Diurnal accumulation of K+-dependent L-asparaginase in leaf of common bean (Phaseolus vulgaris L.)

Abstract

L-Asparaginase (EC 3.5.1.1) activity has been previously reported to fluctuate with the photoperiod in young pea leaves, with higher activity in the light. The present research sought to investigate this phenomenon in developing leaves of common bean (Phaseolus vulgaris L.). There are two genes coding for K+-dependent asparaginase in this species. Expression of PvASPG1 predominates over PvASPG2 in all tissues. The catalytic efficiency of recombinant PvASPG2 was approximately 2-fold lower than that of PvASPG1. Polyclonal antibodies were raised against a specific peptide present in PvASPG1 to use in immunoblotting. In developing seed, asparaginase protein levels in the seed coat stayed constant, whereas levels in cotyledon were lower and progressively declined. In young leaf, asparagine protein levels showed diurnal variation, increasing at the end of the dark period and slowly decreasing during the light period. This was paralleled by changes in activity levels in leaf extracts. These changes accompanied a transient increase in free asparagine concentration at the beginning of the light period. The present results demonstrated that K+-dependent asparaginase activity reaches a maximum level at the transition from dark to light, anticipating dawn, in young leaves of common bean.