| Protected vegetable production, an important part of China’s modern agriculture and vegetables supply in winter have been developed rapidly in recent years. However, tomato (Lycopersicon esculentum Mill.), an important vegetable crop cultivated in Northeastern China, often encounters low night temperature (LNT) stress which resulting in decreased ability of photosynthesis capacity, yield and quality. Therefore, it is of great significance to study the photo-inhibition mechanism of tomato under LNT stress. At present, exogenous calcium application is a low-cost and effective way to alleviate LNT stressed damage and achieve higher yield and good quality.In present, a tomato cultivar’Liaoyuanduoli’was used to study the photo-inhibition mechanism of tomato under LNT and exogenous calcium regulation mechanism. We aim to provide a theoretical basis for the chemical regulation of plant metabolism as well as the alleviation of LNT stress. This work is of importance in both science and practical fields. The main research results are as follows:1. Exogenous calcium pretreatment alleviate PSII photo-inhibition. In present, compared with that of control, light-adapted maximum quantum yield of PSII(Fv’/Fm’), effective quantum yield of PSII [Y(II)], electron transport rate of PSII and qP which reflecting degree of PSII openness decreased significantly under LNT stress, while non-photochemical efficiency of PSII [Y(NPQ)] and photochemical efficiency of PSII [Y(NO)] increased significantly. Decrease of photochemical efficiency of PSII and closure of PSII reaction center which caused by LNT stress result in PSII photo-inhibition. Meanwhile, PSII photo-inhibition aggravated with light increase. Exogenous calcium pretreatment alleviated PSII photo-inhibition by reducing these parameters to some extent. Whereas, EGTA (calcium chelator) and TFP (CaM antagonist) spraying increased these parameters and caused further PSII photo-inhibition damage.Compared with that of control, despite photochemical yield of PSI [Y(I)] decreased under LNT, quantum yield of non-photochemical energy dissipation in PSI due to acceptor side limitation [Y(NA)] had no significant change and quantum yield of non-photochemical energy dissipation in PSI due to donor side limitation[Y(ND)] increased significantly. It implys that LNT did not cause PSI photo-inhibition.2. Effect of exogenous calcium pretreatment on photo-protective mechanism. In this text, effect of exogenous calcium pretreatment on photo-protective mechanism including cyclic electron flow, state transition and xanthophyll cycle were studied. Results showed that Y(CEF) which reflected the level of cyclic electron flow was significantly stimulated by LNT. Exogenous calcium pretreatment promoted Y(CEF), whereas EGTA and TFP reduced it. However it is still higher than that of control on EGTA and TFP-pretreated plants. State transition were reduced by 48.77% on LNT-stressed plants. Compared with LNT-stressed plants, state transition of exogenous calcium pretreatment increased by 31.95%, while EGTA and TFP pretreatment decreased it by 56.63%and 47.93%, respectively. In addition, the extent of de-epoxidation of xanthophyll cycle [(A+Z)/(A+Z+V)] which reflected the condition of xanthophyll was decreased by 2.32% under LNT. Exogenous calcium pretreatment elevated it by 2.32%, however, EGTA and TFP pretreatment decreased it by 2.37% and 0.89%, respectively. These results indicated that LNT stress significantly stimulated cyclic electron flow and significantly inhibited state transition, however, had little effect on xanthophyll cycle of tomato leaves. In conclusion, we suggest that cyclic electron flow is the main way to prevent PSII and PSI from LNT stressed photo-inhibition. Moreover, exogenous calcium pretreatment can alleviate photo-inhibition induced by LNT stress by promoting cyclic electron flow, state transitions and xanthophyll cycle.3. Effect of exogenous calcium pretreatment on trans-thylakoid membrane proton motive force in tomato leaves under LNT. In present, P515 signal which reflecting the trans-thylakoid membrane proton motive force in vivo were measured. Results showed that LNT caused a significantly increase in ApH and a slightly decrease in Acp. Exogenous calcium pretreatment slightly decreased ApH under LNT stress, whereas EGTA and TFP pretreatment slightly increased ApH. However, there had a significant difference between exogenous calcium and EGTA pretreatment. Our observations of faster decay after dark adaptation and a slower decay, after illumination in P515 signal on LNT stressed plant indicated that both the thylakoidmembrane was damaged and ATPase activity was reduced after LNT treatment. Exogenous calcium pretreatment slightly alleviated the damage of thylakoid membranes and ATPase activity while EGTA pretreatment enhanced the damage caused by LNT stress. 4. Effect of exogenous calcium pretreatment on endogenous calcium and reactive oxygen species distribution in guard cells of tomato leaves under LNT. In our study, fluorescent dyes DFCH-DA and Fluo-3 AM were utilized respectively to the observation of reactive oxygen and calcium ions in guard cellsof tomato leaves. Results indicated that LNT caused a substantial accumulation of reactive oxygen species in guard cells. Exogenous calcium pretreatment significantly reduced the fluorescence intensity of reactive oxygen species in the guard cells, while EGTA and TFP pretreatment significantly enhanced the fluorescence intensity of that. Meanwhile, closure of stoma of LNT stressed plants was attributed to accumulation of reactive oxygen species in guard cells. Compared with that of control, calcium of guard cell wall which closing to stoma was increased significantly after LNT treatment. Sprayed with exogenous calcium, calcium content were elevated and accumulated in the chloroplast. EGTA pretreatment decreased calcium fluorescence intensity in guard cell. TFP pretreatment enhanced the accumulation of calcium in guard cell wall which closing to stoma. Determination of different forms of calcium in leaves revealed that there was a significant decrease in pectate-bound calcium contents, phosphate and carbonate calcium as well as oxalate-bound calcium. It suggested that LNT reduced calcium storein vacuole. Exogenous calcium pretreatment increased pectate-bound calcium, phosphate and carbonate calcium, oxalate-bound calcium and calcium silicate by 10.42%,29.09%,16.20%and 18.03%, respectively. It indicated that it improved calcium store and promoted calcium deposition. Gene expression analysis of calcium receptor protein CaM, CDPK and CBL showed that exogenous calcium pretreatment could enhanced their expression under LNT. And, CaM had the most obvious changes. In conclusion, exogenous calcium regulates stomata and photosynthetic apparatus by adjusting the calcium morphology distribution, calcium forms, and Ca2+-CaM signals pathway.5. Effect of exogenous calcium pretreatment on thylakoid membrane proteins of tomato leaves under LNT. In this study, blue-green electrophoresis (BN-PAGE) and polyacrylamide gel electrophoresis (SDS-PAGE) were utilized to isolate and identify thylakoid membrane complex and their subunits. A total of more than 50 membrane protein points were macroscopic. Sixteen selected differentially expressed protein spots were analysis by mass spectrometry. Results showed that LNT caused expression differences in energy absorption and transfer, electron transport, PSII and PSI reaction center, ATPase. Among these proteins, Dl protein, PsbP, CP43, Cytb6, Cytf, and α, β, γ, ε submits of ATP synthase were down-regulated by LNT, and were up-regulated by exogenous calcium while further down-regulated by EGTA and TFP. LHCB1, CP26, A1 and A2 protein of PSI were further up-regulated by exogenous calcium pretreatment. In addition, there were also some proteins involved in the chloroplast DNA and protein modification changed. In conclusion, thylakoid membrane proteins expression of LNT-stressed plants were changed by exogenous calcium ever on translation level or transcription level. |
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