Minisymposium 4: Oxidative stress

Abs # 14005: AtPOS5-2 is a calmodulin-independent NAD⁺ kinase found in Arabidopsis stroma

Presenter:	Guenther, James F, jguenth@utk.edu
Authors	Guenther, James F (A)   Ashley, David W (A)   Choi, Won-Gyu  (A)   Kabachinski, Gregory L (A)   Roberts, Daniel M (A)
Affiliations:	(A): The University of Tennessee

Regulation of cellular pools of nicotinamide adenine nucleotides is mediated by the enzyme NAD⁺ kinase (NADK) which converts NAD⁺ to NADP⁺ using ATP as a phosphodonor. Conversion of NAD⁺ to NADP⁺ is associated with physiological responses such as light regulated shifts in redox co-enzymes during photosynthesis, and also possibly in plant defense as well as regulation of reactive oxygen levels. Biochemical analysis shows two activities of NADK in plants: 1) A Ca²⁺ and calmodulin-dependent activity, and 2) a calmodulin-independent activity. Analysis of transgenic plants that express an ectopic calmodulin that hyperactivates NADK shows elevated ROS production, supporting a role of this enzyme in defense responses. Examination of the Arabidopsis genome reveals three gene products containing predicted NAD kinase domains. These three gene products were termed AtPOS5-1,2,and 3, due to their similarity to the POS5p protein, an NAD⁺(H) kinase involved in oxidative stress-adaption in yeast. All three AtPOS5 proteins possess a conserved carboxyl-terminal NADK domain fused to an extended amino-terminal region which is not conserved in yeast and bacterial NADKs. However, orthologs with high similarity are found in Oryza sativa (OsPOS5). Functional analysis of AtPOS5 and OsPOS5 proteins produced by invitro transcription/translation or by expression of the recombinant proteins in E. coli shows that these enzymes are NADKs with calmodulin-independent activity. Examination of AtPOS5-2 reveals a predicted chloroplast transit peptide at the amino terminus, and Western blotting using an AtPOS5-2 specific antibody displays a immunoreactive band of the predicted molecular weight of AtPOS5-2 in purified stroma fractions of Arabidopsis and spinach. Consistent with this finding, the stromal fraction contains a calmodulin-independent NADK. This activity is stimulated by reduction by redox compounds (DTT, glutathione, and thioredoxin) consistent with the previous finding of light driven increases in stromal NADP⁺/NAD⁺ ratios. The data suggest that AtPOS5-2 is a calmodulin-independent chloroplastic NADK which may be responsible for the light-regulated increase in NADP⁺ associated with photosynthesis. (Supported by National Science Foundation grant MCB-0237219 to DMR)