Regulation of gibberellin 20-oxidase1 expression in spinach by photoperiod

The multifunctional gibberellin (GA) 20-oxidase [GA53, 2-oxoglutarate:oxygen oxidoreductase (20-oxidizing), EC 1.14.11] has been extensively investigated in various species at the genetic and molecular levels, but not at the protein level. Here, we report on expression of GA20ox1 protein in spinach (Spinacia oleracea L.) in response to photoperiod. Polyclonal antibodies were raised against recombinant SoGA20ox1 in a chicken. These antibodies immuno-inhibited the enzymatic activity of the recombinant SoGA20ox1 and immuno-precipitated SoGA20ox1 (43 kDa) isolated from spinach shoot tips. Northern and western analyses showed that the levels of SoGA20ox1 transcript and protein increased in the blades, petioles, young leaves, and tips in response to long-day (LD) conditions. The transcript and protein levels of the SoGA20ox1 gene were up-regulated in the petioles and tips in a time-dependent manner. The estimated number of SoGA20ox1 protein molecules per cell was approximately 13-fold higher in tips grown in LD than in short-day (SD) conditions. The levels of SoGA20ox1 protein gradually decreased in tips when spinach plants grown in LD were transferred to SD conditions. SoGA20ox1 transcripts were detected by in situ hybridization in rapidly growing tissues—such as the shoot apical meristem, leaf and flower primordia, leaflets, and vascular tissues—but not in the expanding subapical region. In petioles, expression of SoGA20ox1 was detected in the companion cells.