Effects Of SlCAS Mediating Ca²⁺ On Alleviate PSⅡ Damage Of Tomato Leaves Under Low Night Temperature

In the northern winter spring cultivation,the facilities vegetables are vulnerable to be damaged by low temperature at night,which affects their photosynthesis.The effects of low night temperature on photosynthesis are various,such as slow photosynthetic electron transfer,photoinhibition and chloroplast morphologic changes.Photosynthesis mainly occurs in chloroplasts,which are important calcium reservoirs in plants.Applying exogenous calcium can make the chloroplasts regular and orderly,relieve the damage of low night temperature to the chloroplasts,and improve the photosynthetic capacity of tomato,but the specific mechanism of the relief is still unclear.In the process of the study of the extracellular calcium receptors on the chloroplast,the study of the calcium receptor（CAS）,which is located in the chloroplast,is emphasized in the process of photosynthesis of plants.In this study,SlCAS were silenced by VIGS.Exogenous calcium pretreatment was followed by low night temperature（Low night temperature,LNT）treatment.By measuring plant CAS expression level,related photosynthetic indexes,and screening and verification of SlCAS interaction proteins,in order to clear the effect of the SlCAS in the mediated exogenous calcium in alleviating photosynthetic damage of tomato leaves under low night temperature.The main results are as follows:1.Bioinformatics analysis and subcellular localization of SlCAS.The protein encoded by this gene was composed of 392 amino acids,the theoretical isoelectric point（PI）is 9.26,and the instability index of the protein was 45.01,indicating that the protein is unstable.This protein has a Rhodanese＿3 domain,the most secondary structure isα-helical structure,including a transmembrane domain,there is no signal peptide,there are several levels of phosphorylation sites.Subcellular localization predicted that the protein is localized in the chloroplast.The subcellular localization vectors of p CAMBIA1300-35S-GFP-CAS was further constructed and expressed in tobacco.The localization of the protein was observed under laser confocal microscope,and the results showed that the protein was indeed localized in chloroplast.2.SlCAS mediated Ca²⁺to alleviate photosynthetic damage of tomato leaves caused by low night temperature.Normal tomato plants pretreated with exogenous calcium and then treated with low night temperature induced strong expression of SlCAS.The p TRV2-CAS plants were pretreated with exogenous calcium and then treated with low night temperature.The results showed that the expression level of CAS in the plants did not change significantly,while the expression level of p TRV2-CK+Ca changed significantly;Compared with p TRV2-CK,Pn,Tr and Gs of p TRV2-CAS were significantly decreased,and Pn,Tr and Gs of p TRV2-CAS were further decreased after low night temperature treatment,while the changes of Ci were contrary to those of Pn,Tr and Gs.Although exogenous calcium pretreatment significantly alleviated the decrease of Pn,Tr and Gs and the increase of Ci under RT（Room temperature,RT）and LNT conditions,it did not recover to the level of PTRV2-CK;Compared with p TRV2-CK,Fv/Fm、Y（Ⅱ）、NPQ、q P、ETR（Ⅰ）、ETR（Ⅱ）and CEF of p TRV2-CAS were decreased,the indicators further reduced after low night temperature treatment;Compared with p TRV2-CK,1-q P and（1-q P）/NPQ were increased.Although exogenous calcium was applied,it could be relieved,but it did not recover to the level of p TRV2-CK.These results indicated that SlCAS plays an important role in mediating Ca²⁺to alleviate PSⅡdamage caused by low night temperature in tomato leaves.3.Screening and validation of SlCAS interacting proteins.SlCAS were found to interact with SlCP24A by Y2H.The interaction between SlCAS and SlCP24A was further verified by Bi FC and LCI.4.Silencing of SlCP24A aggravated photosynthetic damage of tomato leaves under low night temperature.Under the condition of RT,compared with p TRV2-CK,Pn、Fv/Fm、ETR（Ⅰ）、ETR（Ⅱ）and CEF of p TRV2-CP24A were decreased;Pn,Fv/Fm,ETR（Ⅰ）and ETR（Ⅱ）further reduced after LNT,but the CEF increased.These results indicated that SlCP24A plays an important role in response to low night temperature stress,and it may work together with SlCAS to alleviate the PSⅡdamage of tomato leaves caused by low night temperature.