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

Abstract

Summary 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 α‐, β‐ and γ‐classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three α‐ and two β‐classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDβ1 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 µg ml−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, PLDβ1 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 PLDβ1 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 PLDβ1 is a signalling enzyme is discussed.

The role of phospholipase D in plant stress responses

“…reported increased PLD activity in a rice (Oryza sativa) cell-suspension culture treated with an N-acetylchitooligosaccharide elicitor and implied its involvement in ROS production by making use of exogenous application of PA and n-butanol as a PLD inhibitor. Increased PLD activity has also been found in tomato (Lycopersicon esculentum) cell-suspension cultures treated with the fungal elicitor xylanase (van der Luit et al, 2000) and LePLDβ1 was shown to be transcriptionally up-regulated both in tomato cell-suspensions and in leaves (Laxalt et al, 2001). A study of LePLDβ1-silenced cell-suspensions indicated that this isoform is responsible for the increased elicitor-induced PLD activity and implicated its involvement in ROS production, polyphenol oxidase activity and cell-wall remodeling .…”

“…PLD has also been proposed to function as a tether between a protein and membranes (Munnik and Musgrave, 2001). This hypothesis makes use of the twostep transphosphatidylation reaction, and states that the phosphatidyl-PLD intermediate could serve as a transient membrane anchor.…”

“…The tomato cellsuspension culture Msk8 has been used in several studies to investigate plant responses to both biotic stress (Felix et al, 1991;Felix et al, 1999) and abiotic stress (Felix et al, 2000). Our lab has also used this cell-suspension culture in the study of lipid signaling and especially the function of PLD therein (van der Luit et al, 1999;Laxalt et al, 2001;Bargmann et al, 2006). Here, an RNAi construct was used to knock-down LePLDα1 (see Methods).…”

“…The plant PLD family can be partitioned into six classes, the α-, β-, γ-, δ-, ε-and ζ-PLDs, depending on the protein sequence homology and biochemical properties Wang, 2005). Laxalt et al (2001) cloned five members of the PLD family from tomato and examined their expression in different organs of the plant. These authors went on to investigate changes in PLD expression in a tomato cell-suspension culture treated with various stimuli.…”

“…This finding was investigated further by targeting LePLDβ1 for RNAi-mediated gene silencing in tomato cell-suspension cultures and subsequent analysis of the effects of LePLDβ1 knock-down on the response to xylanase . Because LePLDα1 expression is increased upon treatment of tomato cell-suspension cultures with salt (Laxalt et al, 2001), we decided to explore this finding in detail in tomato cell-suspensions and plants as well as Arabidopsis plants.…”