Mechanisms Of MdASMT9-mediated Regulation Of Melatonin Synthesis In Response To Temperature And Low Nitrogen Stress In Apple

China is the world’s largest producer of apples（Malus domestica）,and the Loess Plateau region is one of the largest and optimal apple producing area in China.However,due to the poor soils,excessive application of nitrogen fertilizer is common in this region,which is not conducive to the sustainable development of the apple industry.In addition,temperature is one of the important environmental factors limiting the growth and development of fruit trees.During the growth of apples,they are often subject to chilling,freezing,and heat damage,which seriously affect the yield and quality of fruits.In plants,melatonin not only regulates growth and development,but also plays an important role as an antioxidant in response to abiotic stresses such as low nitrogen,low temperature and high temperature.However,most studies have focused on exogenous melatonin application,and the relationship between endogenous plant melatonin and plant stress resistance has been less studied,especially in perennial fruit trees.In the synthesis of melatonin,N-acetyl-5-hydroxytryptamino-methyltransferase（ASMT）catalyzes the last step of melatonin synthesis and plays an important role in melatonin synthesis.In this study,MdASMT9-overexpressed apple plants were used and found that the overexpression of MdASMT9 increased endogenous melatonin content and improved the tolerance of apple plants to high temperature,low temperature and low nitrogen stress.The main findings were as follows:1.Overexpression of MdASMT9 improved the ability of apple plants to resist high temperature stress.Under high temperature stress,overexpression of MdASMT9 enhanced the activity of plant antioxidant system and reduced the accumulation of ROS.Overexpression of MdASMT9 also protected photosynthetic organs by reducing chloroplast damage,which in turn enhanced photosynthetic capacity.Transcriptome sequencing and physiological data analysis showed that overexpression of MdASMT9 under heat stress promoted the accumulation of soluble carbohydrates（sorbitol,galactose and sucrose）and many amino acids（histidine,arginine,aspartate,proline,glutamine and glycine）in apple plants and improved the adaptability to heat stress.In addition,melatonin promoted the decrease of NCED1/NCED3 gene expression mediated by the transcription factor Md WRKY33,which negatively regulated ABA synthesis,allowing leaves to exhibit greater stomatal opening to maintain proper leaf temperature at high temperatures.2.Overexpression of MdASMT9 enhanced the low temperature tolerance of apple plants.q RT-PCR assay revealed that MdASMT9 transcript levels were significantly induced by cold stress.Seedlings of wild type（WT）and MdASMT9-overexpressed apple plants were treated with 4℃ and it was found that overexpression of MdASMT9 significantly enhanced the activity of antioxidant system and reduced the accumulation of ROS in apple plants.Meanwhile,MdASMT9-overexpressed plants could maintain higher light energy conversion efficiency,improve photoprotection mechanism,and alleviate the damage to photosynthetic system by cold stress.In addition,freezing treatments（-30℃,-35℃,and-40℃）on annual branches of WT and MdASMT9-overexpressed apple plants revealed that the integrity of the membrane system of MdASMT9-overexpressed apple plants was significantly higher than that of WT.3.The transcription factor MdHY5 positively regulated the response of MdASMT9 to cold stress.A b ZIP transcription factor MdHY5 was obtained by yeast one hybridization screening using the MdASMT9 promoter sequence as bait.Electrophoretic mobility shift assay（EMSA）and dual luciferase activity assays showed that MdHY5 is a transcriptional activator of the MdASMT9 and the binding effect of both was enhanced under cold stress.Low temperature treatment of MdHY5-overexpressed and MdHY5-silenced apple calli revealed that both MdASMT9 expression and melatonin content were significantly increased in MdHY5-overexpressed apple calli and significantly decreased in MdHY5-silenced apple calli under cold treatment,indicating that MdHY5 activates the expression of MdASMT9 by specifically binding to its promoter,thereby promoting the synthesis of melatonin under low temperature stress.In addition,the overexpression of MdASMT9 and exogenous melatonin treatment also promoted the expression of MdHY5 and its downstream target genes CBFs under cold stress,which in turn improved the cold tolerance of apple plants.4.Overexpression of MdASMT9 could improve the ability of apple plants to tolerate low nitrogen stress.Under low nitrogen stress,the melatonin content in leaves and roots of MdASMT9-overexpressed apple plants was significantly higher WT,showing better growth status and higher biomass.Meanwhile,overexpression of MdASMT9 could maintain higher light capture capacity and light energy conversion efficiency in apple plants and alleviate the damage to photosynthetic system by low nitrogen stress.Proteome sequencing analysis showed that differential proteins between WT and transgenic apple plants were mainly localized in chloroplasts under low nitrogen stress.COG/KOG functional annotation analysis revealed that many differentially expressed proteins were involved in the transport and metabolism of carbohydrates,lipids and amino acids,energy production and conversion,as well as biosynthesis,transport and metabolism of secondary metabolites.In combination with physiological data,it was found that overexpression of MdASMT9 promoted the tricarboxylic acid cycle（TCA）and positively regulated amino acid metabolism to improve plant resistance to low nitrogen stress.5.The promotion of nitrate uptake by melatonin under low nitrogen stress was dependent on the regulation of nitrate transporters（NRT2.1 and NRT2.4）by MdHY5.The determination of NO₃^-and stable isotopeδ¹⁵N revealed that overexpression of MdASMT9enhanced nitrate uptake in the roots of apple plants.Gene expression analysis revealed that0.5μM exogenous melatonin treatment and overexpression of MdASMT9 promoted the expression of MdHY5,Md NRT2.1 and Md NRT2.4 in apple roots under low nitrogen stress.EMSA and dual luciferase activity assay showed that MdHY5 specifically bind to the promoters of Md NRT2.1 and Md NRT2.4,thus activate their expression.MdHY5 RNAi lines were obtained using Agrobacterium rhizogenes mediated transformation.Under low nitrogen stress,the expression levels of MdHY5,Md NRT2.1 and Md NRT2.4 in WT with exogenous melatonin were significantly higher than those in WT without melatonin application.However,the expression levels of MdHY5,Md NRT2.1 and Md NRT2.4 in MdHY5 RNAi lines with exogenous melatonin were not significantly different from those in MdHY5 RNAi lines without melatonin application.These results showed that melatonin could improve nitrate uptake in apple plants under low nitrogen stress by promoting the expression of MdHY5,which in turn activated the expression of Md NRT2.1 and Md NRT2.4.In addition,the promotion of nitrate uptake by melatonin was dependent on the regulation of MdHY5 on Md NRT2.1 and Md NRT2.4.