Populus Euphratica XTH Mediates Salinity-induced Leaf Succulence And Alleviates Heavy Metal Stress In Tobacco Plants

Salt ions and heavy metals suppress plant growth and development. High salt concentrations lead to water deficit and ion toxicity, which induce oxidative damage in plants. Excess heavy metals, such as cadmium (Cd2+) interferes with a series of physiological processes, including photosynthesis, transpiration, and nutrient balance, resulting in growth retardation and eventually plant death. Being a valuable tree species that survives in saline and alkaline desert sites, Populus euphratica has great potential for genetic improvement in large-scale afforestation. During a prolonged period of salt stress, P. euphratica develops pronounced succulent leaves, which benefits the adaptation of P. euphratica to salt environments. However, the molecular mechanism of salt-induced succulence remains poorly understood. P. euphratica has been shown to be sensitive to Cd2+stress, the acclimation and adaptation processes need to be investigated.Xyloglucan endotransglucosylase/hydrolase (XTH) is considered to be a vital factor controlling cell wall loosening and extensibility. We had cloned the full-length PeXTH gene from leaves of P. euphratica. Real-time PCR assay showed that the expression of PeXTH was up-regulated under salt conditions. Therefore, the PeXTH gene was suggested to involve in salt-induced leaf succulence and thus enhancing salt tolerance of P. euphratica. In addition, XTH is involved in the plant response to heavy metal toxicity. However, the link between XTH and Cd2+stress has not yet been established in higher plants. PeXTH expression was up-regulated by1.2-2.1fold in P. euphratica roots and leaves upon Cd2+exposure (40μM-80μM CdCL2), indicating that the PeXTH gene might play a role in Cd2+tolerance of P. euphratica. In this study, the PeXTH gene was overexpressed into tobacco by transgenic technique. We investigated the functions of PeXTH in salinity and Cd2+tolerance by comparing the leaf anatomy, plant morphology, and physiological traits of PeXTH-transgenic and wild-type tobacco plants. The main results and conclusions are as follows:1. The putative amino acid sequence of PeXTH protein contained the DEIDFEFLG domain, which has been proposed to be the catalytic site of xyloglucan endotrans-glucosylase (XET). In addition, PeXTH localized exclusively to the endoplasmic reticulum and cell wall.2. Purified PeXTH protein was approximately35kD and had XET activity in vitro. The optimum temperature of the enzyme was37℃and the optimum pH was6.0.3. Plants overexpressing PeXTH were more salt tolerant than wild-type tobacco with respect to root and leaf growth, and survival. The increased capacity for salt tolerance was due mainly to the anatomical and physiological alterations caused by PeXTH over-expression. Compared with the wild type, PeXTH-transgenic plants contained36%higher water content per unit area and39%higher ratio of fresh weight to dry weight, a hallmark of leaf succulence. However, the increased water storage in the leaves in PeXTH-transgenic plants was not accompanied by greater leaf thickness but was due to highly packed palisade parenchyma cells and fewer intercellular air spaces between mesophyll cells. In addition to the salt dilution effect in response to NaCl, these anatomical changes increased leaf water-retaining capacity, which lowered the increase of salt concentration in the succulent tissues and mesophyll cells. Moreover, the increased number of mesophyll cells reduced the intercellular air space, which improved carbon economy and resulted in a47%-78%greater net photosynthesis under control and salt treatments. Taken together, the results indicate that PeXTH overexpression enhanced salt tolerance by the development of succulent leaves in tobacco plants without swelling.4. Tobacco plants overexpressing PeXTH were more tolerant to Cd2+stress than wild-type tobacco in terms of root and shoot growth. Transgenic lines accumulated49%-58%less Cd2+in root apical and mature regions, as compared to the wild-type. The less buildup of Cd2+in roots of transgenic lines was the result of lower influx of Cd2+under Cd2+stress. It is noting that transgenic plants displayed56%-87%higher xyloglucan degradation activity (XDA) than the wild type, leading to a25%-27%decline of xyloglucan content in the root cell walls. Therefore, overexpression of PeXTH increased the activity of XDA in transgenic plants, which enhanced the degradation of xyloglucan in the wall. The down-regulated amount of xyloglucan led to less binding sites for Cd2+and thus reduced the root Cd2+uptake and buildup in transgenic plants. Consequently, the Cd2+toxicity was eventually alleviated in transgenic tobacco.