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Identification of ADPR cyclase in Arabidopsis

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ADP -ribosyl cyclase (ADPR cyclase) is involved in calcium homeostasis and shows ubiquitous activity in different tissues of marine invertebrates, amphibians, avians and mammals including humans. ADPR cyclase is a multifunctional enzyme that catalyses NAD and NADP into the two important second massengers cyclic adenosine diphosphoribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) respectively. It also catalyses the hydrolysis of cADPR to produce nicotinamide and ADP ribose. cADPR targets the ryanodine receptor (RyR) present in the sarco/endoplasmic reticulum (ER), whilst NAADP targets the two-pore channel (TPC) in the endolysosomes. Together they act as potent calcium mobilizers in the cell. cADPR elevates (Ca2+)cyt in plants and plays a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid (ABA). Despite evidence for the action of cADPR in plant cells, no proteins with significant similarity to the known ADPR cyclases have been identified in any plant genome database, suggesting that proteins with very low homology or a unique mechanism for cADPR synthesis must exist in plant cells. As the amount of this membrane bound protein is very low in plant cells, we are trying to apply pharmacological approaches using agonists (NaCl, H2O2 , NO and cold water) and antagonists (nicotinamide, 8-Br-cADPR, Mg2+) in order to increase the ADPR cyclase activity. Among the screened agonists, we found that NO is a potential candidate for increasing the activity of ADPR cyclase in Arabidopsis. We confirmed the presence of ADPR cyclase in the cytosol of this plant by the NGD fluorescence-based cycling assay and are trying to develop a new method, reverse cyclase assay, to measure low basal ADPR cyclase activity in Arabidopsis. In addition, we characterized Arabidopsis transformed with 35S:ADPRc by germination and flowering time assays. Although over expression of ADPR cyclase in the plant did not show significant difference in flowering time in SD conditions compared with wild type, a statistically significant difference in leaf number was observed in LD conditions. Furthermore, delayed germination was observed in 35S:ADPRc plants compared to wild-type when treated with ABA confirming the relationship of ABA and cADPR.

This talk is part of the Plant Sciences Research Seminars series.

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