In this paper we provide evidence for involvement of chloroplast as alternate organelle for initiating PCD in plants under light and abiotic stress. In animals, mitochondria are the major source of reactive oxygen species (ROS) and key executioner of programmed cell death (PCD). In plants, however, the primary site of generation of ROS is chloroplast and yet its involvement in PCD has not been worked out in details. We found by Evans blue staining that salt (150 mM NaCl)-treated protoplasts obtained from green seedlings had higher rate of cell death than protoplasts obtained from etiolated seedlings. This indicated that cell death induced by NaCl is accentuated by light. Imposition of salt-stress to green protoplasts generated H2O2. Known hallmarks of PCD i.e., blebbing of cell membrane, loabing in nucleus, nick in DNA were observed in light-exposed salt-treated protoplasts and seedlings. TUNEL-FACS assay demonstrate several DNA nicks in the salt-treated green protoplasts exposed to light. Conversely, salt-treated etiolated protoplasts kept in dark had only a few TUNEL-positive nuclei. Similarly, a substantial numbers of TUNEL positive nuclei were observed in green seedlings due to salt treatment in light. However, salt-treated etiolated seedlings kept in dark had very few TUNEL positive nuclei. Addition of Caspase 3 inhibitor (DAVD-CHO) rescued (~50 %) green protoplasts from salt-stress induced cell death suggesting an involvement of apoptosis like PCD (AL-PCD). Ultra structure studies of chloroplast, mitochondria and nucleus from the leaves obtained from salt treated seedlings at the time point that showed PCD signature, resulted to severe granal de-stacking in chloroplasts while structural integrity of mitochondria was maintained. These studies demonstrate the photo-modulation of salinity-induced PCD in photosynthetic tissues is mainly executed by chloroplasts.
Keywords: Chloroplast; DNA ladder; Oryza sativa; PCD; PORC; Protoplasts; ROS; TUNEL.