Physiological Mechanism Of MdATG5a And MdATG8i In Regulating Water Use Efficiency And Nitrogen Use Efficiency In Apple

China is the largest producer and consumer of apple（Malus×domestica）in the world.The Loess Plateau is the largest and best apple producing area in China.However,the annual precipitation in this region is low and unevenly distributed throughout the annual cycle,therefore long-term drought has become the main factor restricting the apple production in the Loess Plateau.In addition,the poor soil in this area leads to excessive application of nitrogen（N）fertilizer in apple production,which is not conducive to the sustainable development of apple production in the Loess Plateau.As a conserved degradation and recycling mechanism in eukaryotes,autophagy has been reported to participate in plant growth and development and rapid responses to various stresses.However,it is unclear whether autophagy is involved in regulating the adaptive response of plants to long-term stress.In view of the moderate drought and low nitrogen fertilizers utilization efficiency faced by the apple industry in the Loess Plateau,this research focused on exploring the function of autophagy-related genes MdATG8i and MdATG5a in regulating water use efficiency（WUE）and nitrogen use efficiency（NUE）of apple plants under long-term moderate drought stress and and low N stress,respectively.The research provided a theoretical basis for the genetic engineering breeding of apple varieties with high WUE and NUE.The main results are as follows:1.The expression of MdATG8i can be induced by both natural drought and long-term moderate drought stress.MdATG8i positively regulates the natural drought tolerance and the WUE of apple plants under long-term moderate drought stress.Overexpression of MdATG8i improves the tolerance of apple plants under natural drought stress mainly by enhancing the ROS scavenging and autophagic activity.After 80 d of long-term moderate drought stress,MdATG8i-overexpressing（OE）apple plants exhibited higher biomass accumulation and WUE compared with the wild-type（WT）plants.Under long-term drought conditions,the autophagic activity,photosynthetic capacity and osmotic adjustment ability were higher in MdATG8i-OE plants than in WT plants.The results demonstrated that MdATG8i not only regulated stomatal opening of apple plants under long-term moderate drought stress by reducing the ABA content and H₂O₂ levels in guard cells,but also enhanced energy conversion efficiency by alleviating the damage to chloroplast,thus leading to the improved photosynthetic capacity of apple plants under long-term moderate drought stress.In addition,under long-term moderate drought conditions,overexpression of MdATG8i improved the osmotic adjustment by enhancing the accumulation of carbohydrates and amino acids,which eventually contributed to the improved WUE and alleviated growth inhibition caused by long-term drought stress.2.The expression of MdATG5a is also induced by natural drought and long-term moderate drought stress.MdATG5a shows positive effects on regulating apple tolerance under natural drought stress and improving WUE of apple plants under long-term moderate drought stress.The results of Split-Luc demonstrated that MdATG5a can interact with MdATG8i in vivo.MdATG5a not only regulated the tolerance of transgenic plants to natural drought stress and WUE under long-term moderate drought stress through the same mechanism as MdATG8i,but also enhanced water uptake in transgenic plants by promoting root growth and root water conductivity.In addition,MdATG5a can interact with Md ICE1b to mediate the reduction in stomatal density in apple plants under long-term moderate drought stress,thereby reducing the water loss.Furthermore,higher leaf cuticular wax accumulation in the transgenic plants resulted in less non-stomatal water loss.Under long-term moderate drought stress,‘strong absorption’and‘reduced water loss’eventually resulted in higher WUE of transgenic plants than WT.3.MdATG8i can be induced by low N stress in apple,particularly in leaves.Overexpression of this gene can improve the NUE of apple plants under low N conditions.After 90 d of low N treatment,the absorption and content of N in transgenic plants were higher than those in WT,which contributed to enhancing the content of photosynthetic pigments and photosynthetic rate of transgenic apple plants,eventually leading to higher biomass accumulation and NUE.In addition,overexpression of MdATG8i enhanced the activities of NR,Ni R,GS and GOGAT in apple under low nitrogen stress,thus improving the efficiency of nitrogen assimilation and amino acid content of transgenic plants.The increased autophagic activity mediated by MdATG8i promoted the transfer of nitrogen from old leaves to new leaves in apple under low nitrogen stress and therefore improved nitrogen reuse.These results demonstrated that MdATG8i-mediated autophagy improved the NUE of apple plants under low nitrogen stress mainly by promoting the nitrogen absorption,assimilation,and reuse.4.MdATG5a can be induced by low N stress in apple,particularly in roots,and this gene improved the NUE of apple plants under low N stress by positively regulating root growth and N uptake.Under low N conditions,in addition to enhancing autophagic activity,MdATG5a also activated the shikimate pathway to increase the levels of phenylalanine and tryptophan,which in turn promoted the accumulation of flavonoids and IAA in apple roots,which eventually improved root growth of apple plants.In addition,overexpression of MdATG5a enhanced the accumulations of amino acids and soluble sugars in the apple roots under low N conditions,thus providing energy for root growth.The vigorous root growth of transgenic apple enhanced nitrogen absorption,and ultimately increased the NUE of apple plants under low N conditions.In conclusion,all these results demonstrate that MdATG8i and MdATG5a regulate WUE and NUE in apple plants,not only through the same mechanism induced by autophagy,but also through their unique regulatory modalities.