The Relationship Between Dynamic Changes Of ABA Content Regulated By β-glucosidase And Drought Tolerance In Arabidopsis Thaliana

The phytohormone ABA plays crucial roles in various physiological processes including seed dormancy, germination and adaptive responses to environmental stress such as drought, cold and high salt in soil. The content fluctuate of cellular ABA levels allow plants to adapt to the changing physiological and environmental conditions. Cellular ABA is controlled by complex de novo synthesis pathways, hydroxylation and conjugation reactions. ABA glucose ester (ABA-GE) is the dominating form covalent bounding ABA in plants. However, the issue of whether the inactive ABA-GE could re-release and produce the biological active ABA remains to be clarified.We screened a gain-function of ABA-insensitive mutant gai6, the mutation gene encodedβ-glucosidase BGLU10. T-DNA insertion homozygous mutant bglulO showed faster withering and death than WT under drought stress, and stomatal behavior analysis showed that stomata of bglu10 tend to keeping bigger aperure than that of WT, which leads to greater loss of water whether under stress or not. By contrast, the over-expression lines of BGLU10 showed stronger drought-tolerance than WT. In addition, BGLU10-OE lines behaved fewer green cotyledons after germination than WT, while the mutant bglu10 performed better situation after germination and seedling growth under ABA, NaCl and mannitol stress treatments. Determination of ABA content in plants under both normal watering and drought condition showed BGLU10-OE lines hold higher ABA content than that in WT, but ABA content in bglu10 slightly lower than that in WT. The results of expression analyses for marker genes responding to ABA and/or drought showed RAB18, RD29B and RD22 were dramatically induced by dehydration stress, and it worthy noted that the expressions level of these genes were highest in BGLU10-OE lines but lowest in bglulO mutant. The similar expression pattern was found for homeodomain-leucine zipper transcription factor gene ATHB6, which was known significantly up-regulated by ABA. Furthermore, the total P-glucosidase activity is positive correlate to the drought resistance in bglu10 mutant, WT and BGLU10-OE plants. These results also effectively explained the difference between ABA content and drought-tolerance of different plants, suggesting BGLU 10 may have the function of hydrolysing ABA-GE and releasing of free ABA to enhance stress-tolerance response of plants.Fluorescence co-localization result of BGLU10::GFP and y-TIP 1::RFP (a tonoplast marker protein) fusion proteins using transient expression in protoplast of mesophyll cells showed BGLU 10 enzyme protein localized in vacuole, and our experiment also showed the optimal pH is about pH 6.0 of totalβ-glucosidase activity in Arabidopsis, which matched the known vacuolar pH environment. GUS staining analysis of BGLU10 gene organ-specific expression showed that this gene is transcripted in various organizations, including roots, stems, leaves, flowers and siliqua, especially in mature conducting tissues. In addition, BGLU10 was induced by ABA, salt, drought and cold stress, suggesting thatβ-glucosidase BGLU 10 may be involved in variety of stresses and hydrolysis of ABA-GE producing free ABA in plant stress tolerance response.In conclusion, this study provide us a new understanding of ABA level dynamic adjustment mechanism in plants, namely, by fine controlling of free ABA content through collaboratively tuning pathways of ABA de novo synthesis, hydroxylation catabolism, covalent conjugation and releasing free ABA from ABA-GE, so plants can cope with complex and changing stress environment and the developing needs.