Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

Abstract

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.

The Complexity of Enzymic Control of Hydrogen Peroxide Concentration May Affect the Regeneration Potential of Plant Protoplasts

“…5C) basically confirmed our previously reported data for the first 7 d of culture (Siminis et al, 1994). In this work the period of protoplast culture was extended to 14 d. The pattern of CAT activity did not differ between dividing and nondividing protoplasts, although the absolute values were higher in regenerating protoplasts during culture.…”

“…In this work the period of protoplast culture was extended to 14 d. The pattern of CAT activity did not differ between dividing and nondividing protoplasts, although the absolute values were higher in regenerating protoplasts during culture. The ac- tivity staining of native gels (data not shown) confirmed the results already published (Siminis et al, 1994). Light exhibited a stimulatory effect on CAT in regenerating protoplasts (Table 111).…”

“…Mesophyll protoplasts were isolated and cultured as described (Koop and Schweiger, 1985;Katsirdakis and RoubelakisAngelakis, 199213); 4 h of maceration of leaf mesophyll tissue in hydrolytic solution (1% cellulase, 0.5% macerozyme) was used to obtain regenerating protoplasts, and 18 h of maceration in the same hydrolytic solution resulted in recalcitrant protoplasts (Siminis et al, 1994). Unless otherwise specified, the term "regenerating/dividing" is used in the text for protoplasts resulting from the 4 h of maceration and "nonregenerating/nondividing" is used for protoplasts resulting from the 18 h of maceration.…”

“…Toxic oxygen may affect the morphogenic response of cells and other plant explants Roubelakis-Angelakis, 1993, 1994) and its increasing accumulation after protoplast isolation has been indicated as a cause of recalcitrance (Siminis et al, 1994). Cell-wall reconstitution is another crucial step in the regeneration process of protoplasts (Hahne and Hoffmann, 1984;Katsirdakis and Roubelakis-Angelakis, 1992a) and it relies on H,O,-dependent POX activity (Mader et al, 1980).…”

“…Vol. 110, 1996 In this work we have isolated protoplasts from Nicotiana tabacum that exhibit both dividing and nondividing potential, according to Siminis et al (1994). We have followed the changes in the H,O,-dependent enzymic activities in the two systems during a 2-week period of protoplast culture and found that (a) a higher scavenging capability could ensure a better regenerating potential and (b) protoplast isolation induces a modification in the activities of the enzymes related to cell-wall reconstitution.…”