A novel stress-responsive BnaNAL1 transcriptional activator in oilseed rape positively modulates reactive oxygen species production and cell death

Abstract

Programmed cell death (PCD) is an active, genetically controlled process associated with a number of developmental processes including response/adaptation to abiotic and biotic stresses and leaf senescence. Reactive oxygen species (ROS) are important modulators of PCD and leaf senescence. However, the transcriptional control of ROS and PCD in oilseed rape (Brassica napus L.) is largely unknown. In this study, we identified a NAC transcription factor gene BnaNAL1 from oilseed rape, which encodes a transcriptional activator and plays an important role in modulating ROS and cell death. BnaNAL1 was localized exclusively to the nucleus. Expression profiling of the gene showed increased transcript levels in early senescent and mature leaves. Expression of BnaNAL1 resulted in hypersensitive response (HR)-like cell death symptoms. Further analysis of various physiological indices such as diaminobenzidine (DAB) staining, malondialdehyde contents and electrolyte leakage supported the role of BnaNAL1. Furthermore, we also observed an increase in DNA fragmentation and ROS levels. A quantitative reverse transcription PCR (qRT-PCR) analysis indicated genes involved in ROS production (RbohB), cell death (VPE1a, ZEN1), defense (PR2) and leaf senescence (HIN1, ZAT12) as putative downstream targets. We further verified that BnaNAL1 activated promoters of ZEN1, HIN1, ZAT12 and VPEs using a dual luciferase reporter assay. Using electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR assays, we further confirmed that BnaNAL1 could bind to the promoters of ZEN1, ZAT12, HIN1 and four VPEs for direct transcriptional modulation of these genes. Collectively our results demonstrate that BnaNAL1 positively modulates cell death and leaf senescence.