The Synthesis of Polyamines from Methionine in Intact and Disrupted Leaf Protoplasts of Virus-Infected Chinese Cabbage

Abstract

In exploring the role of the chloroplast in the multiplication of turnip yellow mosaic virus, the biosyntheses of the major viral polyamine, spermidine, as well as that of the tetramine, spermine were studied. The synthesis of these polyamines from [2-(14)C]methionine in protoplasts of Chinese cabbage leaf cells derived from healthy plants or those infected by turnip yellow mosaic virus were examined. Populations of protoplasts of infected leaves are homogeneous with respect to containing chloroplast aggregates in contrast to those of healthy leaves. Protoplast preparations have been shown to incorporate methionine into protein, spermidine, and spermine more rapidly than do fresh leaf discs, which also show a very slow utilization of labeled arginine and ornithine into polyamine.Protein synthesis is similar for 4 hours in both healthy and infected protoplasts. Accumulation of labeled spermidine stops after 2 hours in healthy protoplasts but continues in the infected protoplasts. Much of the newly synthesized protein and spermidine is present in the easily sedimentable fraction of the readily disrupted protoplasts.Disrupted and diluted protoplasts have a decreased ability to metabolize methionine to protein and spermidine. The residual synthetic activity is essentially entirely in the easily sedimentable fraction. However, this fraction is unable to synthesize spermine, an activity found in protoplasts and disrupted protoplasts. Disrupted protoplasts contain spermidine synthase (EC 2.5.1.16) and about a quarter of this activity is present in a low-speed sedimentable fraction containing the chloroplasts. The protoplast system is suitable for an analysis of polyamine synthesis in turnip yellow mosaic virus infection and appears particularly suitable for study of the distribution of the enzymes involved.