Physiological and molecular analysis of nitric oxide synthase during bacterial infection of pea (Pisum sativum L.)

In animal cells, the production of NO is catalyzed by nitric oxide synthase (NOS). Although NOS activity has been documented in plants, the process of NO synthesis in plants is not well understood. Isolation of a NOS protein and/or cloning of the corresponding gene will greatly facilitate the understanding of NO synthesis and its role in plant defense against pathogen infection. The objectives of this research were to analyze the physiological and biochemical properties of a NOS-like protein (peaNOS) of pea, to purify and characterize peaNOS, and to clone the genes) encoding peaNOS and relate its expression to NOS activity in pea-bacteria interactions. Induced NOS activity was observed in both the incompatible and compatible bacteria-pea interactions suggesting that NO generation may also be a general response to biotic stress in plants. Antibodies raised against mammalian NOS did not have apparent specificity for isolating a NOS protein from pea. The peaNOS protein was most efficiently extracted under alkaline conditions (pH 8.5 and 9.0). The NOS activity of peaNOS was dependent upon NADPH, Ca2+, and CaM although peaNOS protein lacks binding sites for NADPH and calmodulin (CaM). Analysis of Arabidopsis thaliana genome database identified two gene sequences related to animal NOS, i.e., At4g09680 (similar to NOS of Rattus norvegicus ) and At3g47450 (similar to NOS of Helix pomatia). Gene At4g09680 was not expressed in tissues examined. A partial 784-bp peaNOS cDNA was successfully cloned using RNA template of pea HR tissues. The 784-bp peaNOS cDNA had 50% nucleotide identity to the At3g47450 coding sequence. The correlation of the gene expression of P protein of glycine decarboxylase complex (GDC) of pea and NOS activity during HR was not demonstrated here but the pea P protein gene was highly expressed concomitant with NOS activity during disease development. The NOS-like protein involved in NO production during HR in pea appears to be more related to At3g47450, and is possibly encoded partially by the cloned 784-bp pea cDNA.