Effect Of Cement Dust And Salicylic Acid On Apple Tree And Role Of MdMED81 In Flowering

China’s apple cultivation area and output rank first in the world,accounting for more than50% of the world’s cultivated area and output,which makes a significant contribution to the economic development and the income of fruit farmers.However,Fuji varieties,which account for more than 75% of the cultivated area in China,are easily affected by environmental biotic and abiotic stresses(cement dust deposition)in the process of growth and development,resulting in practical production problems such as difficulty in flower formation and alternate bearing,which seriously affect the high-quality development of China apple industry.In addition,studies have found that the phytohormone salicylic acid(SA)plays an important regulatory role in plant flower induction and flower bud formation.Based on actual production problems such as difficulty in flowering in apple production,this study investigated the effects of abiotic factors such as cement dust deposition and biological factors such as SA on apple leaves,buds,and flowers,and revealed that SA regulates apple flowering induction.The morphological and physiological mechanisms of the buds and the RNA-seq data of SA-treated apical buds were used to clarify the regulatory network that SA regulates the growth,development,and flowering of terminal buds of short shoots(also called the spur buds)in apples.One of Md MED gene family member,Md MED81,was used as a key candidate gene.Overexpressing Md MED81 in Arabidopsis thaliana to elucidate its role in apple flower formation.The above research results are of great theoretical and practical significance for enriching the flowering theory of woody fruit trees and developing a series of flowerpromoting technologies for Fuji apple.1.Effects of cement dust deposition and salicylic acid treatment on leaf quality of apple treesIn this study,the apple tree of ’Changfu 2’ was used as the material,and the long-term cement dust deposition and SA treatment of apple trees were used to clarify the effects of the two treatments on apple leaves,spur buds,and flower formation.The results showed that longterm cement dust deposition significantly affected the growth and development of apple leaves and spur buds,as well as leaf quality.The fresh weight,length and width of leaves and short branch terminal buds decreased significantly in the cement dust deposition treatment,and the analysis results of the leaves and buds showed that the cell area and leaf thickness of the leaves and buds were significantly reduced in the cement dust deposition treatment.The oxidative stress and antioxidant enzyme activity levels of leaves treated with dust deposition significantly increased,but the levels of ascorbic acid,soluble sugars,free amino acids,and pigments decreased.Taken together,it is shown that cement dust is a harmful pollutant that can induce abiotic stress responses in apples and has significant adverse effects on the quality and health of apple leaves and spur buds.In contrast,SA treatment could significantly promote the growth and development of apple leaves,improve leaf quality,and enhance their stress response to environmental stress.For example,SA treatment significantly increased the area,length,and width of apple leaves;SA treatment increased non-enzymatic antioxidants in apple leaves,while enzymatic antioxidants significantly decreased,indicating that it enhanced leaf stress resistance and improved leaf quality and health.In addition,SA treatment significantly promoted the quality and development of apple spur buds,and the expression levels of Md MED80,Md MED81,Md MED3 and Md MED41 genes in leaves were significantly increased under the induction of SA,which may be closely related to the regulation of apple flowering induction by SAmediated photoperiod pathway.2.Effects of salicylic acid on apple flowering and transcriptome analysis of SAtreated spur buds during flowering induction stageIn this study,the difficult-to-flower cultivar ’Changfu 2’ was used as the material to study the effect of SA treatment on apple flowering and the transcriptome analysis of SA treatment at the flowering induction stage of spur buds,in order to clarify the effect of SA on apple flower bud formation,to further explore the key flowering regulation pathways and molecular networks of SA-regulated-formation of flower bud.The results showed that the SA treatment significantly increased the flower formation rate of ’Changfu 2’,and the quality,length and width of the spur buds were significantly greater than those of the control treatment(sprayed with water).In addition,by high performance liquid chromatography(HPLC)analysis showed that SA treatment significantly increased the content of endogenous SA and IAA in apical buds,but significantly down-regulated the levels of gibberellin(GA3 and GA4).Apple flowering may mainly involve the IAA and gibberellin pathways.For example,SA treatment up-regulates the expression of key genes for auxin synthesis(such as Md ASA1 and Md CYP79B3),which in turn promotes IAA and synthesis in the apical buds;but significantly down-regulates the content of gibberellin in spur buds with down-regulating Md GA3(a key enzyme gene for gibberellin synthesis)and up-regulating Md GA2OX1,-2,-8(a key enzyme gene for gibberellin oxidative degradation).It can be seen that the higher IAA and lower gibberellin content in the spur buds are crucial for the formation of flower buds in the apple flower induction stage.In addition,SA treatment significantly increased the flowering genes such as Md MADS-box(such as Md SOC1,MdFUL,MdAGL42,MdAGL42)and Md SPLs,which were directly related to the promotion of apple flower bud formation.3.Functional analysis of apple key candidate Md MED81 gene mediates salicylic acid in flower formationIn this study,based on the evaluation of the effect of SA treatment on apple flowering and the analysis of RNA-seq data of spur buds,it was preliminarily found that members of the apple Md MED family may mediate SA involved in the regulation of apple flowering.Based on this,firstly,83 members of Md MED in apple were identified,and their family gene structure,protein characteristics,location of each member on chromosomes,and phylogenetic structure analysis were systematically analyzed by bioinformatics.The results showed that the members of the Md MED gene family were divided into six subgroups according to the phylogenetic relationship.The candidate key member Md MED81 belonged to the Md MED subgroup-D gene.Md MED genes of subgroup-D were expressed in the roots,stem,leaves and spur buds.The expression of Md MED genes of subgroup-D in spur buds was induced by exogenous SA treatment during the flower induction stage.Among them,the candidate key member Md MED81 was significantly induced by SA.In addition,through subcellular localization analysis,it was found that the Md MED81 gene was mainly located in the nucleus;by obtaining transgenic lines overexpressing A.thaliana OE-Md MED81,it was found that compared with the wild type,all three lines had significant early-flowering and multiflowering expressions.Therefore,the key candidate apple Md MED81 has an important function in the regulation of flowering in response to SA.In addition,the expression level of Md MED81 in apple leaves was found to be significantly correlated with daylight hours,indicating that apple Md MED81 may regulate flower bud formation in response to SA through a photoperiodic pathway.