Elymus nutans Griseb. is a dominant forage in the Qinghai–Tibetan Plateau. However, the combined cold and drought (CD) stress is a major problem inhibiting its growth, development, and yield. Here, the responses of morphological, photosynthetic, osmoregulation levels, and signal transduction under cold, drought, and CD stress were explored. Both cold- and drought-stressed plants showed varying degrees of damage. In addition, CD stress led to more severe damage than single stress, especially in total biomass, photosynthetic capacity, and electron transfer efficiency. The total biomass, net photosynthetic rate, and maximal quantum yield of photosystem II (PSII) photochemistry reduced by 61.47%, 95.80%, and 16.06% in comparison with the control, respectively. Meanwhile, CD stress was accompanied by lower chlorophyll contents, down-regulated expression level of key photosynthetic enzymes (EnRbcS, EnRbcL, and EnRCA), stomatal closure, disrupted chloroplast ultrastructure, and reduced starch content. Furthermore, CD stress induced some adaptability responses in cold- and drought-tolerant E. nutans seedlings. The combined stress provoked alterations in both cold- and drought-related transcription factors and responsive genes. EnCBF12, EnCBF9, EnCBF14, and EnCOR14α were significantly up-regulated under cold or drought stress, and the transcript level of EnCBF3 and EnCBF12 was even 2.94 and 12.59 times higher than control under CD treatment, which indicated the key role of transcription factors activation in coping with CD stress. In addition, the content of soluble sugar, reducing sugar, proline, glycine betaine, and other osmolytes was significantly improved under CD stress. Therefore, we demonstrated that exposure to CD stress led to severe morphological and photosynthetic damage and revealed the acclimation to the cold and drought stress combination via osmotic adjustment and transcription factors activation in the Tibetan wild E. nutans.
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