Increase in reactive oxygen species (ROS) and in senescence-associated gene transcript (SAG) levels during dark-induced senescence of Pelargonium cuttings, and the effect of gibberellic acid

Dark-induced senescence in the leaves of Pelargonium cuttings was manifested in chlorophyll breakdown, and an increase in reactive oxygen species (ROS) levels followed by a subsequent induction of two senescence-associated gene (SAG) transcripts: senescence-related transcription factor PeWRKY6-1 and cystein protease homolog PeSAG12-1. Glutathione applied at the onset of ROS increase, reduced ROS accumulation and prevented the increase in PeSAG12-1 expression. These results suggest that in darkness PeSAG12-1 and maybe PeWRKY6-1 are induced by increases in ROS levels. Since PeWRKY6-1 expression increased concomitantly with that of PeSAG12-1, it most likely does not function as a senescence inducer. Application of gibberellic acid (GA₃) to Pelargonium cuttings before the dark treatment prevented chlorophyll breakdown, ROS increase and PeWRKY6-1 and PeSAG12-1 accumulation. GA₃ also decreased ROS levels when it was applied during the dark period at the onset of ROS accumulation, but not when applied after high ROS levels were evident. The pattern of GA₃ suppression of ROS levels positively correlated with its inhibitory effect on chlorophyll breakdown. However, GA₃ application after ROS accumulation inhibited PeWRKY6-1 and PeSAG12-1 gene expressions, despite high ROS levels in the tissue. Taken together, our results suggest that GA₃ acts to inhibit leaf senescence of Pelargonium, probably not only by reducing ROS levels, but also by interfering with senescence regulation, through an as yet unknown mechanism.