The Role Of Salicylic Acid In Response To Low Temperature Stress In Watermelon Seedlings

Watermelon [Citrullus lanatus(Thunb.) Matsum. & Nakai] originated in the tropics, and it is an important cucurbit crop. Low temperature is the most commonly encountered abiotic stress in early spring protected-watermelons. Salicylic acid(SA) is a phenolic compound and as a signaling molecule, plays an important role in plants resistance to biotic and abiotic stress. However, the mechanism of SA response to chilling stress in the plants is still unclear. In this study, watermelon 97103 was selected as material. Using physiology, molecular biology, genomics and other methods, we examined the effect of endogenous SA on watermelon in chilling temperature and the synthetic pathway, and the role of exogenous SA and endogenous SA in response to chilling in watermelon. At the same time, we studied the change of redox signaling, stress-resistant genes, antioxidant enzyme system and CBF transcription factors, in order to explore the role of SA in response to chilling in watermelon. The main research results are as follows:1. The change of endogenous SA content and its synthetic pathway were investigated in watermelon seedlings exposed to chilling temperature. Chilling stress induced the simultaneous accumulation of free and conjugated SA in watermelon, and relative expression of ClPAL5, ClPAL6, ClPAL7, ClPAL8, ClPAL11 and ClPAL12 significantly increased, transcription level of ClPAL4, ClPAL9 and ClPAL10 increased but not significant; however,just like ClPAL1, ClPAL2 and ClPAL3, ClICS was hardly induced compared with the normal temperature. Consistent with their expressions, PAL and BA2 H activities increased continuously, which suggest that chilling-induced SA production is mainly attributed to the PAL pathway.2. The effect of exogenous SA and inhibitors of SA biosynthesis on watermelon seedlings in response to chilling temperature were studied. Spraying watermelon seedlings with different concentrations of exogenous SA, low concentration(≤5 μM) or high concentration(≥500 μM) of exogenous SA treatment could not improve the resistance of watermelon exposed to chilling, and appropriate concentration(10~100 μM) of SA pretreatment helped to relieve the damage caused by chilling, of which the role of 10 μM SA was most significant. Pretreated with AOPP or PAC, PAL or BA2 H activity was inhibited in watermelon seedlings exposed to chilling, and the chlorophyll fluorescence parameters of Fv/Fm, Y(II), NPQ and Y(NPQ) decreased, but Y(NO) increased, which could be reversed by the application of exogenous SA. However, qP was not affected by pretreated with AOPP or PAC and exogenous SA.3. Redox signaling was studied in watermelon chilling-resistance temperature induced by SA. GSH content, GSSG content, GSH/GSSG, AsA content, DHA content and AsA/DHA were changed by chilling stress, of which GSH/GSSG and AsA/DHA reached a peak at 1 d, then decreased compared with the normal temperature. Sprayed with AOPP, the ratios of GSH/GSSG and AsA/DHA decreased in watermelon exposed to chilling, but recovered by SA, their ratios increased markedly. At the same time, it was found that the transcript levels of antioxidant enzyme genes(tAPX, DHAR, GST and GPX) and HSP70-2 was induced significantly, and the activities of antioxidant enzyme(POD, APX and CAT) were also increased markedly in 3 d as well as the T-AOC in low temperature. These results indicate that the cellular redox status is very sensitive to chilling and can be influenced by SA levels, and antioxidant enzyme genes(tAPX, DHAR, GST and GPX) and HSP70-2 can response to chilling, but later than redox signaling as well as antioxidant enzyme system.4. The relationship between CBF pathway and SA in watermelon seedlings resistance to low temperature was studied. DREB subfamily A-1 of AP2/EREBP transcription factor proteins were identified from Cucurbit Genomics Database, which include 4 members, named CBF1/DREB1B(Cla017719), CBF2/DREB1(Cla011488), CBF3/DREB1A(Cla006212) and CBF4/DREB1D(Cla002330). After spray of AOPP, transcript levels of ClCBF3 and ClCBF4 were up-regulated, while it had no significant impact on ClCBF1 or ClCBF2, compared with no chilling stress, but all of them were down-regulated in SA-treated plants. Similarly, transcript levels of ClLOS4, ClDHN2, ClERD10 and ClLEA14 were up-regulated in AOPP-pretreated plants, and this increase was again compromised in the subsequently SA-treated plants. All these results suggest SA involves in the CBF-dependent responsive pathway, and negatively regulates the CBF-dependent responsive pathway during chilling stress in watermelon.