Changes in Polyphenoloxidase Activity in the Cucumber Mosaic Virus Infected Tobacco Leaves and Its Non-Responsibility for the Changes in Virus Concentration in vivo

Abstract

The purpose of this study is to find the changes in polyphenoloxidase (PPO) activity in cucumber mosaic virus (CMV) infected tobacco leaves at various periods after inoculation, and to find the effect of this enzyme on the changes in symptoms and virus concentration in vivo, which have been described in previous reports.Lower two leaves of tobacco seedlings (Nicotiana tabacunm L. cultivar Bright Yellow) were inoculated with ordinary strain of CMV (CMV-O), in an air-conditioned glasshouse at 25°C. PPO activity in the systemically infected upper three leaves was measured by the rapid method described by Shiroya, at weekly interval for 4 weeks after inoculation. Chlorogenic aid was used as the substrate of PPO reaction. PPO activity in the leaves increased from the 8th day to the 21st day after inoculation, and on the 14th and the 21st day the activities were significantly higher than those in healthy control leaves. On the 21st day, the difference of activities between infected and healthy leaves was highest, and reached 2.5 times on the basis of tissue dry weight. On the 28th day the enzyme activity decreased to the same level as on the 8th day (Fig. 2). These changes in enzyme activity were inversely proportional to the changes in symptoms and virus concentration in vivo.PPO activity in leaf extracts of tobacco plant grown in copper-deficient culture solution is known to decrease to about 1/10-1/25 of control non-deficient leaves, even when the sign of deficiency is not apparent. Tobacco plants were grown in Cu-deficient cuture solution prepared principally by the method of Staut and Arnon and inoculated with CMV-O. About two weeks after inoculation, greyish brown spots appeared on the lower leaves, as a sign of deficiency. During 4 weeks after inoculation, top leaves were harvested at weekly intervals, and homogenized with phosphate buffer (0.1M, pH 7.0) containing 1/30M Na-diethyldithiocarbamate. Infectivity in Cu-deficient and non-deficient leaves was compared by inoculation onto opposite primary leaves of cowpea. Changes in infectivity of Cu-dificient leaves were similar to those of non-deficient leaves (Fig. 5). Very low infectivity and masking of symptoms in newly developed leaves were obtained on the 21st day, even in Cu-deficient plants. The results were confirmed by using standard inoculum of vacuum-freeze dried infected leaf tissues and by serological assay.When Cu-deficient leaves were homogenized with purified yellow strain of CMV, the infectivity was 2.5-5 times higher than that of control inoculum with non-deficient leaves. In homogenate of Cu-deficient leaves, browning of sap was hardly detectable, whereas the non-deficient leaf sap showed strong browning. These results seem to indicate that deficient leaves has very low PPO activity, in agreement with Shiroya's previous finding.It appears that PPO is not directly responsible for the changes in symptoms and CMV concentration in vivo.