| Abstract |
Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts but also, only protoplasts that are able to supply extracellularly H2O2 can actually divide (Siminis et al., 1993, 1994, de Marco and Roubelakis-Angelakis, 1996a, 1996b, 1999). In this work we have attempted to break down the oxidative burst response into individual active oxygen species (AOS), O2-. and H2O2, and generating systems during isolation of regenerating tobacco (Nicotiana tabacum L.) and nonregenerating grapevine (Vitis vinifera L.) mesophyll protoplasts. Wounding of leaf tissue or use of purified cellulase did not elicit AOS production. Use of non-purified cellulase Onozuka during maceration induced a burst of O2-. and H2O2 accumulation in tobacco leaf, while in grapevine significantly lower levels of both AOS were accumulated. When protoplasts isolated with purified cellulase were treated with non-purified cellulase, AOS were also generated with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed; one that showed specificity to NADPH and sensitivity to DPI and was responsible for O2-. production, and a second NAD(P)H activity, sensitive to KCN and NaN3, contributing to the production of both AOS. The first activity probably refers to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grapevine only one AOS generating activity was detected and was responsible for the generation of both AOS. Τobacco protoplasts resulted from non-purified cellulase showed greater O2.- and H2O2, mostly intracellularly, and lower viability and plating efficiency (nonregenerating, NR-), compared to protoplasts isolated with purified cellulase (regenerating, R-). Grapevine protoplasts failed to divide at all treatments (recalcitrant, nonregenerating, NR-). In NR- grapevine protoplasts, AOS accumulation was significantly lower. During a culture period of 8 days, R- and NR- tobacco protoplasts and grapevine protoplasts exhibited lower AOS levels both intra- and extracellularly, compared to freshly isolated protoplasts; higher O2.- and H2O2 the day 8 in NR-ones, was the main difference between R- and NR- tobacco protoplasts. In R-tobacco protoplasts, ASA and GSH predominated, whereas in the NR-ones and grapevine protoplasts, DHA and GSSG did. Furthermore, SOD, APO, MDAR, DHAR, GR and GS-POX activities were significantly greater in R-tobacco than in NR-tobacco and grapevine protoplasts. The increase in SOD and APO was due to the expression of cytoplasmic isoenzymes. Cultured protoplasts from transgenic tobacco plants expressing antisense RNA for cytoplasmic APO, showed the same level of viability and plating efficiency and of enzymic and nonenzymic antioxidants, as the control protoplasts. Protoplast death was induced when APO activity was inhibited up to 45%, with the addition of pCMB in protoplast culture medium. Significant higher endogenous polyamine levels and predominated conjugated forms were found in R-tobacco protoplasts compared to NR- tobacco and especially, grapevine protoplasts. These results suggest that a collapse of defense mechanism against oxidative stress occurred in NR- tobacco and grapevine protoplasts, suggesting that suppressed expression of totipotency in protoplasts is correlated with reduced antioxidant machinery.
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