Monitoring the stability of Rubisco in micropropagated grapevine (Vitis vinifera L.) by two-dimensional electrophoresis

Plants cultured in vitro suffer from several physiological and biochemical impairments due to the artificial conditions of growth, namely the composition of the heterotrophic media. Upon transfer to ex vitro, the higher irradiances, compared to in vitro, can lead to oxidative stress symptoms, which can be counteracted by CO2 concentrations above atmospheric levels. Here we analyse the stability of Rubisco in in vitro grapevine plantlets, and after transfer to ex vitro under four acclimatization treatments: low irradiance (LL, 150 micromol m(-2)s(-1)) and high irradiance (HL, 300 micromol m(-2)s(-1)) in association with CO2 concentrations of 350 (LCO2) and 700 (HCO2) microL L(-1). Proteins were separated with SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis and Rubisco degradation peptides were analysed by immunoblotting with anti-LSU antibodies. These degradation products were present in the leaves of plantlets under both in vitro and ex vitro treatments. Under LCO2 they were maintained for almost all of the 28 days of the acclimatization period, while becoming scarcely detected after 14 days under HCO2 and after 7 days when HCO2 was associated with HL. These results appear to confirm the counteraction of HCO2 concentrations over the oxidative stress eventually caused by HL. The patterns of soluble sugars in acclimatizing leaves under HLHCO2 also gave an indication of a faster acquisition of autotrophic characteristics.