Cloning Of A Cold-Tolerance Related Gene CsLDC And Investigation Of Physiological Mechinisms Underlying Regulation Of Cold-Tolerance By Exogenous Plant Growth Regulators In Cucumis Sativus

Cucumber (Cucumis sativus L.) is one of the typical chilling-sensitive plants and is also being considered as one of the important vegetables. However, low temperature in the early spring and late autumn or winter is the most directly limited factor of cucumber yield and quality. Therefore, it is becoming the focus of current research on cucumber through breeding and culture approaches, and understanding the cold-tolerant mechanism is the basis of breeding for cold-resistant variety. To uncover the chilling tolerant mechanism by which involves growth of cucumber in terms of both physiological and molecular aspects, this study employs a combination of approaches involving plant physiology, biochemistry, molecular biology and functional analysis of target gene to examine the influence of chilling stress on cucumber, while considering 15℃as the stress factor under sub-optimal temperature. Moreover, this sudy investigates the physiological and biochemical mechanisms underlying the chilling-tolerance mediated by important phytohormones in cucumber. Meanwhile, a chilling-tolerant gene of cucumber, CsLDC, was cloned and identified.Specifically, this study is focusing on and has made progress in the followings1. Cloning of a full-length cDNA of Lysine Decarboxylase (CsLDC) from Cucumis sativus and gene expression study of CsLDC in cucumber upon chilling stressHigh quality of RNA is the key for gene expression and downstream studies. It is very difficult to obtain high quality ofRNA from geminating cucumber (Cucumis sativus) seeds using commercial Trizol kit. We improved the Urea-LiCl method by comparing several different protocols. The results clearly showed that we succeeded in obtaining high quality of RNA with clear and intact bands of 28S,18S and 5S, and with an A260/A280 at around 2.0. This RNA can be used not only for cDNA synthesis, but also other downstream studies such as RT-PCR, AFLP, RACE and even Real-time PCR. Moreover, using this improved Urea-LiCl protocol we could get high quality of RNA from seeds of other Cucurbitaceae varieties, including squash, melon and towel guard, and other tissues besides seeds.A full-length cDNA sequence was cloned using RACE (Rapid Amplification of cDNA Ends) from cucumber (Cummis sativus) seeds, based on a 132bp fragment of cucumber cold related tolerance gene (ccrt132) reported previously by our group. This full-length cDNA contains an ORF (Open Reading Frame) of 648 nucleotides encoding a protein with 215 amino acid residues. Alignment analysis has shown that this cDNA sequence and its coded protein have high similarity of nucleotides and homology to reported plant Lysine Decarboxylase, respectively, thus it is named as Cucumis sativus Lysine Decarboxylase (CsLDC). CsLDC contains a highly conserved Lysine_decarboxy superfamily domain with a conserved motif PGGXGTXXE, and the crystal structure of CsLDC is predicted to be highly similar to that of reported Arabidopsis lysine decarboxylase x-ray A chain. The alignment of CsLDC with other plant LDCs suggested a closest relationship of CsLDC with Arabidopsis AtLDC. Moreover, the quantitative data of gene expression conduced by semi-quantitative RT-PCR and quantitative Real-Time PCR demonstrated that CsLDC is highly enhanced in cold-tolerant variety Changchun mici upon chilling stress, but not in cold-sensitive Beijing jietou, suggesting that CsLDC is highly associated with chilling tolerance of cucumber.2. Exogenous polyamines regulate chilling tolerance of cucumber (Cucumis sativus L.) through modulating antioxidative systemUpon chilling treatment, free spermidine (Spd), spermine (Spm) and putrescine (Put) were remarkably increased in the leaves of cv. Changchun mici 1 day after treatment. The induction of Put declined thereafter, whereas Spd and Spmlevels increased steadily. In the leaves of cv. Beijing jietou, Put content was increased only at 1 day after chilling, while Spd content decreased significantly upon chilling treatment and not much changes were detected for Spm. Exogenous PAs increased endogenous content of all three types of PAs, with higher levels in Changchun mici than that in Beijing jietou, and exougenous Spd showed much greater impact on endogenous PAs level than Put. The PAs biosynthetic inhibitor, methyglyoxal-bis-(guanylhydrazone) (MGBG), cancelled the effects of exogenous PAs on the endogenous PA levels, among which the effect of MGBG on chilling-sensitive variety was greater than on chilling-tolerant one. Chilling reduced soluble protein content, the activities of antioxidant enzymes, including superoxidase dismutase (SOD), peroxidase (POD), catalase (CAT) as well as ascorbate peroxidase (APX), and satiability of membrane; however, the reduction on Beijing jietou is much greater than Changchun mici, and the changes could be renovated by exogenous application of Put and Spd. It was also found that pretreatment with Put and Spd diminished the increased electrolyte leakage and malondialdehyde (MDA) content caused by chilling in the leaves of both cultivars. Hoever, the effects caused by PAs mostly depended on variety of cucumber, and pretreatment with MGBG could cancel the effect of PAs. Moreover, histochemical staining and quantitative measurements showed that exogenous application of Put and Spd eliminated but MGBG exaggerated the hydrogen peroxide (H2O2) accumulation caused by chilling stress, especially in leaves of Beijing jietou. Notably, Changchun mici was found to contain higher levels of all three types of endogenous free PAs compared to Beijing jietou. While no significant difference of SOD, POD and CAT activities was found between non-chilling stressed Changchun mici and Beijing jietou seedlings, the former exhibited higher APX activity than the latter. These results indicate that PAs play important roles in the tolerance of cucumber against chilling stress, which is most likely achieved by acting as oxidative machinery against chilling injury.3. Effects of salicylic acid on the antioxidant system and chilling tolerance of Cucumis sativusUpon chilling stress, the endogenous SA levels in the leaves of two different chilling-responsive varieties were obviously higher that that in geminating seeds, and Changchun mici contains higher SA levels in both leaf and germinating seed compared to Beijing jietou. The Proline content and peroxidase (POD) activity in Changchun mici are higher than Beijing jietou, while MDA levels, electrolyte leakage and H2O2 production in the former is much lower than that in the latter. Exogenous application of SA increases significantly POD activity and soluble protein content whereas it decreases MDA content and electrolyte leakage in the leaves under chilling stress. Compared to Beijing jietou, exogenous SA treatment could eliminate the accumulation of H2O2 in leaves of Changchun mici, much greater than in Beijing jietou, upon chilling stress. The data clearly demonstrate that SA production varies due to the difference of chilling responsiveness, and that exogenous SA can enhance the chilling tolerance ability, which might be achieved through modulating antioxidant system in cucumber.