We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O 2 .Ϫ and H 2 O 2 and 7-fold lower levels of O 2 .Ϫ and H 2 O 2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O 2 .Ϫ , resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O 2 .Ϫ and H 2 O 2 were still found in NT-compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.

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The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re‐enter the cell cycle are unknown. Changes in protein profile, indole‐3‐acetic acid (IAA)‐oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine (Vitis vinifera) L. cv. Sultanina) and regenerating tobacco (Nicotiana tabacum) L. cv. Xanthi). Using [35S]‐methionine. SDS‐PAGE and two‐dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA‐oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.

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Ferric chelate reduction (FCR) activity of roots and leaves was determined in the grapevine rootstock 140 Ruggeri under iron (Fe)-deficient and Fe-sufficient conditions. Micropropagated plants were subjected to 0 or 40 μm Fe(III) EDTA in hydroponic culture. After 10 days of treatments, Fe-deficient plants had a lower level of root FCR activity and chlorophyll content compared with Fe-sufficient plants. Iron supply (20 μm) to Fe-deficient plants caused a rapid increase of root FCR activity (five times higher than initial values) and plants restored leaf chlorophyll content, whereas those not supplied with Fe retained reducing activity at low levels. In Fe-sufficient plants, root FCR activity decreased gradually when Fe was removed from the nutrient solution, reaching the same low levels as the Fe-deficient plants. Iron-sufficient plants displayed constitutively elevated root-reducing capacity for 3 weeks in culture under Fe-sufficient conditions. In Fe-deficient plants, root FCR activity was confined at the apical region of the lateral roots, whereas in Fe-treated plants, activity was detected along almost the whole length of the lateral roots. Root hair development, from the aspect of length and density, proved to be independent of the Fe status of rootstock plants. Leaf FCR activity was measured in mesophyll protoplasts from in vitro-growing Fe-deficient and Fe-sufficient plants. Mesophyll protoplasts reducing activity in Fe-deficient plants appeared to be decreased 43.5% or 40.3%, on a protoplast number or protein basis, respectively, in comparison with that in protoplasts from Fe-sufficient plant leaves. The results suggest that Fe is one of the agents inducing root and leaf FCR activity in grapevine rootstock 140 Ruggeri, altering furthermore its localization pattern in roots.