CHARACTERIZATION OF NUCLEASE ACTIVITIES AND DNA FRAGMENTATION INDUCEDUPON HYPERSENSITIVE RESPONSE CELL-DEATH AND MECHANICAL-STRESS

Programmed cell death (PCD) is activated during the response of multic ellular organisms to some invading pathogens. One of the key aspects o f this process is the degradation of nuclear DNA which is thought to f acilitate the recycling of DNA from dead cells. The PCD of tobacco pla nts (genotype NN) infected with tobacco mosaic virus (TMV) is accompan ied by the induction of nuclease activities and the cleavage of nuclea r DNA to fragments of about 50 kb. We examined the correlation between the increase in nuclease activities and the fragmentation of nuclear DNA during TMV- and bacteria-induced PCD in tobacco. We found that the increase in nuclease activities did not always correlate with fragmen tation of nuclear DNA. Thus, in addition to pathogens that induce PCD, mechanical injury and infiltration of leaves with 1 M sucrose or bact eria that did not induce PCD also resulted in an increase in nuclease activities. Analysis of nuclease activities in total leaf extracts, nu clear extracts, and intercellular fluid !i.e., apoplast) revealed that at least four different nuclease activities are induced during PCD in tobacco; of these at least three appear to be secreted into the inter cellular fluid. Although the latter were also induced in response to t reatments that did not result in DNA fragmentation, they may function in the recycling of plant DNA during late stages of PCD when the integ rity of the plasma membrane is compromised. This suggestion is support ed by the finding that DNA degradation occurred late during TMV-induce d PCD in tobacco. In addition, the finding of induced nuclease activit ies in the intercellular fluid raises the possibility that they may se rve a protective function by degrading the DNA of invading pathogens.