The Mechanisms Of Alfalfa In Response To Drought And Salt Stresses And Functional Analysis Of MtPHD6 Gene

Drought and salt are two of the main environmental stresses limiting plant growth, development, productivity and distribution. Water deficit and soil salinization severely threat the development of sustainable agriculture. In addition, with the increased population and serious environment pollution, it is difficult for the global agriculture to meet the world’s food and energy requirements. Therefore, how to utilize the stress-affected lands reasonably is an important and urgent problem to be resolved for sustainable agriculture.As an annual or perennial herbaceous plant, medicago has been regarded as "the king of grass" with abundant amino acid and protein contents. Because of the higher drought and salt-tolerance, it has been widely cultivated in China, including coastal saline areas and northwestern arid and semi-arid regions. Although medicago can be grown well under moderate stress conditions, its productivity and distribution are limited at some extent under severe stress conditions, except some relative high tolerant varieties. Researches showed that medicago has a large diversity of varieties with different tolerance to stresses. Accordingly, it is important to select varieties with relatively higher stress tolerance and identify the function of stress responsive genes for the development of novel medicago varieties with enhanced stress resistance and higher productivity and expanding the cultivation areas. In the present study, we firstly compared and screened preliminarily the drought and salt-tolerance of different alfalfa (M. sativa) varieties. Then, two varieties with contrasting stress tolerance were selected and assayed at the physiological, morphological and transcriptional levels. In addition, the MtPHD6 gene were cloned from the leaf of M. truncatula variety Jemalong A17 and transferred into the Arabidopsis plants. The transgenic plants were tested for tolerance to Mannitol, NaCl and ABA. The main research results were as follows:(1) Drought-tolerant Longdong and sensitive Algonquin were selected from 10 alfalfa varieties, and comparatively assayed at the physiological, morphological and transcriptional levels. Under control condition, the tolerant Longdong with smaller leaf size and lower stomata density showed less water loss than the sensitive Algonquin. After exposing to drought stress, Longdong showed less severe cell membrane damage, more proline and ascorbate (ASC) contents and less accumulation of reactive oxygen species (ROS) than Algonquin. Moreover, significantly higher antioxidant enzymes activities after drought treatment were found in Longdong when compared with Algonquin. In addition, transcriptional expression analysis showed that Longdong exhibited significantly higher transcripts of drought-responsive genes such as MtDehyd, MsNAC, MtCBF4 and MsHSP23 in leaf and root under drought stress condition. These results indicated that Longdong variety was more drought -tolerant than Algonquin variety as evidenced by less leaf firing, more lateral root number, higher relative aboveground/underground biomass per plant and survival rate after re-watering.(2) We selected the salt-tolerant 211609 and sensitive Xinjiang Daye from 14 alfalfa varieties. After 250 mM NaCl stress for 18 d,211609 exhibited higher leaf water content (LWC) and soluble protein content, less severe cell membrane damage and lower accumulation of reactive oxygen species (ROS) than Xinjiang Daye which exhibited lower GSH content and less antioxidant enzyme activities under salt stress. In addition, significantly higher expression levels of NHX1, ZFG, CBF4 and HSP23 genes were observed in 211609 than those in Xinjiang Daye upon exposure to salt stress. The above results indicated that 211609 showed higher tolerance to salt stress than Xinjiang Daye through regulation of physiological and transcriptional pathways.(3) The MtPHD6 gene was successfully cloned and transferred into Arabidopsis plants. The two lines, MtPHD6-4 and MtPHD6-7 with relatively higher expression were selected and tested the tolerance to mannitol, NaCl and ABA. Although no significant difference was observed in radicle emergence between the transgenic seeds and wild-type, the ratio of green cotyledon in transgenic plants was significantly higher than that in wide-type under high stress conditions.Collectively, alfalfa varieties showed different tolerance to drought and salt stresses. Through the comparative analysis at morphological, physiological and molecular levels, we concluded that Longdong variety exhibited higher tolerance to drought than Algonquin variety; and variety 211609 was more salt-tolerant than variety Xinjiang Daye. Moreover, the transgenic plants with overexpressing MtPHD6 gene could grow better than the wild under different high stress conditions. The above results were expected to provide some scientific basis for alfalfa breeding programs with improved stress tolerance in future.