The compositions and contents of soluble sugars highly determine the flavor and quality of fleshy fruits.In the present study,we found that the overexpression of transcription factor MdWRKY126 localized on the nucleus...The compositions and contents of soluble sugars highly determine the flavor and quality of fleshy fruits.In the present study,we found that the overexpression of transcription factor MdWRKY126 localized on the nucleus enhanced sucrose concentration while decreased fructose and glucose concentration in transgenic apple calli and ripening tomato fruits.To comprehensively understand the effects of the MdWRKY126 on the content of various soluble sugars in apple and tomato fruits,enzyme activities and related essential genes associated with the sugar metabolism and transportation pathway in MdWRKY126-overrexpressed apple and tomato lines were analyzed.The results indicated that the overexpression of MdWRKY126 upregulated sucrose phosphate synthase(SPS)activity and the gene expression levels of SPS and sucrose transporter SUT,which was conducive to a large accumulation of sucrose in fruit cells.Meanwhile,MdWRKY126 overexpression downregulated the activity of enzymes involved in sucrose decomposition including cell wall invertase(CWINV),sucrose synthase(SUSY)and the corresponding gene expressions,as well as inhibited the expression levels of hexose transporter(HTs)and tonoplast sugar transporter(TSTs)that transport hexose into vacuoles,resulting in a reduced hexose level in apple calli and tomato fruit.These findings enrich our understanding of the metabolism and regulation of soluble sugars in apple fruits.展开更多
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/i...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.展开更多
Cadmium(Cd)stress is a serious threat to apple growth and development.Ethylene response factors(ERFs)are a major family of transcription factors(TFs)that play a key role in the resistance to Cd stress.In this study,we...Cadmium(Cd)stress is a serious threat to apple growth and development.Ethylene response factors(ERFs)are a major family of transcription factors(TFs)that play a key role in the resistance to Cd stress.In this study,we found that the ERF TF Md ERF114 was induced in response to Cd stress.The overexpression of Md ERF114 in apple(Malus domestica)roots reduced the accumulation of Cd in the plants and enhanced their tolerance to Cd stress.Yeast one-hybrid(Y1H)assays,dual-luciferase assays,and electrophoretic mobility shift assays indicated that MdERF114 directly binds to the promoter of MdATG16 and activates its expression to increase autophagic activity,which leads to higher resistance to Cd stress.In addition,MdMYB306 interacts with MdERF114 and enhances the resistance to Cd stress by promoting the binding of MdERF114 to the promoter of MdATG16.Our findings reveal an important mechanism by which MdMYB306-MdERF114-MdATG16 influences the resistance of apple to Cd stress.展开更多
The pivotal role of N^(6)-methyladenosine(m^(6)A)demethylases in regulating plant stress responses has been widely explored;however,the function of apple m^(6)A demethylases under heat stress and fixed-carbon starvati...The pivotal role of N^(6)-methyladenosine(m^(6)A)demethylases in regulating plant stress responses has been widely explored;however,the function of apple m^(6)A demethylases under heat stress and fixed-carbon starvation is unclear.In this study,the apple RNA demethylase gene family was identified,and the demethylase gene MdALKBH1A was selected for further analysis.Using liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis,we demonstrated that MdALKBH1A is the m^(6)A demethylase of apple.Moreover,transgenic‘Micro Tom’tomato plants overexpressing MdALKBH1A were more sensitive to high temperature,probably due to the decreased antioxidant ability,increased membrane lipid peroxidation and reduced plasma membrane stability.However,these tomato plants overexpressing MdALKBH1A were more resistant to fixed-carbon starvation,as evidenced by the improved plasma membrane stability,enhanced photosynthetic rates and elevated autophagic activity.In summary,our results highlight the crucial role played by Md ALKBH1A in the response of apple plants to high-temperature stress and fixedcarbon starvation.展开更多
UGT(UDP-dependent glycosyltransferase)family members are mainly involved in the modification of secondary metabolites,as well as plant stress responses.Previously,we identified the UGT gene MdUGT88F4 from Malus spp.In...UGT(UDP-dependent glycosyltransferase)family members are mainly involved in the modification of secondary metabolites,as well as plant stress responses.Previously,we identified the UGT gene MdUGT88F4 from Malus spp.In order to verify whether it had biological function in apple plants,we generated transgenic apple plants overexpressed MdUGT88F4 and treated them with salt stress.The results showed that it conferred enhanced salt stress tolerance in transgenic‘GL-3’apple(Malus domestica)plants.The expression of MdUGT88F4 was induced by salt stress,and overexpression of this gene alleviated the inhibitory effects of salt stress on the growth of apple plants.After 15 days of salt stress treatment(100 mmol·L^(-1)NaCl),necrotic spots were present on the leaves of wild-type(WT)plants,and none were observed on the leaves of transgenic plants overexpressing MdUGT88F4(OX).The relative electrolyte leakage and malondialdehyde content were lower and the total chlorophyll content and the relative water content were higher in OX plants than in WT plants.The photosynthetic efficiency was higher in OX plants than in WT plants under salt stress,which was consistent with their larger stomatal aperture;this might stem from a reduction in the content of abscisic acid.The production of reactive oxygen species was lower and the activities of antioxidant enzymes were higher in OX plants than in WT plants.In addition,OX plants accumulated less Na^(+)but more K^(+)than WT plants,and the expression of several genes involved in Na^(+)transport was upregulated in OX plants.MdUGT88F4 failed to promote the accumulation of isosalipurposide in vivo,and it was involved in isosalipurposide synthesis.Most of the flavonoid metabolites accumulated to a greater degree in OX plants than in WT plants.In summary,our results show that MdUGT88F4 positively regulates the salt stress response in apple plants,possibly by affecting stomatal movement,as well as the accumulation of ions and flavonoids.Our findings enhance our understanding of the metabolic mechanisms by which UGT proteins ameliorate the effects of salt stress in plants.展开更多
High temperature is an abiotic stress factor that threatens plant growth and development.Autophagy in response to heat stress involves the selective removal of heat-induced protein complexes.Previously,we showed that ...High temperature is an abiotic stress factor that threatens plant growth and development.Autophagy in response to heat stress involves the selective removal of heat-induced protein complexes.Previously,we showed that a crucial autophagy protein from apple,MdATG18a,has a positive effect on drought tolerance.In the present study,we treated transgenic apple(Malus domestica)plants overexpressing MdATG18a with high temperature and found that autophagy protected them from heat stress.Overexpression of MdATG18a in apple enhanced antioxidase activity and contributed to the production of increased beneficial antioxidants under heat stress.Transgenic apple plants exhibited higher photosynthetic capacity,as shown by the rate of CO_(2) assimilation,the maximum photochemical efficiency of photosystem II(PSII),the effective quantum yield,and the electron transport rates in photosystems I and II(PSI and PSII,respectively).We also detected elevated autophagic activity and reduced damage to chloroplasts in transgenic plants compared to WT plants.In addition,the transcriptional activities of several HSP genes were increased in transgenic apple plants.In summary,we propose that autophagy plays a critical role in basal thermotolerance in apple,primarily through a combination of enhanced antioxidant activity and reduced chloroplast damage.展开更多
Fruits from three cultivars of apple(Malus × domestica Borkh.)—‘McIntosh’,‘Gala’and‘Mutsu’—were harvested from the exterior and interior of the tree canopy.Peel and flesh tissues were sampled separately t...Fruits from three cultivars of apple(Malus × domestica Borkh.)—‘McIntosh’,‘Gala’and‘Mutsu’—were harvested from the exterior and interior of the tree canopy.Peel and flesh tissues were sampled separately to determine how the position of the fruit on the tree might affect the levels of the primary and secondary metabolites in the fruit.Fruit from the outer-canopy had a higher fresh weight and a higher soluble solids content compared with inner-canopy fruit.Both the flesh and peel of the outer-canopy fruit had higher concentrations of soluble sugars and sugar alcohols,but lower starch concentrations than the inner-canopy fruit.Canopy position did not significantly affect malic acid concentrations,except in the peel of‘McIntosh’and the flesh of‘Mutsu’.Although levels of ascorbic and succinic acids were higher in the peel of the outer-canopy fruit,the responses of other organic acids to canopy position depended on tissue type and cultivar.Except for histidine,lysine,threonine and glycine,most amino acids accumulated at higher concentrations in the inner-canopy fruit.By contrast,levels of phenolic compounds from both the peel and flesh were significantly higher in the outer-canopy fruit.The significant effects of location within the canopy on both primary metabolites and secondary metabolites demonstrate the importance of light exposure on apple fruit quality.展开更多
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 cons...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.展开更多
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 ...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.展开更多
Fruit with stripes,which are generally longitudinal,can occur naturally,but the bioprocesses underlying this phenomenon are unclear.Previously,we observed an atypical anthocyanin distribution that caused red-striped f...Fruit with stripes,which are generally longitudinal,can occur naturally,but the bioprocesses underlying this phenomenon are unclear.Previously,we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport“Red Zaosu”(Pyrus bretschneideri Rehd.).In this study,comparative transcriptome analysis of the sport and wild-type“Zaosu”revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin(GA)2-beta-dioxygenase 8,PbGA2ox8.Consistently,decreased methylation was also observed in the promoter region of PbGA2ox8 from“Red Zaosu”compared with“Zaosu”.Moreover,the GA levels in“Red Zaosu”seedlings were lower than those in“Zaosu”seedlings,and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in“Red Zaosu”.Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin.Moreover,the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches(VBs)in“Red Zaosu”.Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves.We propose a fruit-striping mechanism,in which the abnormal overexpression of PbGA2ox8 in“Red Zaosu”induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.展开更多
Flavonoids play important roles in regulating plant growth and development.In this study,three kaempferol 3-O-glycosides were identi fi ed and mainly accumulated in fl owers but not in leaves or fruits of Malus.In Mal...Flavonoids play important roles in regulating plant growth and development.In this study,three kaempferol 3-O-glycosides were identi fi ed and mainly accumulated in fl owers but not in leaves or fruits of Malus.In Malus,fl ower petal color is normally white,but some genotypes have red fl owers containing anthocyanin.Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes.The white fl ower Malus genotypes had better-developed seeds than the red fl ower genotypes.In fl owers,the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin,but decreased that of kaempferol 3-O-glycosides.After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds.Exogenous application ofdifferent fl avonoid compounds suggested that kaempferol 3-O-glycosides,especially kaempferol 3-O-rhamnoside,regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides.It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin,the Rho of plants(ROP)GTPases,calcium and the phosphoinositides signaling pathway.With the inhibition of auxin transport,the transcription levels of Heat Shock Proteins(HSPs)and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling,suggesting that HSPs and ROP GTPases were downstream of auxin signaling,but upstream of calcium signaling.In summary,kaempferol glycoside concentrations in pistils correlated with auxin transport,the transcription of HSPs and ROP GTPases,and calcium signaling in pollen tubes,culminating in changes to pollen tube growth and seed set.展开更多
Drought stress can negatively impact apple fruit quality and yield.Apple microRNAs(miRNAs)participate in apple tree and fruit development,as well as in biotic stress tolerance;however,it is largely unknown whether the...Drought stress can negatively impact apple fruit quality and yield.Apple microRNAs(miRNAs)participate in apple tree and fruit development,as well as in biotic stress tolerance;however,it is largely unknown whether these molecules are involved in the drought response.To identify drought-responsive miRNAs in Malus,we first examined the drought stress tolerance of ten F1 progenies of R3(M.×domestica)×M.sieversii.We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants.The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions.We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions.Under drought stress,61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants,respectively.We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis.We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes.Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes.Finally,we confirmed roles of two miRNAs in apple response to mannitol.Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species,thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress.展开更多
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...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.展开更多
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 drou...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.展开更多
Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acet...Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acetic acid,IAA),an endogenous hormone in plants,is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens.Gretchen Hagen3(GH3)is one of the early/primary auxin response genes.The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F.solani by treating MdGH3-2/12 RNAi plants with F.solani.The results show that under F.solani infection,RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage.After inoculation with F.solani,MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase.This led to the inhibition of free IAA combining with amino acids,resulting in excessive free IAA accumulation.This excessive free IAA altered plant tissue structure,accelerated fungal hyphal invasion,reduced the activity of antioxidant enzymes(SOD,POD and CAT),increased the reactive oxygen species(ROS)level,and reduced total chlorophyll content and photosynthetic ability,while regulating the expression of PR-related genes including PR1,PR4,PR5 and PR8.It also changed the contents of plant hormones and amino acids,and ultimately reduced the resistance to F.solani.In conclusion,these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F.solani and ARD.展开更多
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-...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.展开更多
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 prote...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.展开更多
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 expressi...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.展开更多
Phytohormones play crucial roles in fruit set regulation and development.Here,gibberellins(GA4+7),but not GA3,induced pear parthenocarpy.To systematically investigate the changes upon GA4+7 induced pear parthenocarpy,...Phytohormones play crucial roles in fruit set regulation and development.Here,gibberellins(GA4+7),but not GA3,induced pear parthenocarpy.To systematically investigate the changes upon GA4+7 induced pear parthenocarpy,dynamic changes in histology,hormone and transcript levels were observed and identified in unpollinated,pollinated and GA4+7-treated ovaries.Mesocarp cells continued developing in both GA4+7-treated and pollinated ovaries.In unpollinated ovaries,mesocarp cells stopped developing 14 days after anthesis.During fruit set process,GA4+7,but not GA1+3,increased after pollination.Abscisic acid(ABA)accumulation was significantly repressed by GA4+7 or pollination,but under unpollinated conditions,ABA was produced in large quantities.Moreover,indole-3-acetic acid biosynthesis was not induced by GA4+7 or pollination treatments.Details of this GA–auxin–ABA cross-linked gene network were determined by a comparative transcriptome analysis.The indole-3-acetic acid transport-related genes,mainly auxin efflux carrier component genes,were induced in both GA4+7-treated and pollinated ovaries.ABA biosynthetic genes of the 9-cis-epoxycarotenoid dioxygenase family were repressed by GA4+7 and pollination.Moreover,directly related genes in the downstream parthenocarpy network involved in cell division and expansion(upregulated),and MADS-box family genes(downregulated),were also identified.Thus,a model of GA-induced hormonal balance and its effects on parthenocarpy were established.展开更多
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 a...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.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.32172521)the Excellent Youth Science Foundation of Heilongjiang Province(Grant No.YQ2023C006)+1 种基金the China Postdoctoral Science Foundation(Grant No.2023MD744175)the Talent Introduction Programof Northeast Agricultural University of China,and the Collaborative Innovation System of the Agricultural Bioeconomy in Heilongjiang Province,China.
文摘The compositions and contents of soluble sugars highly determine the flavor and quality of fleshy fruits.In the present study,we found that the overexpression of transcription factor MdWRKY126 localized on the nucleus enhanced sucrose concentration while decreased fructose and glucose concentration in transgenic apple calli and ripening tomato fruits.To comprehensively understand the effects of the MdWRKY126 on the content of various soluble sugars in apple and tomato fruits,enzyme activities and related essential genes associated with the sugar metabolism and transportation pathway in MdWRKY126-overrexpressed apple and tomato lines were analyzed.The results indicated that the overexpression of MdWRKY126 upregulated sucrose phosphate synthase(SPS)activity and the gene expression levels of SPS and sucrose transporter SUT,which was conducive to a large accumulation of sucrose in fruit cells.Meanwhile,MdWRKY126 overexpression downregulated the activity of enzymes involved in sucrose decomposition including cell wall invertase(CWINV),sucrose synthase(SUSY)and the corresponding gene expressions,as well as inhibited the expression levels of hexose transporter(HTs)and tonoplast sugar transporter(TSTs)that transport hexose into vacuoles,resulting in a reduced hexose level in apple calli and tomato fruit.These findings enrich our understanding of the metabolism and regulation of soluble sugars in apple fruits.
基金supported by the National Key Research and Development Program of China(Grant No.2018YFD1000307)the National Natural Science Foundation of China(Grant No.32172529)+2 种基金the Special Funds for Major Scientific and Technological Innovation from Shaanxi Province(Grant No.2020zdzx03-0101)the Earmarked Fund for China Agriculture Research System(Grant No.CARS-27)China Postdoctoral Science Foundation(Grant Nos.2017M610657,2018T111108)。
文摘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.
基金supported by the earmarked fund for the China Agriculture Research System(CARS-27)the National Natural Science Foundation of China(31972389)the Key S&T Special Projects of Shaanxi Province,China(2020zdzx03-01-02)。
文摘Cadmium(Cd)stress is a serious threat to apple growth and development.Ethylene response factors(ERFs)are a major family of transcription factors(TFs)that play a key role in the resistance to Cd stress.In this study,we found that the ERF TF Md ERF114 was induced in response to Cd stress.The overexpression of Md ERF114 in apple(Malus domestica)roots reduced the accumulation of Cd in the plants and enhanced their tolerance to Cd stress.Yeast one-hybrid(Y1H)assays,dual-luciferase assays,and electrophoretic mobility shift assays indicated that MdERF114 directly binds to the promoter of MdATG16 and activates its expression to increase autophagic activity,which leads to higher resistance to Cd stress.In addition,MdMYB306 interacts with MdERF114 and enhances the resistance to Cd stress by promoting the binding of MdERF114 to the promoter of MdATG16.Our findings reveal an important mechanism by which MdMYB306-MdERF114-MdATG16 influences the resistance of apple to Cd stress.
基金supported by the earmarked fund for the China Agricultural Research System(CARS-27)。
文摘The pivotal role of N^(6)-methyladenosine(m^(6)A)demethylases in regulating plant stress responses has been widely explored;however,the function of apple m^(6)A demethylases under heat stress and fixed-carbon starvation is unclear.In this study,the apple RNA demethylase gene family was identified,and the demethylase gene MdALKBH1A was selected for further analysis.Using liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis,we demonstrated that MdALKBH1A is the m^(6)A demethylase of apple.Moreover,transgenic‘Micro Tom’tomato plants overexpressing MdALKBH1A were more sensitive to high temperature,probably due to the decreased antioxidant ability,increased membrane lipid peroxidation and reduced plasma membrane stability.However,these tomato plants overexpressing MdALKBH1A were more resistant to fixed-carbon starvation,as evidenced by the improved plasma membrane stability,enhanced photosynthetic rates and elevated autophagic activity.In summary,our results highlight the crucial role played by Md ALKBH1A in the response of apple plants to high-temperature stress and fixedcarbon starvation.
基金supported by the Natural Science Basic Research Program of Shaanxi(Grant No.2023-JC-YB-170)the earmarked fund for the China Agricultural Research System(Grant No.CARS-27)the Key S&T Special Projects of Shannxi Province(Grant No.2020zdzx03-01-02)。
文摘UGT(UDP-dependent glycosyltransferase)family members are mainly involved in the modification of secondary metabolites,as well as plant stress responses.Previously,we identified the UGT gene MdUGT88F4 from Malus spp.In order to verify whether it had biological function in apple plants,we generated transgenic apple plants overexpressed MdUGT88F4 and treated them with salt stress.The results showed that it conferred enhanced salt stress tolerance in transgenic‘GL-3’apple(Malus domestica)plants.The expression of MdUGT88F4 was induced by salt stress,and overexpression of this gene alleviated the inhibitory effects of salt stress on the growth of apple plants.After 15 days of salt stress treatment(100 mmol·L^(-1)NaCl),necrotic spots were present on the leaves of wild-type(WT)plants,and none were observed on the leaves of transgenic plants overexpressing MdUGT88F4(OX).The relative electrolyte leakage and malondialdehyde content were lower and the total chlorophyll content and the relative water content were higher in OX plants than in WT plants.The photosynthetic efficiency was higher in OX plants than in WT plants under salt stress,which was consistent with their larger stomatal aperture;this might stem from a reduction in the content of abscisic acid.The production of reactive oxygen species was lower and the activities of antioxidant enzymes were higher in OX plants than in WT plants.In addition,OX plants accumulated less Na^(+)but more K^(+)than WT plants,and the expression of several genes involved in Na^(+)transport was upregulated in OX plants.MdUGT88F4 failed to promote the accumulation of isosalipurposide in vivo,and it was involved in isosalipurposide synthesis.Most of the flavonoid metabolites accumulated to a greater degree in OX plants than in WT plants.In summary,our results show that MdUGT88F4 positively regulates the salt stress response in apple plants,possibly by affecting stomatal movement,as well as the accumulation of ions and flavonoids.Our findings enhance our understanding of the metabolic mechanisms by which UGT proteins ameliorate the effects of salt stress in plants.
基金supported by the National Key Research and Development Program of China(2018YFD1000300)the Earmarked Fund for China Agriculture Research System(CARS-27).
文摘High temperature is an abiotic stress factor that threatens plant growth and development.Autophagy in response to heat stress involves the selective removal of heat-induced protein complexes.Previously,we showed that a crucial autophagy protein from apple,MdATG18a,has a positive effect on drought tolerance.In the present study,we treated transgenic apple(Malus domestica)plants overexpressing MdATG18a with high temperature and found that autophagy protected them from heat stress.Overexpression of MdATG18a in apple enhanced antioxidase activity and contributed to the production of increased beneficial antioxidants under heat stress.Transgenic apple plants exhibited higher photosynthetic capacity,as shown by the rate of CO_(2) assimilation,the maximum photochemical efficiency of photosystem II(PSII),the effective quantum yield,and the electron transport rates in photosystems I and II(PSI and PSII,respectively).We also detected elevated autophagic activity and reduced damage to chloroplasts in transgenic plants compared to WT plants.In addition,the transcriptional activities of several HSP genes were increased in transgenic apple plants.In summary,we propose that autophagy plays a critical role in basal thermotolerance in apple,primarily through a combination of enhanced antioxidant activity and reduced chloroplast damage.
基金This work was supported by the Cornell Agricultural Experiment Station and New York Apple Research and Development Program。
文摘Fruits from three cultivars of apple(Malus × domestica Borkh.)—‘McIntosh’,‘Gala’and‘Mutsu’—were harvested from the exterior and interior of the tree canopy.Peel and flesh tissues were sampled separately to determine how the position of the fruit on the tree might affect the levels of the primary and secondary metabolites in the fruit.Fruit from the outer-canopy had a higher fresh weight and a higher soluble solids content compared with inner-canopy fruit.Both the flesh and peel of the outer-canopy fruit had higher concentrations of soluble sugars and sugar alcohols,but lower starch concentrations than the inner-canopy fruit.Canopy position did not significantly affect malic acid concentrations,except in the peel of‘McIntosh’and the flesh of‘Mutsu’.Although levels of ascorbic and succinic acids were higher in the peel of the outer-canopy fruit,the responses of other organic acids to canopy position depended on tissue type and cultivar.Except for histidine,lysine,threonine and glycine,most amino acids accumulated at higher concentrations in the inner-canopy fruit.By contrast,levels of phenolic compounds from both the peel and flesh were significantly higher in the outer-canopy fruit.The significant effects of location within the canopy on both primary metabolites and secondary metabolites demonstrate the importance of light exposure on apple fruit quality.
基金funded by the earmarked fund for the Natural Science Foundation of China(Grant No.31601715)the China Agriculture Research System(Grant No.CARS-27)+2 种基金the China Postdoctoral Science Foundation(Grant No.2016M602875)the Fundamental Research Funds for the Central Universities(Grant No.2452016025)the Start-up Funds of Northwest A&F University(Grant No.2452016142).
文摘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.
基金This work was supported by the State Key Program of the National Natural Science Foundation of China(31330068)the Young Scientists Fund of the National Natural Science Foundation of China(31601735)the earmarked fund for the China Agriculture Research System(CARS-27).
文摘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.
基金supported by the National Natural Science Foundation of China(31572086,31171925,and 31401845)the China Agriculture Research System(CARS 28-45).
文摘Fruit with stripes,which are generally longitudinal,can occur naturally,but the bioprocesses underlying this phenomenon are unclear.Previously,we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport“Red Zaosu”(Pyrus bretschneideri Rehd.).In this study,comparative transcriptome analysis of the sport and wild-type“Zaosu”revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin(GA)2-beta-dioxygenase 8,PbGA2ox8.Consistently,decreased methylation was also observed in the promoter region of PbGA2ox8 from“Red Zaosu”compared with“Zaosu”.Moreover,the GA levels in“Red Zaosu”seedlings were lower than those in“Zaosu”seedlings,and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in“Red Zaosu”.Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin.Moreover,the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches(VBs)in“Red Zaosu”.Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves.We propose a fruit-striping mechanism,in which the abnormal overexpression of PbGA2ox8 in“Red Zaosu”induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.
基金This work was funded by the National Key R&D Program(2018YFD1000200)the National Natural Science Foundation(31972366)of China。
文摘Flavonoids play important roles in regulating plant growth and development.In this study,three kaempferol 3-O-glycosides were identi fi ed and mainly accumulated in fl owers but not in leaves or fruits of Malus.In Malus,fl ower petal color is normally white,but some genotypes have red fl owers containing anthocyanin.Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes.The white fl ower Malus genotypes had better-developed seeds than the red fl ower genotypes.In fl owers,the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin,but decreased that of kaempferol 3-O-glycosides.After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds.Exogenous application ofdifferent fl avonoid compounds suggested that kaempferol 3-O-glycosides,especially kaempferol 3-O-rhamnoside,regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides.It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin,the Rho of plants(ROP)GTPases,calcium and the phosphoinositides signaling pathway.With the inhibition of auxin transport,the transcription levels of Heat Shock Proteins(HSPs)and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling,suggesting that HSPs and ROP GTPases were downstream of auxin signaling,but upstream of calcium signaling.In summary,kaempferol glycoside concentrations in pistils correlated with auxin transport,the transcription of HSPs and ROP GTPases,and calcium signaling in pollen tubes,culminating in changes to pollen tube growth and seed set.
基金supported by the National Natural Science Foundation of China(31622049)the Project of Shaanxi Provincial Youth Science and Technology Star Plan(2015kjxx-14)the Key Program of the National Natural Science Foundation of China(31330068).
文摘Drought stress can negatively impact apple fruit quality and yield.Apple microRNAs(miRNAs)participate in apple tree and fruit development,as well as in biotic stress tolerance;however,it is largely unknown whether these molecules are involved in the drought response.To identify drought-responsive miRNAs in Malus,we first examined the drought stress tolerance of ten F1 progenies of R3(M.×domestica)×M.sieversii.We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants.The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions.We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions.Under drought stress,61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants,respectively.We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis.We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes.Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes.Finally,we confirmed roles of two miRNAs in apple response to mannitol.Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species,thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress.
基金financially supported grants from the National Natural Science Foundation of China(31972391,32172529)the China Agriculture Research System of MOF and MARA(CARS-27)the Special Funds for Major Scientific and Technological Innovation from Shaanxi Province(2020zdzx03-01-01).
文摘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.
基金supported by grants from the Natural Science Foundation of Hebei Province(Grant No.C2022204086)the Hebei Apple Innovation Team of Modern Agricultural Industry Technology System(Grant No.HBCT2021100211)the National Natural Science Foundation of China(Grant No.32072524).
文摘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.
基金supported by the Earmarked Fund for the China Agriculture Research System(CARS-27)the Key Science and Technology Special Projects of Shaanxi Province,China(2020zdzx03-01-02).
文摘Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acetic acid,IAA),an endogenous hormone in plants,is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens.Gretchen Hagen3(GH3)is one of the early/primary auxin response genes.The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F.solani by treating MdGH3-2/12 RNAi plants with F.solani.The results show that under F.solani infection,RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage.After inoculation with F.solani,MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase.This led to the inhibition of free IAA combining with amino acids,resulting in excessive free IAA accumulation.This excessive free IAA altered plant tissue structure,accelerated fungal hyphal invasion,reduced the activity of antioxidant enzymes(SOD,POD and CAT),increased the reactive oxygen species(ROS)level,and reduced total chlorophyll content and photosynthetic ability,while regulating the expression of PR-related genes including PR1,PR4,PR5 and PR8.It also changed the contents of plant hormones and amino acids,and ultimately reduced the resistance to F.solani.In conclusion,these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F.solani and ARD.
基金This work was supported in part by the National Natural Science Foundation of China(No.31372038 to M.L.)Cornell Agricultural Experiment Station.
文摘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.
基金supported by the National Natural Science Foundation of China(31572106 and 31622049).
文摘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.
基金This work was supported by the Program for the National Natural Science Foundation of China(No.31372038)by the earmarked fund for the China Agriculture Research System(CARS-28).
文摘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.
基金This work was supported by the China Agriculture Research System(CARS-29-40)Weinan Experimental Station foundation of Northwest A&F University.
文摘Phytohormones play crucial roles in fruit set regulation and development.Here,gibberellins(GA4+7),but not GA3,induced pear parthenocarpy.To systematically investigate the changes upon GA4+7 induced pear parthenocarpy,dynamic changes in histology,hormone and transcript levels were observed and identified in unpollinated,pollinated and GA4+7-treated ovaries.Mesocarp cells continued developing in both GA4+7-treated and pollinated ovaries.In unpollinated ovaries,mesocarp cells stopped developing 14 days after anthesis.During fruit set process,GA4+7,but not GA1+3,increased after pollination.Abscisic acid(ABA)accumulation was significantly repressed by GA4+7 or pollination,but under unpollinated conditions,ABA was produced in large quantities.Moreover,indole-3-acetic acid biosynthesis was not induced by GA4+7 or pollination treatments.Details of this GA–auxin–ABA cross-linked gene network were determined by a comparative transcriptome analysis.The indole-3-acetic acid transport-related genes,mainly auxin efflux carrier component genes,were induced in both GA4+7-treated and pollinated ovaries.ABA biosynthetic genes of the 9-cis-epoxycarotenoid dioxygenase family were repressed by GA4+7 and pollination.Moreover,directly related genes in the downstream parthenocarpy network involved in cell division and expansion(upregulated),and MADS-box family genes(downregulated),were also identified.Thus,a model of GA-induced hormonal balance and its effects on parthenocarpy were established.
基金supported by the National Key Research and Development Program of China(2018YFD1000100)the National Natural Science Foundation of China(31622049,31872080,and 31660565).
文摘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.
