Exogenous ATP enhance signal response of suspension cells of transgenic rice (Oryza sativa L.) expressing maize C 4 -pepc encoded phosphoenolpyruvate carboxylase under PEG treatment

Abstract

The aim of this study was to analyze the biochemical and molecular responses of suspension cells of transgenic rice (Oryza sativa L.) over-expressing the maize C 4 -pepc gene encodes phosphoenolpyruvate carboxylase (PEPC) (PC line) to drought stress by exogenous ATP. Suspension cells of PC and wild-type (WT) rice cultured in N6 basic medium were subjected to the following treatments: a 24-h drought treatment imposed by 20% (m/v) polyethylene glycol 6000 (PEG6000 treatment); an exogenous adenosine 5′-triphosphate treatment (ATP; 1 mmol L−1); and a combined exogenous ATP + PEG6000 treatment. We measured DNA fragmentation, cell viability, lipid peroxidation, antioxidant enzyme activities, PEPC activity, ATP content, and the levels of Ca2+, H2O2, and NO using spectrophotometric methods and fluorescent dyes, and quantified the transcript levels of stress-related genes such as NAC1 (one of the transcription factors from NAC (NAM/ATAF/CUC) family), cytochrome P450 (P450) and programmed cell death (PCD) under these treatments. Compared with WT cells, PC cells showed more stable cellular activity, lower lipid peroxidation, enhanced antioxidant enzyme activity, higher levels of endogenous ATP level, PEPC activity, and transcript levels of C 4 -pepc and the transcription factor NAC1 under drought stress. While the content of H2O2 and the transcript levels of P450 and PCD were decreased and kept at the low levels during the same treatments. Adding ATP, PC reinforced these early signaling effects response to PEG6000 treatment. Based on the results, the exogenous ATP triggers a series of signaling cascade reactions for enhancing antioxidant protection in PC cells during the drought treatment, conferring to the drought-tolerance.