Overexpression of auxin/indole-3-acetic acid gene MdIAA24 enhances Glomerella leaf spot resistance in apple(Malus domestica)

Auxin is throughout the entire life process of plants and is involved in the crosstalk with other hormones,yet its role in apple disease resistance remains unclear.In this study,we investigated the function of auxin/indole-3-acetic acid(IAA)gene Md IAA24 overexpression in enhancing apple resistance to Glomerella leaf spot(GLS)caused by Colletotrichum fructicola(Cf).Analysis revealed that,upon Cf infection,35S::Md IAA24 plants exhibited enhanced superoxide dismutase(SOD)and peroxidase(POD)activity,as well as a greater amount of glutathione(reduced form)and ascorbic acid accumulation,resulting in less H_(2)O_(2)and superoxide anion(O_(2)^(-))in apple leaves.Furthermore,35S::Md IAA24 plants produced more protocatechuic acid,proanthocyanidins B1,proanthocyanidins B2 and chlorogenic acid when infected with Cf.Following Cf infection,35S::Md IAA24 plants presented lower levels of IAA and jasmonic acid(JA),but higher levels of salicylic acid(SA),along with the expression of related genes.The overexpression of Md IAA24 was observed to enhance the activity of chitinase andβ-1,3-glucanase in Cfinfected leaves.The results indicated the ability of Md IAA24 to regulate the crosstalk between IAA,JA and SA,and to improve reactive oxygen species(ROS)scavenging and defense-related enzymes activity.This jointly contributed to GLS resistance in apple.

Overexpression of the transcription factor MdWRKY115 improves drought and osmotic stress tolerance by directly binding to the MdRD22 promoter in apple

Abiotic stress reduces plant yield and quality.WRKY transcription factors play key roles in abiotic stress responses in plants,but the molecular mechanisms by which WRKY transcription factors mediate responses to drought and osmotic stresses in apple(Malus×domestica Borkh.)remain unclear.Here,we functionally characterized the apple GroupⅢWRKY gene MdWRKY115.qRT-PCR analysis showed that MdWRKY115 expression was up-regulated by drought and osmotic stresses.GUS activity analysis revealed that the promoter activity of MdWRKY115 was enhanced under osmotic stress.Subcellular localization and transactivation assays indicated that MdWRKY115 was localized to the nucleus and had a transcriptional activity domain at the N-terminal region.Transgenic analysis revealed that the overexpression of MdWRKY115 in Arabidopsis plants and in apple callus markedly enhanced their tolerance to drought and osmotic stresses.DNA affinity purification sequencing showed that MdWRKY115 binds to the promoter of the stress-related gene MdRD22.This binding was further verified by an electrophoretic mobility shift assay.Collectively,these findings suggest that MdWRKY115 is an important regulator of osmotic and drought stress tolerance in apple.

Genome-wide identification and function analysis of the sucrose phosphate synthase MdSPS gene family in apple

Sucrose phosphate synthase(SPS)is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase(SPP)for sucrose synthesis,and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.However,studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.In the present study,a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.The gene structures and their promoter cis-elements,protein conserved motifs,subcellular localizations,physiological functions and biochemical properties were analyzed.A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication(WGD)and segmental duplication played vital roles in MdSPS gene family expansion.The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.Furthermore,three SPS gene subfamilies were classified based on phylogenetic relationships,and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.In addition,a major gene related to sucrose accumulation(MdSPSA2.3)was identified according to the highly consistent trends in the changes of its expression in four apple varieties(‘Golden Delicious’,‘Fuji’,‘Qinguan’and‘Honeycrisp’)and the correlation between gene expression and soluble sugar content during fruit development.Furthermore,the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

4-methylumbelliferone (4-MU) enhances drought tolerance of apple by regulating rhizosphere microbial diversity and root architecture

The dwarfing rootstocks-mediated high-density apple orchard is becoming the main practice management.Currently,dwarfing rootstocks are widely used worldwide,but their shallow root system and drought sensitivity necessitate high irrigation requirements.Here,the root transcriptome and metabolome of dwarfing(M9-T337,a drought-sensitive rootstock)and vigorous rootstocks(Malus sieversii,a drought-tolerant species,is commonly used as a rootstock)showed that a coumarin derivative,4-Methylumbelliferon(4-MU),was found to accumulate significantly in the roots of vigorous rootstock under drought condition.When exogenous 4-MU was applied to the roots of dwarfing rootstock under drought treatment,the plants displayed increased root biomass,higher root-to-shoot ratio,greater photosynthesis,and elevated water use efficiency.In addition,diversity and structure analysis of the rhizosphere soil microbial community demonstrated that 4-MU treatment increased the relative abundance of putatively beneficial bacteria and fungi.Of these,Pseudomonas,Bacillus,Streptomyces,and Chryseolinea bacterial strains and Acremonium,Trichoderma,and Phoma fungal strains known for root growth,or systemic resistance against drought stress,were significantly accumulated in the roots of dwarfing rootstock after 4-MU treatment under drought stress condition.Taken together,we identified a promising compound—4-MU,as a useful tool,to strengthen the drought tolerance of apple dwarfing rootstock.

Overexpression of MdFRK2 enhances apple drought resistance by promoting carbohydrate metabolism and root growth under drought stress

Soluble sugars function not only as the energy and structural blocks supporting plants,but also as osmoregulators and signal molecules during plant adaptation to water deficit.Here,we investigated drought resistance in transgenic apple(Malus×domestica)overexpressing MdFRK2,a key gene regulating fructose content and sugar metabolism.There is no obvious phenotypic difference between MdFRK2-overexpressing transgenic plants and WT plants under the well-watered condition.However,the transgenic plants and the grafted plants using MdFRK2-overexpressing rootstock exhibited improved tolerance to drought stress.Overexpression of MdFRK2 significantly promoted the growth of root system under drought stress.RNA sequencing showed that under drought stress,genes involved in sugar metabolism,transcription regulation,signal transduction or hormone metabolism were differentially expressed in MdFRK2 transgenic plants.Consistent with the gene expression profile,the activities of enzyme(SDH,FRK and NI)involved in sugar metabolism in the roots of MdFRK2 transgenic plants were significantly higher than those of untransformed control plants after drought stress.Under drought stress,overexpression of MdFRK2 promoted the accumulation of IAA,and decreased the contents of ABA and CK in apple root system.In conclusion,these results suggest that MdFRK2 can promote the growth of apple roots under drought stress by regulating sugar metabolism and accumulation,hormone metabolism and signal transduction,and then resist drought stress.

The m6A reader MhYTP2 regulates the stability of its target mRNAs contributing to low nitrogen tolerance in apple (Malus domestica)

Studies have shown that the m6A reader primarily affects genes expression by participating in the regulation of mRNA localization,splicing,degradation,translation,and other metabolic processes.Previously,we discovered that the apple(Malus domestica)m6A reader MhYTP2 bound with and destabilized m6A-modified MdMLO19 mRNA.In addition,it enhanced the translation efficiency of m6A-modified mRNA of MdGDH1L,encoding a glutamate dehydrogenase,which confers resistance to powdery mildew.In this study,we report the function of MhYTP2 in the regulation of resistance to low nitrogen(N).The overexpression of MhYTP2 enhances the resistance of apple to low N.We show that MhYTP2 binds with and stabilizes the mRNAs of MdALN,which participates in the allantoin catabolic process and cellular response to N starvation in apple;MdPIDL,which participates in root hair elongation;MdTTG1,which is involved in the differentiation process of trichomes;and MdATG8A,which is a core participant in the regulation of autophagy.In addition,MhYTP2 accelerates the degradation of MdRHD3 mRNA,which regulates root development.RNA immunoprecipitation-seq and electrophoretic mobility shift assays show that the mRNAs of MdALN,MdATG8A,MdPIDL,MdTTG1,and MdRHD3 are the direct targets of MhYTP2.Overexpressing or knocking down the above genes in MhYTP2 overexpressing plants dismisses the function of MhYTP2 under low N,suggesting the role of MhYTP2 is dependent on those genes.Together,these results demonstrate that MhYTP2 enhances the resistance of apple to N deficiency by affecting the stability of the bound mRNAs.

Insights into the molecular mechanisms underlying responses of apple trees to abiotic stresses

Apple(Malus×domestica)is a popular temperate fruit crop worldwide.However,its growth,productivity,and quality are often adversely affected by abiotic stresses such as drought,extreme temperature,and high salinity.Due to the long juvenile phase and highly heterozygous genome,the conventional breeding approaches for stress-tolerant cultivars are time-consuming and resource-intensive.These issues may be resolved by feasible molecular breeding techniques for apples,such as gene editing and marker-assisted selection.Therefore,it is necessary to acquire a more comprehensive comprehension of the molecular mechanisms underpinning apples’response to abiotic stress.In this review,we summarize the latest research progress in the molecular response of apples to abiotic stressors,including the gene expression regulation,protein modifications,and epigenetic modifications.We also provide updates on new approaches for improving apple abiotic stress tolerance,while discussing current challenges and future perspectives for apple molecular breeding.

Overexpression of MdATG18a in apple improves resistance to Diplocarpon mali infection by enhancing antioxidant activity and salicylic acid levels

Marssonina apple blotch,caused by Diplocarpon mali,is one of the most serious diseases of apple.Autophagy plays a key role in pathogen resistance.We previously showed that MdATG18a has a positive influence on drought tolerance.Herein,we describe how overexpression(OE)of MdATG18a enhances resistance to D.mali infection,probably because less H2O2 but more salicylic acid(SA)is accumulated in the leaves of OE apple plants.Expression of chitinase,β-1,3-glucanase,and SA-related marker genes was induced more strongly by D.mali in OE lines.Transcript levels of other important MdATG genes were also drastically increased by D.mali in OE plants,which indicated increased autophagy activities.Taken together,these results demonstrate that OE of MdATG18a enhances resistance to infection by D.mali and plays positive roles in H2O2-scavenging and SA accumulations.Our findings provide important information for designing strategies which could induce autophagy to minimize the impact of this disease on apple production.

Transcriptome analysis reveals the effects of alkali stress on root system architecture and endogenous hormones in apple rootstocks

Soil alkalinity is a major factor that restricts the growth of apple roots.To analyze the response of apple roots to alkali stress, the root structure and endogenous hormones of two apple rootstocks, Malus prunifolia (alkali-tolerant) and Malus hupehensis (alkali-sensitive), were compared. To understand alkali tolerance of M. prunifolia at the molecular level, transcriptome analysis was performed. When plants were cultured in alkaline conditions for 15 d, the root growth of M. hupehensis with weak alkali tolerance decreased significantly. Analysis of endogenous hormone levels showed that the concentrations of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in M. hupehensis under alkali stress were lower than those in the control. However, the trend for IAA and ZR in M. prunifolia was the opposite. The concentration of abscisic acid (ABA) in the roots of the two apple rootstocks under alkali stress increased, but the concentration of ABA in the roots of M. prunifolia was higher than that in M. hupehensis. The expression of IAA-related genes ARF5, GH3.6, SAUR36, and SAUR32 and the Cytokinin (CTK)-related gene IPT5 in M. prunifolia was higher than those in the control, but the expression of these genes in M. hupehensis was lower than those in the control. The expression of ABA-related genes CIPK1 and AHK1 increased in the two apple rootstocks under alkali stress, but the expression of CIPK1 and AHK1 in M. prunifolia was higher than in M. hupehensis. These results demonstrated that under alkali stress, the increase of IAA, ZR, and ABA in roots and the increase of the expression of related genes promoted the growth of roots and improved the alkali tolerance of apple rootstocks.

Sugar metabolism and accumulation in the fruit of transgenic apple trees with decreased sorbitol synthesis

Both sorbitol and sucrose are synthesized in source leaves and transported to fruit for supporting fruit growth in tree fruit species of the Rosaceae family.In apple(Malus domestica),antisense suppression of aldose-6-phosphate reductase,the key enzyme for sorbitol synthesis,significantly decreased the sorbitol concentration but increased the sucrose concentration in leaves,leading to a lower sorbitol but a higher sucrose supply to fruit in these plants.In response to this altered carbon supply,the transgenic fruit had lower concentration of sorbitol and much higher concentration of glucose but similar levels of fructose,sucrose,and starch throughout fruit development relative to the untransformed control.Activities of sorbitol dehydrogenase,fructokinase,and sucrose phosphate synthase were lower,whereas activities of neutral invertase,sucrose synthase,and hexokinase were higher in the transgenic fruit during fruit development.Transcript levels of MdSOT1,MdSDHs,MdFK2,and MdSPS3/6 were downregulated,whereas transcript levels of MdSUC1/4,MdSUSY1-3,MdNIV1/3,MdHKs,and MdTMT1 were upregulated in the transgenic fruit.These findings suggest that the Sucrose cycle and the sugar transport system are very effective in maintaining the level of fructose and provide insights into the roles of sorbitol and sucrose in regulating sugar metabolism and accumulation in sorbitol-synthesizing species.

The apple DNA-binding one zinc-finger protein MdDof54 promotes drought resistance

DNA-binding one zinc-finger(Dof)proteins constitute a family of transcription factors with a highly conserved Dof domain that contains a C2C2 zinc-finger motif.Although several studies have demonstrated that Dof proteins are involved in multiple plant processes,including development and stress resistance,the functions of these proteins in drought stress resistance are largely unknown.Here,we report the identification of the MdDof54 gene from apple and document its positive roles in apple drought resistance.After long-term drought stress,compared with nontransgenic plants,MdDof54 RNAi plants had significantly shorter heights and weaker root systems;the transgenic plants also had lower shoot and root hydraulic conductivity,as well as lower photosynthesis rates.By contrast,compared with nontransgenic plants,MdDof54-overexpressing plants had higher photosynthesis rates and shoot hydraulic conductivity under long-term drought stress.Moreover,compared with nontransgenic plants,MdDof54-overexpressing plants had higher survival percentages under short-term drought stress,whereas MdDof54 RNAi plants had lower survival percentages.MdDof54 RNAi plants showed significant downregulation of 99 genes and significant upregulation of 992 genes in response to drought,and 366 of these genes were responsive to drought.We used DAPseq and ChIP-seq analyses to demonstrate that MdDof54 recognizes cis-elements that contain an AAAG motif.Taken together,our results provide new information on the functions of MdDof54 in plant drought stress resistance as well as resources for apple breeding aimed at the improvement of drought resistance.

Increased activity of MdFRK2, a high-affinity fructokinase, leads to upregulation of sorbitol metabolism and downregulation of sucrose metabolism in apple leaves

To investigate the functions of fructokinase(FRK)in apple(Malus domestica)carbohydrate metabolism,we cloned the coding sequences of MdFRK1 and MdFRK2 from the‘Royal Gala’apple.The results showed that MdFRK2 expression was extremely high in shoot tips and young fruit.Analyses of heterologously expressed proteins revealed that MdFRK2 had a higher affinity for fructose than did MdFRK1,with Km values of 0.1 and 0.62 mM for MdFRK2 and MdFRK1,respectively.The two proteins,however,exhibited similar Vmax values when their activities were significantly inhibited by high concentrations of fructose.MdFRK2 ectopic expression was associated with a general decrease in fructose concentration in transgenic lines.In leaves,increased FRK activity similarly resulted in reduced concentrations of glucose and sucrose but no alterations in sorbitol concentration.When compared with those in the untransformed control,genes involved in sorbitol synthesis(A6PR)and the degradation pathway(SDH1/2)were significantly upregulated in transgenic lines,whereas those involved in sucrose synthesis(SPS1)and other degradation processes(SUSY4,NINV1/2,and HxK2)were downregulated.The activity of enzymes participating in carbohydrate metabolism was proportional to the level of gene expression.However,the growth performance and photosynthetic efficiency did not differ between the transgenic and wild-type plants.These results provide new genetic evidence to support the view that FRK plays roles in regulating sugar and sorbitol metabolism in Rosaceae plants.

Linkage map and QTL mapping of red flesh locus in apple using a R1R1×R6R6 population

The red flesh in apple fruit is a desired trait by consumers and it is associated to the anthocyanin content,which is mainly controlled by MdMYB10 with a R6 promoter.In this study,a high-density linkage group was constructed using the‘Fuji’x‘Red3’population which contained homozygous alleles R1R1 and R6R6,respectively.The linkage group consists of 7630 SNPs along 17 linkage groups,spanning 2270.21 cM,with an average density of 0.30 cM permarker.The cyanidin-3-galactoside concentration was used as the phenotypic data in QTL analysis.Moreover,one QTL peak which was flaked by two markers,marker2187260 to marker2173766,with LOD scores of 4.49 was detected.This QTL ranged from 0 to 40.79 cM on the top of linkage group(LG16).In addition one candidate molecular marker(marker2175442)in this QTL was identified,which was significant correlated with the flesh cyanidin-3-galactoside concentration.These genetic findings enrich the breeding basis of fruit flesh coloration in apple.

Physiological evaluation of nitrogen use efficiency of different apple cultivars under various nitrogen and water supply conditions

Nitrogen(N) deficiency is a common problem for apple(Malus×domestica) production in arid regions of China.However,N utilization efficiency(NUE) of different apple cultivars grown under low N conditions in arid regions has not been evaluated.In this study,NUE was assessed for one-year-old seedlings of six apple cultivars,Golden Delicious,Qinguan,Jonagold,Honeycrisp,Fuji and Pink Lady,grafted onto Malus hupehensis Rehd.rootstocks.Four treatments were used,including control water with control N(CWCN),limited water with control N(LWCN),control water with low N(CWLN) and limited water with low N(LWLN).Our results showed that growth indices such as biomass,plant height and stem diameter,and photosynthetic rate of all cultivars decreased in the order CWCN>CWLN>LWCN>LWLN.When subjected to LWLN treatment,Qinguan showed better growth and photosynthetic characters than other tested cultivars.Additionally,Qinguan and Pink Lady had higher NUE,while Honeycrisp and Jonagold had lower NUE,based on the determination of biomass,photosynthetic parameters,chlorophyll content,the maximal photochemical efficiency of PSII(Fv/Fm),15 N and N contents.

Regulation of phenylpropanoid biosynthesis by MdMYB88 and MdMYB124 contributes to pathogen and drought resistance in apple

MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress.In this study,using a metabolomic approach,we identified differentially accumulated phenylpropanoid and flavonoid metabolites in MdMYB88/124 transgenic RNAi plants under control and long-term drought stress conditions in apple roots.We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions.Using Electrophoretic Mobility Shift Assay(EMSA)and ChIPquantitative PCR(qPCR)analyses,we found that MdMYB88 positively regulates the MdCM2 gene,which is responsible for phenylalanine biosynthesis,through binding to its promoter region.Under long-term drought conditions,MdMYB88/124 RNAi plants consistently accumulated increased amounts of H2O2 and MDA,while MdMYB88 and MdMYB124 overexpression plants accumulated decreased amounts of H2O2 and MDA.We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of MdMYB88 and MdMYB124 transgenic apple plants after long-term drought stress.We found that metabolites responsible for plant defense,including phenylpropanoids and flavonoids,accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions.We further demonstrated that MdMYB88/124 RNAi plants were more sensitive to Alternaria alternata f.sp.mali and Valsa mali,two pathogens that currently severely threaten apple production.In contrast,MdMYB88 and MdMYB124 overexpression plants were more tolerant to these pathogens.The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124,further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance,and provided information concerning molecular aspects of their roles in disease resistance.

Genome-wide identification,molecular evolution, and expression divergence of the hexokinase gene family in apple

Hexokinase(HXK)is the first irreversible catalytic enzyme in the glycolytic pathway,which not only provides energy for plant growth and development but also serves as a signaling molecule in response to environmental changes.However,the evolutionary pattern of the HXK gene family in apple remains unknown.In this study,a total of nine HXK genes were identified in the Malus×domestica genome GDDH13 v1.1.The physiological and biochemical properties,exonintron structures,conserved motifs,and cis-elements of the MdHXK genes were determined.Predicted subcellular localization indicated that the MdHXK genes were mainly distributed in the mitochondria,cytoplasm,and nucleus.Gene duplication revealed that whole-genome duplication(WGD)and segmental duplication played vital roles in MdHXK gene family expansion.Theωvalues of pairwise MdHXK genes indicated that this family was subjected to strong purifying selection during apple domestication.Additionally,five subfamilies were classified,and recent/old duplication events were identified based on phylogenetic tree analysis.Different evolutionary rates were estimated among the various HXK subfamilies.Moreover,divergent expression patterns of the MdHXK genes in four source-sink tissues and at five different apple fruit developmental stages indicated that they play vital roles in apple fruit development and sugar accumulation.Our study provides a theoretical basis for future elucidation of the biological functions of the MdHXK genes during apple fruit development.

Overexpression of MdMIPS1 enhances drought tolerance and water-use efficiency in apple

Myo-inositol and its derivatives play important roles in the tolerance of higher plants to abiotic stresses, and myo-inositol-1-phosphate synthase(MIPS) is the rate-limiting enzyme in myo-inositol biosynthesis. In this study, we found that increased myo-inositol biosynthesis enhanced drought tolerance in MdMIPS1-overexpressing apple lines under shortterm progressive drought stress. The effect of myo-inositol appeared to be mediated by the increased accumulation of osmoprotectants such as glucose, sucrose, and proline, and by the increased activities of antioxidant enzymes that eliminate reactive oxygen species. Moreover, enhanced water-use efficiency(WUE) was observed in MdMIPS1-overexpressing apple lines under long-term moderate water deficit conditions that mimicked the water availability in the soil of the Loess Plateau. Enhanced WUE may have been associated with the synergistic regulation of osmotic balance and stomatal aperture mediated by increased myo-inositol biosynthesis. Taken together, our findings shed light on the positive effects of MdMIPS1-mediated myo-inositol biosynthesis on drought tolerance and WUE in apple.

Response of carbohydrate metabolism-mediated sink strength to auxin in shoot tips of apple plants

Auxin(indole-3-acetic acid, IAA) has a considerable impact on the regulation of plant carbohydrate levels and growth, but the mechanism by which it regulates sugar levels in plants has received little attention. In this study, we found that exogenous IAA altered fructose(Fru), glucose(Glc), and sucrose(Suc) concentrations in shoot tips mainly by regulating MdSUSY1, MdFRK2, MdHxK1 and MdSDH2 transcript levels. Additionally, we used 5-year-old ’Royal Gala’ apple trees to further verify that these genes play primary roles in regulating sink strength. The results showed that MdSUSY1, MdFRK2, MdHxK1/3 and MdSDH2 might be major contributors to sink strength regulation. Taken together, these results provide new insight into the regulation of the carbohydrate metabolism mechanism, which will be helpful for regulating sink strength and yield.

Overexpression of MdATG8i improves water use efficiency in transgenic apple by modulating photosynthesis, osmotic balance, and autophagic activity under moderate water deficit

Water deficit is one of the major limiting factors for apple(Malus domestica)production on the Loess Plateau,a major apple cultivation area in China.The identification of genes related to the regulation of water use efficiency(WUE)is a crucial aspect of crop breeding programs.As a conserved degradation and recycling mechanism in eukaryotes,autophagy has been reported to participate in various stress responses.However,the relationship between autophagy and WUE regulation has not been explored.We have shown that a crucial autophagy protein in apple,MdATG8i,plays a role in improving salt tolerance.Here,we explored its biological function in response to long-term moderate drought stress.The results showed that MdATG8i-overexpressing(MdATG8i-OE)apple plants exhibited higher WUE than wild-type(WT)plants under long-term moderate drought conditions.Plant WUE can be increased by improving photosynthetic efficiency.Osmoregulation plays a critical role in plant stress resistance and adaptation.Under long-term drought conditions,the photosynthetic capacity and accumulation of sugar and amino acids were higher in MdATG8i-OE plants than in WT plants.The increased photosynthetic capacity in the OE plants could be attributed to their ability to maintain optimal stomatal aperture,organized chloroplasts,and strong antioxidant activity.MdATG8i overexpression also promoted autophagic activity,which was likely related to the changes described above.In summary,our results demonstrate that MdATG8i-OE apple lines exhibited higher WUE than WT under long-term moderate drought conditions because they maintained robust photosynthesis,effective osmotic adjustment processes,and strong autophagic activity.

Ectopic expression of AANAT or HIOMT improves melatonin production and enhances UV-B tolerance in transgenic apple plants

Melatonin is involved in plant responses to various environmental stresses.Although many studies have demonstrated that the tolerance of plants to stress is improved by exogenous melatonin,the role of endogenous melatonin metabolism in the response of apples to UV-B stress remains unclear.Here,the human melatonin biosynthesis-related enzyme genes AANAT or HIOMT were transformed into‘GL-3'apple,and the transgenic lines were treated with UV-B stress.The ectopic expression of AANAT or HIOMT significantly increased the melatonin content in apples.After UV-B stress,the tolerance of apple lines with ectopic expression of AANAT or HIOMT was markedly improved.The decrease in chlorophyll fluorescence,the generation of reactive oxygen species and the shrinkage of stomata caused by UV-B stress were alleviated by AANAT or HIOMT ectopic expression.In addition,the total phenolic content was markedly increased in the transgenic lines compared with the WT(wild type).The increase in phenolic compounds was related to the increase in benzoic acid,hydroxycinnamic acid,dihydrochalcones and flavanols,among which increases in chlorogenic acid,phloridzin and procyanidin B1 content were most prominent.Furthermore,the transgenic lines did not only promote the expression of genes related to phenolic synthesis under UV-B stress,but they also increased the accumulation of phenolic compounds by inhibiting the expression of MdPPO and MdPOD related to phenolic degradation.In summary,our results demonstrate that AANAT-or HIOMT-mediated melatonin synthesis improved the tolerance of apples to UV-B stress,mainly by scavenging reactive oxygen species,increasing photosynthetic capacity and increasing total phenolic content.