Covalent dimerization of ribulose bisphosphate carboxylase subunits by UV radiation

Abstract

The effect of UV radiation (UV-A, UV-B and UV-C) on ribulose bisphosphate carboxylase from a variety of plant species was examined. The exposition of plant leaves or the pure enzyme to UV radiation produced a UV-dependent accumulation of a +5 kDa polypeptide (P65). Different approaches were utilized to elucidate the origin and structure of P65: electrophoretic and fluorographic analyses of 35S-labelled ribulose bisphosphate carboxylase exposed to UV radiation and immunological experiments using antibodies specific for P65, for the large and small subunits of ribulose bisphosphate carboxylase and for high-molecular-mass aggregates of the enzyme. These studies revealed that P65 is a dimer, formed by the covalent, non-disulphide linkage of one small subunit with one large subunit of ribulose bisphosphate carboxylase. For short periods of time (< 1 h), the amount of P65 formed increased with the duration of the exposure to the UV radiation and with the energy of the radiation applied. Prolonged exposure to UV radiation (1-6 h) resulted in the formation of high-molecular-mass aggregates of ribulose bisphosphate carboxylase. Formation of P65 was shown to depend on the native state of the protein, was stimulated by inhibitors of enzyme activity, and was inhibited by activators of enzyme activity. A UV-independent accumulation of P65 was also achieved by the in vitro incubation of plant crude extracts. However, the UV-dependent and the UV-independent formation of P65 seemed to occur by distinct molecular mechanisms. The UV-dependent accumulation of P65 was immunologically detected in all species examined, including Lemna minor, Arum italicum, Brassica oleracea, Triticum aestivum, Zea mays, Pisum sativum and Phaseolus vulgaris, suggesting that it may constitute a universal response to UV radiation, common to all photo-synthetic tissues.

Sugarcane (Saccharum sp. Hybrid) Propagated in Headspace Renovating Systems Shows Autotrophic Characteristics and Develops Improved Anti-oxidative Response

“…Western blot analysis was performed by electrophoretic transfer of proteins, separated by SDS-PAGE, to a nitrocellulose membrane (Millipore) and probed with the respective antibodies using the procedure described by Ferreira et al [16]. RNA Isolation and cDNA Preparation for Real-Time PCR Total RNA from leaves was extracted by adapting the method described by Chang et al [9].…”

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

“…The acrylamide concentration in the stacking gel was 5% (w/v), and was 12.5% in the separation gel (w/v), as previously described (Ferreira et al, 1996). Samples were prepared for electrophoresis by boiling for 3 min in the presence of 2% (w/v) SDS and 0.1 M b-mercaptoethanol.…”

“…Western blot analysis was performed by electrophoretic transfer of proteins, separated either by SDS-PAGE or by 2-DE, to a nitrocellulose membrane (Millipore) and probing with rabbit anti-LSU Rubisco immunoglobulin G. The reaction with antibodies was carried out according to Ferreira et al (1996). Relative quantification of LSU was obtained by using the software Quantity one (Bio-Rad).…”

UV-B Photoreceptors, Their Role in Photosignaling, Physiological Responses, and Abiotic Stress in Plants

“…UV radiations have negative effects on germinating pollen tubes leading to lowered crop yields (Gao et al 2004 ;Koti et al 2004 ). UV absorption also causes a net inhibition of photosynthesis in many species of plants caused primarily by reduction in RUBISCO levels and by disruption of PSII (Ferreira et al 1996 ;Galmes et al 2013 ;Teramura and Sullivan 1994 ;Vass et al 1996 ). The UV-B radiations have the capacity to modify nucleic acids (phototransformation) producing cyclobutane pyrimidine dimers (CPD), pyrimidine-pyrimidinone dimers (Ballare et al 1996 ;Landry et al 1997 ;Maueler et al 1994 ;Takahashi et al 2011 ), and aromatic amino acid residues of protein molecules (Vass et al 1996 ).…”