The role of circadian and calcium signalling in the enhanced abiotic stress tolerance of Arabidopsis lines with altered NAD metabolism.

Hypothesis: Altered NAD metabolism increases plant tolerance to abiotic stress by altering the cellular concentration of cyclic adenosine diphosphate ribose (cADPR) and consequently, Ca signalling. Abiotic stresses are major limitation on the yield of field crops, which is 30-70% of theoretical maximum. Genetic manipulation to increase tolerance to stress will increase food security and bioenergy crop production. Bayer BioScience, as part of a strategy of innovative biotechnological solutions, have identified modification of NAD metabolism as a target for engineering improved tolerance to abiotic stresses. Arabidopsis and Brassica with reduced poly(ADP-ribose) polymerase (PARP) activity due to overexpression of hairpin PARP constructs or the application of metabolic inhibitors of PARP activity have increased tolerance to oxidative, high light and drought stress (De Block et al (2005) Plant J. 41, 95-106; Vanderauwera et al (2007) PNAS 104, 15150-15155). PARP is activated in response to stresses and DNA damage. PARP cleaves NAD to catalyse the addition of poly(ADP)ribose to proteins. Extreme consumption of NAD by PARP can lead to necrotic cell death. Inhibition of PARP might increase stress tolerance by reducing the consumption of NAD, thereby reducing the demand for mitochondrial respiration and reducing the production of reactive oxygen species (De Block et al, 2005). Recently, Bayer have identified another possible explanation for the increased stress tolerance. Plants expressing hairpin constructs of PARP2 have elevated levels of the hormone abscisic acid (ABA) and expression of both ABA- and cADPR-induced genes (Vanderauwera et al 2007). cADPR is a small intracellular signalling molecule that elevates cytosolic-free Ca concentration and participates in ABA signalling (Wu et al (1997) Science 278:2126-2129). Recently, the Webb lab demonstrated that cADPR is part of the circadian signalling network of plants (Dodd et al. 2007 Science 318, 1789 -1792). cADPR is generated by ADP ribosyl cyclase which, like PARP, consumes NAD. It is proposed that low PARP activity increases stress tolerance by stimulating cADPR production as a consequence of elevated NAD, leading to stimulation of Ca-sensitive stress-response pathways (Vanderauwera et al 2007). Lines with reduced PARP1/2 expression and activity have been previously identified (De Block et al (2005) Plant J. 41, 95-106). Levels of cADPR will be measured in these lines before and after a range of abiotic stress (ROS, high light, NaCl and cold). PARP knockdown lines will be transformed with aequorin and cameleon YC3.6, recombinant reporters of Ca to measure the consequence on abiotic Ca-signalling. Additionally, the effect on circadian function will be assayed by measuring circadian rhythms of leaf movement and Ca in PARP knockdown lines. The Webb laboratory demonstrated that the circadian clock modulates cold signalling (Dodd et al 2006 Plant J.48, 962 - 973) and Dr Hannah has recently demonstrated that cold disrupts circadian function affecting cold-responsive gene expression (Bieniawska et al (2008) Plant Physiology 147:263-279). Systems analysis of the publicly available transcriptome of plants with altered PARP activity will probe further the consequences of altered PARP activity on cADPR/Ca-sensitive signalling networks. First level analysis will identify the overlap with transcriptomes of plants with known cADPR and Ca signalling deficiencies and also the overlap of stress and circadian activated transcriptomes. A relational gene network will be constructed based on co-expression in multiple data sets. Hypotheses concerning key target genes affected by PARP knockdown will be examined using appropriate reverse genetic tools. Assays of PARP activity and systems approaches will be performed at Bayer, Gent. cADPR and Ca measurements, circadian assays and systems analysis will be performed in Cambridge.