specificity of their expression has been examined by RT-PCR [94,95]. NPC5 is highly specific to flowers and NPC4 is expressed highly both in flowers and siliques.…”