Over Expression Mitochondrial Heat Shock Protein70Suppresses Programmed Cell Death In Rice Suspension Cells

Programmed cell death (PCD) plays an important role on plant growth and development, disease resistance and the interactions between plant and its environment. Mitochondrion, as one of the key organelles, regulates the main progress of PCD. The preview study established a salt stress-induced PCD model in rice root tip cells, while mtHSP70was up-regulated more than2-fold in mitochondrial proteome after salt stress. In this study, we found that heat shock treatment resulted in some typic PCD progresses in rice protoplasts. A series of relevant studies had carried out to investigate the functions of mtHSP70in PCD. The vector control (VC) and over-expression (OX) mtHSP70rice protoplasts from different suspension cell lines were researched under the treatment of heat shock at48℃for15min and returned to the normal conditon, the both types of protoplasts showed the specific PCD features, such as DNA Ladder, nuclear condense and deformation, TUNEL positive reaction and mitochondrial membrane potential decrease. It was proved that the chromatin condensation, nuclei deformation and genome DNA would degradated under heat shock via Hoechst33342, but the chromatin condensation in OX protoplasts was similar to VC at6h. It was found that the number of TUNEL positive nuclei of OX protoplasts was less than VC under the heat shock treatment, the positive rates of VC and OX were90%and70%, indicating that the activities of Ca2+/Mg2+-dependent endonuclease were decreased. Results indicated that, comparing to VC protoplasts, OX were resistant to PCD in many fields. For OX protoplasts, the thermo treatment could induce the appearance of DNA Ladder at4h, while appeared at2h in VC. Death rate test under H2O2treatment revealed the similar tendency to heat shock treatment. The death rate of heat shock in VC approached63.2%at6h, but OX was39.3%, while100mM H2O2treatment made the death rates of VC and OX rising to91%and43%respectively. Cytochrome c released from mitochondria could be detected immediately after heat shock; the optical density of VC was calculated more than1.0, while OX showed less than0.7.H2DCFDA staining suggested that OX protoplasts could avoid the injuries of the oxidative burst as the ROS level remained stability and up-regulated slowly, while the VC protoplasts occurrent bimodal in prophase. The specific probe JC-1displayed that mitochondrial membrane potential decreased fast when heat shock from5min to15min, and OX protoplasts were slower than VC, indicating, attributed to the over expression of mtHSP70, the excellent integrity and low mitochondrial membrane injuries of the OX protoplasts. The Caspase-3-like was specifically activated after heat shock, OX protoplasts showed significantly less Caspase-3-like activity than the VC. The RFU of VC protoplasts was more than8-fold to OX. It indicated mtHSP70over expression may inactivate Caspase-3-like by maintaining the mitochondrial membrane potential.All the results above suggested that mtHSP70may delay the plant PCD by maintaining mitochondrial membrane potential and inhibiting cellular ROS bursts in rice suspenssion cells.