Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species

Abstract

A strategy for elucidating specific molecular targets of calcium and calmodulin in plant defense responses has been developed. We have used a dominant‐acting calmodulin mutant (VU‐3, Lys to Arg115) to investigate the oxidative burst and nicotinamide co‐enzyme fluxes after various stimuli (cellulase, harpin, incompatible bacteria, osmotic and mechanical) that elicit plant defense responses in transgenic tobacco cell cultures. VU‐3 calmodulin differs from endogenous plant calmodulin in that it cannot be methylated post‐translationally, and as a result it hyperactivates calmodulin‐dependent NAD kinase. Cells expressing VU‐3 calmodulin exhibited a stronger active oxygen burst that occurred more rapidly than in normal control cells challenged with the same stimuli. Increases in NADPH level were also greater in VU‐3 cells and coincided both in timing and magnitude with development of the active oxygen species (AOS) burst. These data show that calmodulin is a target of calcium fluxes in response to elicitor or environmental stress, and provide the first evidence that plant NAD kinase may be a downstream target which potentiates AOS production by altering NAD(H)/NADP(H) homeostasis.

Regulation of the Arabidopsis Transcriptome by Oxidative Stress

“…Calmodulin is a calcium-binding protein that may well have a pivotal role in stress tolerance. Intracellular calcium concentrations increase in response to oxidative stress (Price et al, 1994), and calcium influx is required for the activation of ROS generation (Schwacke and Hager, 1992;Baker et al, 1993;Desikan et al, 1997;Harding et al, 1997). Furthermore, NADPH oxidase, a potential ROSgenerating enzyme, contains EF hand calciumbinding motifs (Desikan et al, 1998b;Keller et al, 1998), and at least one of the NADPH oxidase genes is induced by H 2 O 2 (Desikan et al, 1998b).…”

“…Moreover, a calmodulin has been shown to mediate between calcium and ROS generation in tobacco (Nicotiana tabacum) cells undergoing the hypersensitive response (HR). Calmodulin is a regulator of NAD kinase, which generates NADPH for NADPH oxidase activity (Harding et al, 1997). Thus, a significant amount of cross talk occurs between ROS and calcium, and both these signaling molecules mediate cross tolerance to a variety of stresses (Bowler and Fluhr, 2000).…”

Cloning and Characterization of Two NAD Kinases from Arabidopsis. Identification of a Calmodulin Binding Isoform

“…Nevertheless, there is considerable interest in plant NADKs mostly arising from the observation that plants possess both calmodulin (CaM)-independent and CaM-regulated NADK isoforms (Roberts and Harmon, 1992;Harding et al, 1997;Pou De Crescenzo et al, 2001). Indeed, NADK was the first CaM-regulated enzyme identified in plants (Muto and Miyachi, 1977;Anderson et al, 1980) and remains among the most studied at the biochemical level (Zielinski, 1998).…”

“…CaM is a ubiquitous and critical Ca 21 sensor found in all eukaryotes where it regulates a diverse array of targets involved in cytoskeletal rearrangements, signaling, metabolism, transcription, and ion homeostasis (Snedden and Fromm, 2001;Yang and Poovaiah, 2003). In plants, a CaM-dependent NADK has been suggested to play a metabolic role (Jarrett et al, 1982) and to participate in Ca 21 -mediated cellular defense against invading pathogens by helping to provide reductant (indirectly) for the NADPH-dependent oxidative burst (Harding et al, 1997). It is interesting to note that early reports suggested NADKs found in neutrophils (Williams and Jones, 1985) and sea urchin eggs (Epel et al, 1981;Iwasa and Mohri, 1983), both systems capable of generating a substantial oxidative burst, are activated by CaM.…”

Changes in Cell Ca2+ Distribution in Loquat Leaves and Its Effects on Cold Tolerance

“…It plays an important role in the signal reception, transduction and responses of plant to the external biotic and abiotic stresses (Guo et al, 2004;Snedden and Fromm, 2001). The role of calcium has been implicated in plant responses to cold tolerance (Braam and Davis, 1990), mechanical stimulation (Botella and Arteca, 1994;Braam and Davis, 1990), drought stress (Shao et al, 2008) and oxidative stress (Harding et al, 1997). Calcium also assists in maintaining stabilization and integrity of cell membrane and its function (Msadek et al, 1998), reducing lipid peroxidation (Zeng et al, 1987), and inducing gene expression and synthesis of specific proteins (Gao and Chen, 2002;Kim et al, 2009).…”