Comparative transcriptome analysis reveals key genes and pathways in response to Alternaria alternata apple pathotype infection

Apple leaf spot,caused by the Alternaria alternata apple pathotype(AAAP),is an important fungal disease of apple.To understand the molecular basis of resistance and pathogenesis in apple leaf spot,the transcriptomes of two apple cultivars‘Hanfu'(HF)(resistant)and‘Golden Delicious'(GD)(susceptible)were analyzed at 0,6,18,24 and 48 h after AAAP inoculation by RNA-Seq.At each time point,a large number of significantly differentially expressed genes(DEGs)were screened between AAAP-inoculated and uninoculated apple leaves.Analysis of the common DEGs at four time points revealed significant differences in the resistance of‘HF'and‘GD'apple to AAAP infection.RLP,RNL,and JA signal-related genes were upregulated in both cultivars to restrict AAAP development.However,genes encoding CNLs,TNLs,WRKYs,and AP2s were only activated in‘HF'as part of the resistance response,of which,some play major roles in the regulation of ET and SA signal transduction.Further analysis showed that many DEGs with opposite expression trends in the two hosts may play important regulatory roles in response to AAAP infection.Transient expression of one such gene MdERF110 in‘GD'apple leaves improved AAAP resistance.Collectively,this study highlights the reasons for differential resistance to AAAP infection between‘HF'and‘GD'apples which can theoretically assist the molecular breeding of disease-resistant apple crops.

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.

The role of the auxin-response genes MdGH3.1 and MdSAUR36 in bitter pit formation in apple

Apples often exhibit bitter pits in response to metabolic disorders during ripening and storage;however, the mechanisms underlying the bitter pit(BP) development remain unclear. Here, metabolome and transcriptome analyses were performed to investigate BP pulp of 'Fuji'. Two auxin-response genes, MdGH3.1 and MdSAUR36, were screened. Their expression as well as the auxin content in BP pulp were found to be higher than those in healthy pulp(P < 0.01). In the field, excess CO(NH2)2increased the incidence of BP. Moreover, the auxin content and MdGH3.1 expression increased in apples after nitrogen fertilization. On Day 30 before harvest, the two genes were transiently transferred to the fruit, and 20.69% and 23.21% of BP fruits were harvested. After 10 μmol·L-1auxin was infiltrated at low pressure into postharvest fruit, the increase in MdGH3.1 expression occurred earlier than that in MdSAUR36. MdGH3.1 increased the expression of MdSAUR36, but MdSAUR36 did not increase expression of MdGH3.1. Therefore, we suggest that MdGH3.1 acts upstream of MdSAUR36 during BP formation and that these genes induce BP formation by regulating auxin and phenylpropanoid biosynthesis.

Transcriptional regulation of MdPIN7 by MdARF19 during gravityinduced formation of adventitious root GSA in self-rooted apple stock

Self-rooted apple stock is widely used for apple production.However,the shallowness of the adventitious roots in self-rooted apple stock leads to poor performance in the barren orchards of China.This is because of the considerable difference in the development of a gravitropic set-point angle(GSA)between self-rooted apple stock and seedling rootstock.Therefore,it is crucial to study the molecular mechanism of adventitious root GSA in self-rooted apple stock for breeding self-rooted and deep-rooted apple rootstock cultivars.An apple auxin response factor MdARF19 functioned to establish the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.MdARF19 bound directly to the MdPIN7 promoter,activating its transcriptional expression and thus regulating the formation of the adventitious root GSA in 12-2 self-rooted apple stock.However,MdARF19 influenced the expression of auxin efflux carriers(MdPIN3 and MdPIN10)and the establishment of adventitious root GSA of self-rooted apple stock in response to gravity signals by direct activation of MdFLP.Our findings provide new information on the transcriptional regulation of MdPIN7 by auxin response factor MdARF19 in the regulation of the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.

Silencing of early auxin responsive genes MdGH3-2/12 reduces the resistance to Fusarium solani in apple

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.

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.

Isolation of Trichoderma virens 6PS-2 and its effects on Fusarium proliferatum f. sp. Malus domestica MR5 related to apple replant disease in China

Apple replant disease(ARD) is primarily caused by biotic factors that seriously inhibits the development of apple industry. Therefore, the use of biological control measures to inhibit the main pathogens(such as Fusarium spp.) that cause ARD is of great significance to the sustainable development of the apple industry. Trichoderma virens 6PS-2, which exhibited antagonism toward a variety of pathogens, was screened from the rhizosphere soils of healthy apple trees(Malus robusta) in different replanted orchards in the Yantai and Zibo Cities, Shandong Province, China. Its fermentation extract inhibited the growth of pathogenic Fusarium proliferatum f. sp. Malus domestica MR5, which was proportional to the concentration. These substances also increased the hairy root volume and growth of Arabidopsis thaliana lateral roots. The phenotype of Malus hupehensis seedlings and microbial community structure in rhizosphere soils in greenhouse experiment using Highthroughput sequencing were analyzed, and the field experiment with grafted apple trees were used for further verification. Compared with the application of potato dextrose broth(PDB) medium, application of 6PS-2 spore suspension directly to replanted soils could improve the growth of M. hupehensis seedlings as well as the elongation of grafted apple trees. Concomitant decreases in the gene copy number of Fusarium and increases in the culturable bacteria/fungi were also observed in the greenhouse and field experiments. The abundance of Trichoderma,Bacillus, and Streptomyces increased significantly, but that of Fusarium, Pseudarthrobacter, and Humicola decreased. The content of esters, phenols,furans, and amino acids in root exudates of M. hupehensis seedlings increased, which significantly inhibited the multiplication of Fusarium, but was positively correlated with Bacillus and Trichoderma. In summary, T. virens 6PS-2 not only directly inhibits the activity of pathogenic Fusarium but also secrets secondary metabolites with antifungal and growth-promoting potential. In addition, 6PS-2 spore suspension can also promote the growth of plants to a certain extent, and change the soil microbial community structure of rhizosphere soils. It is believed that T. virens 6PS-2 has the potential for the alleviation of apple replant disease(ARD) in China.

Biochar alleviates apple replant disease by reducing the growth of Fusarium oxysporum and regulating microbial communities

Apple replant disease(ARD)negatively affects plant growth and reduces yields in replanted orchards.In this study,biochar was applied to apple replant soil with Fusarium oxysporum.Our aim was to investigate whether biochar could promote plant growth and alleviate apple replant disease by reducing the growth of harmful soil microorganisms,changing soil microbial community structure and improving the soil environment.This experiment included five treatments:apple replant soil(CK),methyl bromide fumigation apple replant soil(FM),replant soil with biochar addition(2%),replant soil with F.oxysporum spore solution(8×10^(7)spores·mL^(-1)),and replant soil with biochar and F.oxysporum spore solution addition.Seedling biomass,the activity of antioxidant enzymes in the leaves and roots,and soil environmental variables were measured.Microbial community composition and community structure were analyzed using 16SrDNA and ITS2 gene sequencing.Biochar significantly reduced the abundance of F.oxysporum and increased soil microbial diversity and richness.Biochar also increased the soil enzyme activities(urease,invertase,neutral phosphatase,and catalase),the biomass(plant height,fresh weight,dry weight)and the activity of antioxidant enzymes(superoxide dismutase,peroxidase,and catalase).The root indexes of apple seedlings was also increased in replant soil by biochar.In sum,biochar promoted the growth of plants,improved the replant soil environment,and alleviated apple replant disease.

Potassium alleviated high nitrogen-induced apple growth inhibition by regulating photosynthetic nitrogen allocation and enhancing nitrogen utilization capacity

There is a close relationship between potassium(K)and nitrogen(N).However,the roles of K under high N conditions remain unclear.Using a hydroponics approach,we monitored the morphological,physiological,and molecular changes in M9T337 apple(Malus domestica)rootstocks under different nitrate(10 and 30 mmol·L^(-1)NO_(3)^(-))and K supply(0.5,6,10,and 20 mmol·L_(-1)K^(+))conditions.Results revealed that high nitrate inhibited the root growth of M9T337 rootstocks,downregulated the expressions of K transporter genes(MdPT5,MdHKT1,and MdATK1),and reduced the net NO3-and K+influx at the surface of roots,thereby resulting in an N/K imbalance in rootstocks.Further investigation showed that 10 mmol·L^(-1)K increased the activity of N metabolic enzymes(NR,GS,NiR,and GOGAT),upregulated the expressions of genes related to nitrate uptake and transport(MdNRT1.1,MdNRT1.2,MdNRT1.5,and MdNRT2.4),promoted15N transport from the roots to the shoots,optimized leaf N distribution,and improved photosynthetic N utilization efficiency under high nitrate conditions.These results suggest that the negative effects of high nitrate may be related to the N/K imbalance and that reducing N/K in plants by increasing K supply level can effectively alleviate the inhibition of N assimilation by high nitrate stress.

Improved multi-scale inverse bottleneck residual network based on triplet parallel attention for apple leaf disease identification

Accurate diagnosis of apple leaf diseases is crucial for improving the quality of apple production and promoting the development of the apple industry. However, apple leaf diseases do not differ significantly from image texture and structural information. The difficulties in disease feature extraction in complex backgrounds slow the related research progress. To address the problems, this paper proposes an improved multi-scale inverse bottleneck residual network model based on a triplet parallel attention mechanism, which is built upon ResNet-50, while improving and combining the inception module and ResNext inverse bottleneck blocks, to recognize seven types of apple leaf(including six diseases of alternaria leaf spot, brown spot, grey spot, mosaic, rust, scab, and one healthy). First, the 3×3 convolutions in some of the residual modules are replaced by multi-scale residual convolutions, the convolution kernels of different sizes contained in each branch of the multi-scale convolution are applied to extract feature maps of different sizes, and the outputs of these branches are multi-scale fused by summing to enrich the output features of the images. Second, the global layer-wise dynamic coordinated inverse bottleneck structure is used to reduce the network feature loss. The inverse bottleneck structure makes the image information less lossy when transforming from different dimensional feature spaces. The fusion of multi-scale and layer-wise dynamic coordinated inverse bottlenecks makes the model effectively balances computational efficiency and feature representation capability, and more robust with a combination of horizontal and vertical features in the fine identification of apple leaf diseases. Finally, after each improved module, a triplet parallel attention module is integrated with cross-dimensional interactions among channels through rotations and residual transformations, which improves the parallel search efficiency of important features and the recognition rate of the network with relatively small computational costs while the dimensional dependencies are improved. To verify the validity of the model in this paper, we uniformly enhance apple leaf disease images screened from the public data sets of Plant Village, Baidu Flying Paddle, and the Internet. The final processed image count is 14,000. The ablation study, pre-processing comparison, and method comparison are conducted on the processed datasets. The experimental results demonstrate that the proposed method reaches 98.73% accuracy on the adopted datasets, which is 1.82% higher than the classical ResNet-50 model, and 0.29% better than the apple leaf disease datasets before preprocessing. It also achieves competitive results in apple leaf disease identification compared to some state-ofthe-art methods.

ALL ABOUT APPLES!

Crunch!Apples are a sweet and delicious fruit.There are over 7,500 different kinds!The fruit is usually red,but it can be green or other colors.Applesaren't expensive.They make a great snack.You can eat a whole apple.You can slice it,too.They're also good with other foods.Do you like apples?

Extracellular and intracellular infection of Botryosphaeria dothidea and resistance mechanism in apple cells

Apple ring rot,which is caused by Botryosphaeria dothidea,severely affects apple production.The mechanisms employed in apple cells against B.dothidea remain unknown.In this research,the pathogen infection mode and the relationship between cell death and disease resistance in‘Fuji’/B.dothidea interaction pathosystem were investigated.By using transmission electron microscopy(TEM),our research showed that the pathogen infects apple cells both intracellularly and extracellularly.However,compared with that in immature fruit,the incidence of hyphae in the interior of mature apple fruit cells increased dramatically,suggesting that cell wall-mediated penetration resistance could be important in apple resistance against B.dothidea.TEM ultrastructural characterization identified the nuclear morphology of programmed cell death induction in both apple fruit and callus cells under B.dothidea infection.Overexpression of MdVDAC2(MDP0000271281),which encodes an outer-membrane localized anion channel protein in mitochondria,significantly promoted cell death under B.dothidea infection and simultaneously inhibited pathogen infection,suggesting that cell death represents a disease resistance mechanism in apple against B.dothidea infection.Furthermore,BdCatalase(KAF4307763),a cytochromeP450 family protein BdCYP52A4(KAF4300696),and subtilisin-domain containing proteinswere identified fromB.dothidea-secreted proteins,which suggested the potential involvement of active oxygen species and phytoalexins in combating B.dothidea infection and triggering or dampening apple resistance.Collectively,our research suggested that cell wall-mediated penetration resistance,programmed cell death machinery and microbial effector-interrelated signaling were among strategies recruited in apple to combat B.dothidea.The current research laid the foundation for further investigations into resistance mechanisms in apple.

Isolation of phloridzin-degrading,IAA-producing bacterium Ochrobactrum haematophilum and its effects on the apple replant soil environment

We isolated and identified a bacterium that could produce IAA and degrade phloridzin in the rhizosphere soil of healthy replanted apple(the rootstock is M9T337 and the scion is Yanfu 3),providing a theoretical basis for reducing the obstacles associated with apple replant disease(ARD).Isolates were screened using Salkowski colorimetry and screening medium for phloridzin.The isolate of interest(W6)was identified as Ochrobactrum haematophilum based on morphological analysis,physiological and biochemical tests,and 16S rDNA sequencing.In a laboratory experiment,W6 produced auxin and promoted the growth of Arabidopsis thaliana roots,and its degradation rate of 100 mg.L^(-1 )phloridzin was 62.0%.In a pot experiment,W6 significantly reduced the phenolic acid contents of replanted soil,lowered the abundance of the harmful fungus Fusarium solani,and increased soil enzyme activities,thereby improving the micro-ecological environment of replant soil.W6 increased the root antioxidant enzyme activity and leaf photosynthetic pigment content of replanted Malus hupehensis Rehd.seedlings,effectively alleviating the decrease in net photosynthetic rate,transpiration rate and stomatal conductance caused by ARD.In a field experiment,W6 also promoted the growth of replanted apple(the rootstock is M9T337 and the scion is Yanfu 3)saplings.Therefore,W6 can promote apple growth and degrade phenolic acids,and it can be used as an effective treatment for the reduction of ARD.

HATCH评分、APPLE评分在心房颤动患者射频消融术后复发的预测价值研究

目的探讨HATCH评分、APPLE评分在心房颤动(房颤)患者射频消融术后复发的预测价值。方法选取2018年5月至2020年6月于秦皇岛市第一医院心血管内科行射频消融术治疗的105例房颤患者的临床资料进行回顾性分析,观察手术时间、X线曝光时间、消融成功率及复发率;且根据术后3个月后是否出现持续时间≥30 s的快速性房性心律失常分为复发组(n=36)和未复发组(n=69),分析影响复发的危险因素,评估HATCH评分、APPLE评分及两项联合评估房颤患者术后复发的AUC值、敏感度、特异度。结果105例房颤患者均顺利完成射频消融术,消融成功率为65.71%,复发率为34.29%。复发组与未复发组在性别、年龄、病程、合并高血压、糖尿病、心力衰竭、估算的肾小球滤过率(eGFR)、持续性房颤、左心室大小、左室射血分数(LVEF)、右室前后径、升主动脉内径、肺动脉主干内径、室内隔厚度、左室后壁厚度、主动脉瓣口血流速度、肌酐等资料比较中,差异均无统计学意义(P>0.05);而在左心房大小、E峰、HATCH评分、APPLE评分等比较中,差异具有统计学意义(P<0.05)。二元Logistic回归分析显示,左心房大小、E峰、HATCH评分、APPLE评分是影响手术治疗后复发的独立危险因素(P<0.001)。ROC曲线分析显示,HATCH评分、APPLE评分及两项联合评估房颤患者术后复发的AUC值分别为(0.747、0.807、0.927,P<0.05);敏感度分别为44.40%、69.40%、94.40;特异度分别为91.30%、85.50%、84.10%。结论HATCH评分、APPLE评分预测房颤患者射频消融术后复发具有一定价值,且两项联合有利于提高敏感度,能为识别术后复发提供指导。

Effects of water stress on quality and sugar metabolism in'Gala'apple fruit

Sugar plays an important role in apple fruit development,appearance and quality as well as contributing to a plant’s water stress response.Trehalose and the trehalose biosynthetic metabolic pathways are part of the sugar signaling system in plants,which are important regulator of water stress response in apple.The effect of water stress treatments applied to apple trees and the corresponding effects of ABA on developmental fruit quality were examined for indicators of fruit quality during fruit development.The results indicated that the severe water stress treatment(W2)occurring after the last stage of fruit cell division caused a decrease in the color and size of fruit.The moderate water stress(W1)occurring after the last stage of fruit cell enlargement(S2)caused an increase in the content of fructose and sorbitol while the apple fruit shape was not affected.These changes in sugar are related to the activity of sugar metabolic enzymes.While the enzymatic activity of vacuolar acid invertase(vAINV)was higher,that of sucrose-phosphate synthase(SPS)was lower in water stress treated fruit throughout the developmental period.This indicates that enhanced sucrose degradation and reduced sucrose synthesis leads to an overall reduced sucrose content during times of drought.Thus,water stress reduced sucrose content.Whereas the content of endogenous trehalose and ABA were the highest in water stress treated fruit.A moderate water stress(W1)imposed on apple trees via water restriction(60%–65%of field capacity)after the fruit cell enlargement phase of fruit development yielded sweeter fruit of higher economic value.

Comparison of transcriptome and metabolome analysis revealed differences in cold resistant metabolic pathways in different apple cultivars under low temperature stress

Freezing injury in winter is an important abiotic stress that seriously affects plant growth and development.Deciduous fruit trees resist freezing injury by inducing dormancy.However,different cultivars of the same species have different cold resistance strategies.Little is known about the molecular mechanism of apple trees in response to freezing injury during winter dormancy.Therefore,in this study,1-year-old branches of the cold-resistant cultivar‘Hanfu’(HF)and the cold-sensitive cultivar‘Changfuji No.2’(CF)were used to explore their cold resistance through physiological,biochemical,transcriptomics,and metabolomics analyses.Combining physiological and biochemical data,we found that HF had a stronger osmotic regulation ability and antioxidant enzyme activity than CF,as well as stronger cold resistance.The functional enrichment analysis showed that both cultivars were significantly enriched in pathways related to signal transduction,hormone regulation,and sugar metabolism under freezing stress.In addition,the differentially expressed genes(DEGs)encoding galactinol synthase,raffinose synthase,and stachyose synthetase in raffinose family oligosaccharides(RFOs)metabolic pathways were upregulated in HF,and raffinose and stachyose were accumulated,while their contents in CF were lower.HF accumulated 4-aminobutyric acid,spermidine,and ascorbic acid to scavenge reactive oxygen species(ROS).While the contents of oxidized glutathione,vitamin C,glutathione,and spermidine in CF decreased under freezing stress,consequently,the ability to scavenge ROS was low.Furthermore,the transcription factors apetala 2/ethylene responsive factor(AP2/ERF)and WRKY were strongly induced under freezing stress.In summary,the difference in key metabolic components of HF and CF under freezing stress is the major factor affecting their difference in cold resistance.The obtained results deepen our understanding of the cold resistance mechanism in apple trees in response to freezing injury during dormancy.

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.

Farmers’precision pesticide technology adoption and its influencing factors:Evidence from apple production areas in China

The research aimed to understand farmers’willingness to adopt(WTA)and willingness to pay(WTP)for precision pesticide technologies and analyzed the determinants of farmers’decision-making.We used a two-stage approach to consider farmers’WTA and WTP for precision pesticide technologies.A survey of 545 apple farmers was administered in Bohai Bay and the Loess Plateau in China.The data were analyzed using the double-hurdle model.The results indicated that 78.72%of respondents were willing to apply precision pesticide technologies provided by service organizations such as cooperatives and dedicated enterprises,and 69.72%were willing to buy the equipment for using precision pesticide technologies.The results of the determinant analysis indicated that farmers’perceived perceptions,farm scale,cooperative membership,access to digital information,and availability of financial services had significant and positive impacts on farmers’WTA precision pesticide technologies.Cooperative membership,technical training,and adherence to environmental regulations increased farmers’WTP for precision pesticide technologies.Moreover,nonlinear relationships between age,agricultural experience,and farmers’WTA and WTP for precision pesticide technology services were found.

Functional analysis of MdSUT2.1,a plasma membrane sucrose transporter from apple

Sugar content is a determinant of apple(Malus×domestica Borkh.)sweetness.However,the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive.Herein,this study reported the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apples.The MdSUT2.1 gene encoded a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts.MdSUT2.1 was highly expressed in fruit and was positively correlated with sucrose accumulation during apple fruit development.Moreover,complementary growth assays in a yeast mutant validated the sucrose transport activity of MdSUT2.1.MdSUT2.1 overexpression in apples and tomatoes resulted in significant increases in sucrose,fructose,and glucose contents compared to the wild type(WT).Further analysis revealed that the expression levels of sugar metabolism-and transport-related genes SUSYs,NINVs,FRKs,HXKs,and TSTs increased in apples and tomatoes with MdSUT2.1 overexpression compared to WT.Finally,unlike the tonoplast sugar transporters MdTST1 and MdTST2,the promoter of MdSUT2.1 was not induced by exogenous sugars.These findings provide valuable insights into the molecular mechanism underlying sugar accumulation in apples.

Comparison of cell wall changes of two different types of apple cultivars during fruit development and ripening

Fruit development and ripening is a complex procedure(Malus×domestica Borkh.)and can be caused by various factors such as cell structure,cell wall components,and cell wall hydrolytic enzymes.In our study,we focused on the variations in fruit firmness,cell wall morphology and components,the activity of cell wall hydrolytic enzymes and the expression patterns of associated genes during fruit development in two different types of apple cultivars,the hard-crisp cultivar and the loose-crisp cultivar.In this paper,the aim was to find out the causes of the texture variations between the different type cultivars.Cell wall materials(CWMs),hemicellulose and cellulose content were strongly associated with variations in fruit firmness during the fruit development.The content of water soluble pectin(WSP)and chelator soluble pectin(CSP)gradually increased,while the content of ionic soluble pectin(ISP)showed inconsistent trends in the four cultivars.The activities of polygalacturonase(PG),β-galactosidase(β-gal),cellulase(CEL),and pectate lyase(PL)gradually increased in four cultivars.And the activities of PG,β-gal,and CEL were higher in‘Fuji’and‘Honeycrisp’fruit with the fruit development,while the activity of PL of‘Fuji’and‘Honeycrisp’was lower than that of‘ENVY’and‘Modi’.Both four cultivars of fruit cells progressively became bigger as the fruit expanded,with looser cell arrangements and larger cell gaps.According to the qRT-PCR,the relative expression levels of MdACO and Mdβ-gal were notably enhanced.Our study showed that there were large differences in the content of ISP and hemicellulose,the activity of PL and the relative expression of Mdβ-gal between two different types of apple cultivars,and these differences might be responsible for the variations in the texture of the four cultivars.