Mutation in cysteine bridge domain of the g-subunit affects light regulation of the ATP synthase in Arabidopsis

Abstract

The catalytic regulation of the chloroplast ATP synthase is considerably more intricate than tits counterpart ATP synthase of mitochondria and bacteria. The light-dependent activation of chloroplast ATP synthase involves several steps including redox modulation of a cysteine bridge in g -subunit which is unique to the chloroplast enzyme. However, it is unclear exactly what role redox modulation plays in the overall regulation of the chloroplast enzyme. In order to investigate the significance of redox modulation we used site-directed mutagenesis and plant transformation to change the regulatory bridge domain within g -subunit in Arabidopsis. We designed four mutations in the redox active domain designated as C199S, C205S, C199S/C205S and deletion. These were transformed into wild type Arabidopsis (Columbia ecotype) plants. The expression levels of transgenes and original genes in T0 plants were identified by RT-PCR following by restriction digestion. The results showed the transgenes were extensively overexpressed behind a constitutive 35S promoter, compared with the endogenous g -subunit gene, indicating that g -subunit in ATP synthase of transgenic plants originated predominately from the transgenes. We monitored the activation and redox modulation of the chloroplast ATP synthase by measuring D A518 relaxation kinetics of the dark-adapted transgenic mutant plants after a 3 s of preillumination with 45 µmol quanta m-2 s-1 red light and 4 min of re-dark adaptation. The D A518 relaxation kinetics of the transgenic mutant plants confirmed interference with redox modulation in transgenic plants. Our ongoing work is focusing on TMR-maleimide labeling of cysteine residues to quantify the extent of substitution of the thiol group in g -subunit of the transformed plants and investigating the physiological consequences of the loss of redox modulation.