Study On The Function And Mechanism Of MdHB-7 And MdHB7-like In Apple On Drought And Salt Tolerance

Apple(Malus domestica)is an important fruit throughout the world,and China is the largest producer and consumer of apple.The Loess Plateau is the eugenic and main producing area for apple in China.However,stresses such as drought and salt limit the apple sustainable cultivation in this area.The homeodomain-leucine zipper(HD-Zip)transcription factors play important roles in the regulation of drought and salt tolerance in plants,but their specific functions in the regulation of apple drought and salt response remain unclear.The previous research of our research group found that the expression of some HD-Zip family genes was significantly up-regulated in the long-term drought transcriptome of apple plant.Therefore,the members of the apple HD-Zip family were identified genome-wide,and two genes MdHB-7 and MdHB7-like,which significantly responded to drought and salt stress,were screened.Subsequently,the functions and molecular mechanisms of MdHB-7 and MdHB7-like in apple in response to drought and salt stress were systematically analyzed by apple genetic transformation and related physiological and molecular techniques.The main results are documented as follows.1.Genome-wide identification of the HD-Zip gene family in apple.A total of 59 genes were confirmed to be members of the HD-Zip family in apple.Conserved domain analysis showed that they all contained HD and Zip domains.Phylogenetic analysis showed that the HD-Zip gene family was classified into four major subfamilies.Synteny analysis of the apple HD-Zip genes indicated that segmental duplications have likely contributed to the expansion and evolution of these genes.Long-term moderate drought transcriptome analysis revealed that MdHB-7 and MdHB7-like were enriched in up-regulated differentially expressed genes.Promoter cloning and sequence analysis revealed that MdHB-7 and MdHB7-like promoters contained several putative abiotic stress-related cis-elements.The transcript levels of MdHB-7 and MdHB7-like were up-regulated in leaves under drought,salinity,or ABA treatments,indicating that these two genes may play important roles in the stress response of apple plants.MdHB-7 and MdHB7-like were expressed in the nucleus and had transcriptional activation activity.2.MdHB-7 plays a pivotal role in drought tolerance and water use efficiency in transgenic apple.We created transgenic apple plants that exhibited either MdHB-7overexpression or RNAi-induced MdHB-7 suppression.Subsequently,GL-3 and MdHB-7transgenic plants were subjected to short-term drought treatment.Under normal conditions,there were no significant differences in growth status or physiological indicators between transgenic and GL-3 plants.Under drought conditions,compared with GL-3,overexpression transgenic plants showed less morphological damage and improved physiological indicators related to stress damage,whereas suppression of MdHB-7 expression had the opposite effect.MdHB-7 enhances drought tolerance in transgenic plants by promoting drought-induced endogenous ABA accumulation,stomatal closure and reactive oxygen species(ROS)scavenging.In addition,overexpression of MdHB-7 enhanced long-term moderate drought adaptation and improved water use efficiency of apple plants.3.Apple MdHB7-like plays a pivotal role in drought tolerance and water-use efficiency.We created transgenic apple plants that exhibited either MdHB7-like overexpression or RNAi-induced MdHB7-like suppression.Subsequently,GL-3 and MdHB7-like transgenic plants were subjected to short-term drought treatment.Comparison of drought phenotypes and several physiological indicators indicated that transgenic apple with upregulated expression of MdHB7-like showed enhanced drought tolerance,whereas MdHB7-like RNAi lines were more sensitive to drought.Further analysis showed that MdHB7-like reduced stomatal density by negatively regulating the expression of genes positively regulating stomatal development(Md EPFL9.1 and Md EPFL9.2);and promoted drought-induced endogenous ABA accumulation to induce stomatal closure;and also activated the antioxidant system to scavenge reactive oxygen species(ROS)by promoting the expression of antioxidant enzymes,which in turn improved the stomatal density of transgenic plants.Further study showed that the overexpression of MdHB7-like significantly increased water use efficiency of transgenic plants under long-term moderate drought conditions.4.MdHB-7 and MdHB7-like confers tolerance to salinity in transgenic apple(Malus domestica).GL-3,MdHB-7 and MdHB7-like transgenic apple plants were treated with Na Cl in hydroponic and soil culture systems.Comparison of salt stress phenotypes and several physiological indicators indicated that MdHB-7 or MdHB7-like overexpression transgenic apple plants enhanced salt tolerance.MdHB-7 and MdHB7-like enhanced the tolerance of transgenic plants to salt stress by maintaining a higher photosynthetic rate,reducing the accumulation of ROS and Na+,and promoting the accumulation of proline.