Abstract
Coconut palm (Cocos nucifera L.), is a versatile crop with multifold uses, important for the livelihood and security of people in Kerala. Kerala Root (wilt) disease (KWD) is a major production constraint causing an estimated yield loss of 968 million nuts in southern India. Affected palms show bending of leaflets (flaccidity), foliar yellowing, and marginal necrosis. Phytoplasma have been observed to be associated with this disease by electron (EM) and transmission microscopy. Pathogenesis induces biochemical and physiological changes in host plants that may leads to the production of ractive oxygen species (ROS) such as superoxide anion, hydrogen peroxide. As an initial part, extracts from healthy and diseased palm were screened for NADPH oxidase activity to know the level of superoxide anion production and to analyze the hypersensitive response developed in palm due to phytoplasma infection. NOX activity ranged from 132 U/mg protein in healthy to 373.5 U/mg in diseased. Subsequently, NADPH oxidase (NOX) of Kerala wilt diseased coconut palms was extracted and purified through ammonium sulfate, gel filtration and HPLC chromatography. The purification factor for NOX was 856 fold with 5.2% yield. The enzyme was characterized for thermal stability, pH and kinetic parameters. The molecular mass of purified NOX was approximately 49 kDa estimated by gel filtration chromatography. The purity was checked by ingel showing a single prominent band. The pH optimum was found to be 7.0. The enzyme had a temperature optimum at 30°C and was relatively stable at 70ºC with 48% loss of activity. Inhibitors such as Diphenyleneiodonium (DPI), sodium diethyldithiocarbamate (NaDCC) and sodium azide (NaN3) significantly inhibit NOX activity. NADPH was found to be the efficient substrate for NOX considering the Vmax/km ratio. The data obtained in this study may help to understand the properties of NOX related to superoxide anion production due to hypersensitive response of the palm. Further works are designed to trace the ROS cycle in the KWD palm and to design a biocontrol as remedy for the disease.
