Salt Stress-induced Programmed Cell Death And Via Ca²⁺ Mediated Regulator Mechanism In Banana Root

Banana（Musa nana Lour.）is an important crop in China.In terms of production,banana cultivation in my country is most suitable for tropical and subtropical regions,such as high temperature and less rain,rising groundwater level,backward seawater irrigation,soil salinization is becoming more and more serious,and banana is a salt sensitive crop.The root system is the first organ of plants to feel the stress.Under salt stress,plants can regulate the response of plants through active programmed cell death in root tissue,and adjust physiological state to adapt to salt stress.Calcium ia an significant factor to control programmed cell death.However,the mechanism by which salt stress induces programmed cell death and calcium mechanism has not been reported yet.In this research,500 mmol/L Na Cl was used to simulate salt stress,and 5mmol/L Ca Cl₂ was added as exogenous Ca²⁺substance to treat the roots of M.Paradisiaca seedlings,and the peroxidase activity,reactive oxygen species content,malondialdehyde content,soluble protein content and chlorophyll fluorescence parameters were measured to explore the conditions in the roots of M.Paradisiaca seedlings,we used the DNA Ladder technique to detect the programmed cell death in the roots of M.Paradisiaca seedlings;Using Western blotting technique to detect the release of cytochrome c from mitochondria in the roots of M.Paradisiaca seedlings;RNA-Seq technology was used to analyze the whole transcription level of programmed cell death in the early and late stages of M.Paradisiaca seedling roots,so as to explore the possible pathway of exogenous Ca²⁺regulating programmed cell death.The results are as follows:（1）Exogenous Ca²⁺significantly increased the activities of cat,CAT,POD and SOD in M.Paradisiaca seedling roots under salt stress by 42.86%,37.72%and49.12%at 24 h compared with salt stress,significantly reduced the accumulation of H₂O₂,O₂^-and MDA in M.Paradisiaca seedling roots under salt stress by 21.48%,20.16%and 17.5%at 24 h compared with salt stress,and significantly increased the activity of SOD in M.Paradisiaca seedling roots under salt stress by 8,12 and 24 h.The accumulation of soluble protein was 47.17%,19.74%and 49.18%higher than that under salt stress,respectively;the chlorophyll fluorescence parameters Fv/Fm,Y（II）,r ETR,I_K and r ETR_max of M.Paradisiaca seedlings under salt stress were significantly increased,which were 21.27%,34.57%,35.65%,69.54%and 60.29%higher than that under salt stress at 24 h,respectively.（2）Under high salt condition,programmed cell death could be induced in M.Paradisiaca seedling roots.Under the condition of 500 mmol/L Na Cl treatment for2 h,a few positive cells appeared in M.Paradisiaca seedling roots by tuenl detection,which indicated the early programmed cell death of M.Paradisiaca seedling roots;under the condition of 500 mmol/L Na Cl treatment for 22 h,a large number of DNA specific breakages appeared in the roots of banana seedlings.The agarose gel electrophores is showed 200bp ladders,indicating the late stage of programmed cell death in the roots of M.Paradisiaca seedlings.（3）Exogenous Ca²⁺significantly promoted the release of Cytochrome c from mitochondria to cytoplasm in M.Paradisiaca seedling roots under salt stress.The content of Cytochrome c in mitochondria decreased significantly at 2 h,but increased significantly in cytoplasm.The content of Cytochrome c in mitochondria almost did not change at 4 h.（4）When M.Paradisiaca seedlings were treated with exogenous Ca²⁺for 22 h under salt stress,the number of differentially expressed genes and up-regulated differentially expressed genes increased in Ca²⁺dependent signaling pathway in response to salt stress.There were six differentially expressed genes in SOS signal transduction,five of which were up-regulated and one down regulated.There are 5up-regulated and 1 down-regulated differential genes in the SOS signaling pathway.There are 5 up-regulated and 2 down-regulated differential genes in the CDPK pathway.There are 10 differential genes in the ABA signaling pathway,all of which are up-regulated.There are 9 differentially expressed genes in phospholipid signal pathway,of which 6 are up-regulated and 3 are down regulated.（5）Exogenous Ca²⁺enhanced the differential expression of metacaspase gene in M.Paradisiaca seedling roots under salt stress.Nine differentially expressed genes were up-regulated at 22 h,and the genes of Mu MC-4,Mu MC-5 and Mu MC-8 with Ca²⁺dependent catalytic activity were also up-regulated.It is predicted that the regulation of exogenous Ca²⁺on PCD in banana seedling roots under salt stress may be related to metacaspase.In conclusion,exogenous Ca²⁺can improve the physiological response of M.Paradisiaca seedling roots under salt stress,which can effectively reduce salt stress damage.Exogenous Ca²⁺can accelerate the desorb of Cytochrome c from the root nucleus of M.Paradisiaca seedlings under salt stress;exogenous Ca²⁺can increase the differences in Ca²⁺-dependent pathway related genes under salt stress expression;exogenous Ca²⁺can increase the differential expression of metacaspase related genes related to PCD under salt stress It is speculated that metacaspase may be an important pathway for exogenous Ca²⁺to regulate programmed cell death induced by salt stress in M.Paradisiaca seedling roots.