Functional Characterization Of An Apple Autophagy-related Gene MdATG18a In Response To Different Stresses

Apple is one of the main fruits in the world and occupies an important position in the world's fruit production.The Northwest Loess Plateau is the largest and best apple production area in China,but the less rainfall,poor soil and diseases in the area restrict the production of apples.Autophagy is a highly conserved process of intracellular macromolecular degradation which can circulate excess or damaged cell components during stress or growth.Autophagy is also involved in the degradation of proteins and organelles.It plays an important role in leaf senescence,nutrition starvation and oxidative stress.There are few reports on the identification and function analysis of autophagy-related genes?ATG?in Malus.In this thesis,we first identified an autophagy-related gene MdATG18a from apple genome and explored its role in response to leaf senescence and various stresses.Overexpressed MdATG18a transgenic apple,tomato and Arabidopsis were obtained by transgenic technology.The plants were subjected to various adversity treatments to identify their function and mechanism under various stresses.The main results obtained are as follows:1.ORF of MdATG18a is a complete 1281bp of nucleotides encoding a polypeptide of426 amino acids.The deduced protein contains two WD40 domains.Phylogenetic analysis confirmed that MdATG18a clusters with Arabidopsis members AtATG18a.When transiently expressed in onion epidermal cells,the MdATG18a–GFP fusion protein was localized in the nucleus and cell membrane.Prediction of an 1824 bp promoter region cloned from apple genome DNA indicated several key cis-regulatory elements involved in plant responses to abiotic stresses such as LTR,MBS and ARE.MdATG18a was expressed in all tested tissues and showed obvious up-regulation under leaf senescence,drought,heat,oxidative stress,nitrogen starvation or ER stress.Taken together,our findings suggest that MdATG18a plays a role in responses to leaf senescence and abiotic stresses,which might be indicative of autophagy involved in mechanism regulation for stress tolerance in apple.2.Overexpression of MdATG18a in tomato and apple plants markedly enhanced their tolerance to drought.Compared to the wild type?WT?,the relative water content and survival rate of transgenic tomato plants were significantly higher,MDA content and electrolyte leakage of transgenic tomato plants were lower under drought stress.In transgenic apple plants,the relative water content and chlorophyll content were higher than WT,and the MDA and electrolyte leakage were lower than WT under drought stress.Under drought conditions,the photosynthesis rate and antioxidant capacity were significantly elevated in OE apple lines when compared with the untransformed WT.The percentage of insoluble protein in proportion to total protein and H₂O₂ content was lower and less oxidized protein accumulated in the OE lines than in the WT under drought stress.Transcript levels of major apple ATG genes were more strongly up-regulated in transgenic MdATG18a OE lines than in the WT.Transmission electron microscopy analysis showed that transgenic apple plants accumulated more autophagosomes under drought stress.These results demonstrate that overexpression of MdATG18a enhances plant tolerance to drought stress,probably because of greater autophagosome production and a higher frequency of autophagy.Those processes suggesting that autophagy plays important roles in the drought response.3.Overexpression of MdATG18a in Arabidopsis and apple both improved tolerance to N-depletion and caused a greater accumulation of anthocyanin.The increased anthocyanin concentration in transgenic apple was possibly due to up-regulating flavonoid biosynthetic and regulatory genes?MdPAL,MdCHS,MdCHI,MdANS,MdUFGT and MdMYB1?,and higher soluble sugars concentration.MdATG18a overexpression enhanced starch degradation with up-regulating amylase gene?MdAM1?,and up-regulated sugar metabolism related genes?MdSS1,MdHXKs,MdFK1 and MdNINVs?.Furthermore,MdATG18a functioned in nitrate uptake and assimilation by up-regulating nitrate reductase MdNIA2 and three high-affinity nitrate transporters MdNRT2.1/2.4/2.5.MdATG18a overexpression also elevated other important MdATG genes expression and autophagosomes formation under N-depletion,which play key contributions to above changes.Together these results demonstrate that overexpression of MdATG18a enhances tolerance to N-deficiencies and plays positive roles in anthocyanin biosynthesis through greater autophagic activity.4.Overexpression of MdATG18a enhances resistance to D.mali infection.Less H₂O₂ but more SA was detected in OE lines after D.mali infection.Expression of chitinase,?-1,3-glucanase,and SA-related marker genes was induced more strongly by D.mali in those lines.Transcript levels of other important MdATG genes were also drastically induced by D.mali in OE plants.Taken together,these results demonstrate that overexpression of MdATG18a enhances resistance to D.mali infection and has positive roles in H₂O₂-scavenging and SA accumulations.