Physiological and Molecular Analysis of Nitric Oxide Synthase During Bacterial Infection of Pea (Pisum sativum L.)

Abstract

Nitric oxide (NO) and reactive oxygen species are two key components in the induction of the hypersensitive response (HR) during plant defense against pathogen infection. 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 the NOS protein and/or cloning of the corresponding gene will greatly facilitate the understanding of NO synthesis and its role in plant defense. The objectives of this research were to analyze the physiological and biochemical properties of a NOS-like protein (peaNOS) of pea (Pisum sativum L.), to purify and characterize peaNOS, and to clone the gene(s) encoding peaNOS and relate its expression to NOS activity in pea-bacteria interactions. The application of abiotic agents that induce systemic defense in plants [copper chloride, Actiguard®, Triton-X100 and salicylic acid (SA)] to pea leaves did not induce NOS activity and verified reports that NO and NOS function upstream of SA in the signaling pathway of defense responses. Maximum (two-fold) NOS activity was detected three hours before the onset of HR in pea leaves infiltrated with incompatible bacteria (Ralstonia solanacearum), which is consistent with the effect of NO in the activation of HR after interaction with H2O2. The compatible bacteria (Pseudomonas syringae pv pisi) induced NOS activity significantly, 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 and utility for isolating peaNOS and should be used with caution in non-mammalian systems. The peaNOS protein was most efficiently extracted under alkaline conditions (pH 8.5 and 9.0) as compared to the neutral conditions (pH 7.4-7.5) in animal systems. Precipitation of the peaNOS protein with various concentrations of ammonium sulfate, sodium citrate and sodium chloride caused rapid loss of NOS activity. The peaNOS protein did not bind to 2',5'-ADP-Sepharose and calmodulin (CaM)-agarose indicating that the protein lacks binding sites for NADPH and CaM. Cloning of a peaNOS gene based on mammalian NOS was unsuccessful suggesting that the structure of peaNOS gene may be significantly different from mammalian NOS. Analysis of the Arabidopsis thaliana genome database identified two gene sequences related to animal NOS, i.e., accessions At4g09680 (similar to NOS of Rattus norvegicus) and At3g47450 (similar to NOS of Helix pomatia). Gene At4g09680 is probably not expressed since attempts to clone cDNA of this gene using reverse transcription-polymerase chain reaction (RT-PCR) consistently failed, even when RNA of Arabidopsis was used as a template. A potential expressed peaNOS gene was successfully cloned using RNA template of pea HR tissues in RT-PCR. The 784-bp peaNOS cDNA sequence had 50% nucleotide identity to the At3g47450 coding sequence and had no other significant match in the database. The correlation of the gene expression of P protein of glycine decarboxylase complex (GDC) of pea (peaP) and NOS activity during HR in pea was not demonstrated here but peaP 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 sequence, and is possibly encoded partially by the cloned 784-bp pea cDNA.