Altered NAD metabolism to enhance plant stress tolerance

Abiotic stress limits crop yield to 30-70% of its theoretical maximum. Bayer BioScience have identified modification of NAD metabolism as a target for engineering improved stress tolerance, particularly via down-regulation of polyADP-ribose polymerase (PARP) (De Block et al 2005 Plant J. 41, 95-106; Vanderauwera et al 2007 PNAS 104, 15150-15155). Nicotinamide (NIC) is a product and inhibitor of many NAD consuming enzymes, including PARP, sirtuins (SIRT), which are histone deacetylases and ADP ribosyl cyclase that generates cyclic ADP ribose (cADPR), a Ca2+ agonist. Addition of NIC inhibits abscisic acid signalling, stress-induced Ca2+ signalling and increases the period of the circadian clock by 3 h in plants (Dodd et al 2007 Science 318, 1789-1792) and mammals (Nakahata et al 2008 Cell 134, 329-340). In mammals, it is proposed that NAD synthesis from NIC via the salvage pathway oscillates in a circadian manner to regulate SIRT1 activity and circadian gene expression (Nakahata et al 2009 Science, 324. 654-657). NIC lengthens clock period independently of SIRT1 activity (Nakahata et al 2008 Cell 134, 329-340), suggesting an additional mechanism. In plants, we proposed that cADPR regulates the clock through a Ca2+-dependent pathway (Dodd et al. 2007 Science 318, 1789 -1792), which may also operate in mammals (Ikeda et al 2003 Neuron 38, 253-263). NIC is thus a powerful tool to dissect the role of NAD metabolism in circadian clock function and stress tolerance. We will identify mutants with altered responses to NIC. Ethyl methanesulfonate-mutagenized Arabidopsis seedlings carrying both the CHLOROPHYLL A/B BINDING PROTEIN2:luciferase and CAULIFLOWER MOSIAC VIRUS 35S:AEQUORIN reporters will be screened for altered circadian response to NIC by measuring CAB2:luc activity in a photon counting camera. Comparison of luminescence intensity of NIC-treated individuals at 96 h in continuous light (LL) with treated and untreated WT seedlings will be sufficient to identify candidate mutant phenotypes because NIC treated plants will be 12 h phase shifted (3 h period increase x 4 days). We will screen up to 6000 individuals per week (200 seedlings measurement-1, 6 measurements day-1). Candidates lines will be validated via automated imaging of circadian rhythms of CAB2:luc in LL +/- NIC treatment. Bone fide mutants will be crossed to circadian mutants to identify allelism to known clock genes. In a parallel high throughput, low complexity screen for NIC-response mutants, we will identify mutants with altered sensitivity to NIC-induced inhibition of germination. Mutations will be mapped using simultaneous mapping and mutation identification by deep sequencing (Schneeberger et al (2009) Nat Methods 6: 550-551) funded by Bayer CropScience. If costs are prohibitive, we will use recombinant mapping coupled to sequencing of candidate genome regions. Mutant lines will be further screened for stress tolerance phenotypes, including cold and drought responses, alterations in stress-induced gene expression, alterations in Ca2+ signalling and changes in NAD and cADPR levels. Once a mutated gene is known, further alleles will be sought from the stock centres and characterised. Complementation studies and backcrossing will be used to confirm genetic and phenotypic relationships. Based on the results from stress tolerance phenotyping and novelty, the most interesting 1 or 2 genes will be selected for detailed analysis within this project. Depending on the identity of the mutated gene, appropriate biochemical and molecular assays will be performed to determine the mode of action in the circadian and stress signalling networks. E.g deactylase mutants will prompt studies focusing on chromatin remodelling. Screening and associated plant work, physiological characterisation and analysis of gene function will be performed at Cambridge. Stress tolerance phenotyping, marker analysis/sequencing for gene identification will be performed at Bayer, Gent.