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Glomerella leaf spot (GLS), a fungal disease caused by Colletotrichum fructicola, severely affects apple (Malus domestica) quality and yield. In this study, we found that the transcription factor MdWRKY71 was significantly induced by C. fructicola infection in the GLS‐susceptible apple cultivar Royal Gala. The overexpression of MdWRKY71 in apple leaves resulted in increased susceptibility to C. fructicola, whereas RNA interference of MdWRKY71 in leaves showed the opposite phenotypes. These findings suggest that MdWRKY71 functions as a susceptibility factor for the apple—C. fructicola interaction. Furthermore, MdWRKY71 directly bound to the promoter of the salicylic acid (SA) degradation gene Downy Mildew Resistant 6 (DMR6)‐Like Oxygenase 1 (DLO1) and promoted its expression, resulting in a reduced SA level. The sensitivity of 35S:MdWRKY71 leaves to C. fructicola can be effectively alleviated by knocking down MdDLO1 expression, confirming the critical role of MdWRKY71‐mediated SA degradation via regulating MdDLO1 expression in GLS susceptibility. In summary, we identified a GLS susceptibility factor, MdWRKY71, that targets the apple SA degradation pathway to promote fungal infection. [ABSTRACT FROM AUTHOR]

Valsa mali causes Valsa canker, one of the most destructive diseases on apple trees, leading to severe losses for the apple industry in China. Considering the development of fungicide resistance and the harmful effects of chemical residues, it is urgent to identify alternatives to control this disease. Trans‐cinnamic acid (t‐CA), a compound with good antibacterial, antitumour and anti‐inflammatory properties, is widely used in food, medicine and other industries. However, the antifungal activity of t‐CA against V. mali and its regulatory role in apple defence against biotic stresses are unclear. Based on a metabolomic assay, we found that Malus yunnaensis (Valsa canker‐resistant) twigs infected with V. mali dramatically accumulated t‐CA. Exogenous application of t‐CA effectively inhibited V. mali growth on potato dextrose agar. The EC50 value of t‐CA inhibiting mycelial growth was 200 μg/mL. Malus prunifolia (Valsa canker‐susceptible) leaves and twigs pretreated with t‐CA had significantly enhanced V. mali resistance. The t‐CA application increased salicylic acid but reduced jasmonic acid levels in leaves and twigs. Moreover, the contents of phenolic acids and flavonoids increased in t‐CA‐treated samples. In addition, t‐CA increased the activities of phenylalanine ammonia‐lyase, β‐1,3‐glucanase and chitinase. These results indicate that t‐CA plays a significant role in inhibiting V. mali growth and priming apple defence. [ABSTRACT FROM AUTHOR]

Summary: Understanding the molecular regulation of plant cold response is the basis for cold resistance germplasm improvement. Here, we revealed that the apple histone deacetylase MdHDA6 can perform histone deacetylation on cold‐negative regulator genes and repress their expression, leading to the positive regulation of cold tolerance in apples. Moreover, MdHDA6 directly interacts with the transcription factor MdTCP15. Phenotypic analysis of MdTCP15 transgenic apple lines and wild types reveals that MdTCP15 negatively regulates cold tolerance in apples. Furthermore, we found that MdHDA6 can facilitate histone deacetylation of MdTCP15 and repress the expression of MdTCP15, which positively contributes to cold tolerance in apples. Additionally, the transcription factor MdTCP15 can directly bind to the promoter of the cold‐negative regulator gene MdABI1 and activate its expression, and it can also directly bind to the promoter of the cold‐positive regulator gene MdCOR47 and repress its expression. However, the co‐expression of MdHDA6 and MdTCP15 can inhibit MdTCP15‐induced activation of MdABI1 and repression of MdCOR47, suggesting that MdHDA6 suppresses the transcriptional regulation of MdTCP15 on its downstream genes. Our results demonstrate that histone deacetylase MdHDA6 plays an antagonistic role in the regulation of MdTCP15‐induced transcriptional activation or repression to positively regulate cold tolerance in apples, revealing a new regulatory mechanism of plant cold response. [ABSTRACT FROM AUTHOR]

Hail, known as an agricultural meteorological disaster, can substantially constrain the growth of the apple industry. Presently, apple orchards use a variety of colored (photo-selective) hail nets as a preventative measure. However, it is unclear which color proves most effective for apple orchards. This study provides a systematic investigation of the impact of four photo-selective colored hail nets (white, blue, black, and green; with white being the control) on the microenvironment of apple orchards, fruit tree development, fruit quality, and yield over a two-year period (2020–2021). Different photo-selective nets do not evidently alter the intensity of light, although the nets' shading effects decrease in the order from black to green to blue. Among them, blue nets increased the proportion of blue light, while green nets enhanced the proportion of green light. On the other hand, black, green, and blue nets diminished the proportion of red and far-red light. Such photo-selective nets effectively lowered soil temperature but did not have an impact on relative humidity and air temperature. Encasing apple trees with blue nets promoted growth, increasing shoot length, thickness, leaf area, and water content, while simultaneously decreasing leaf thickness. Black nets had comparable effects, although the impacts of green nets were inconsistent. Different photo-selective nets did not significantly influence the leaf shape index or overall chlorophyll content. However, black and green nets reduced the chlorophyll a/b ratio, while blue nets slightly boosted this ratio. Additionally, blue nets proved beneficial for apple trees' photosynthesis. With the employment of a principal component analysis and comprehensive evaluation, this study concludes that blue nets offer the most favorable environmental conditions for apple growth while protecting apple orchards against hail, compared to black, white, and green nets. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a conserved degradation pathway in eukaryotes that is required for recycling unwanted intracellular components, maintaining homeostasis, and coping with biotic and abiotic stresses. Pathogens have evolved to subvert autophagic machinery by secreting host cell-entering effector proteins. Here, we provided evidence that an apple autophagy-related gene MdATG8i, activated autophagy and contributed to resistance against Valsa canker caused by Valsa Mali (Vm) when being overexpressed in apple. MdATG8i interacted with a plastid elongation factor Tu (MdEF-Tu) which became insoluble and aggregated during Vm infection and was degraded through the autophagy pathway. Intriguingly, we identified a highly-induced effector secreted from Vm, Vm1G-1794, which competitively interacted with MdATG8i, suppressed autophagy, and depleted MdEF-Tu out of MdATG8i complexes. The formation of stable MdEF-Tu aggregates caused by Vm1G-1794 promoted the susceptibility of apple to Vm. Overall, our study demonstrated that MdATG8i contributed to Vm resistance by targeting and degrading MdEF-Tu, and Vm1G-1794 competed with MdEF-Tu to target MdATG8i and prevent MdEF-Tu degradation, thus favoring infection. Abbreviations: 35S: cauliflower mosaic virus 35S promoter; AIM: ATG8-interacting motif; ATG8–PE: ATG8 conjugated with phosphatidylethanolamine; BiFC: biomolecular fluorescence complementation; Con A: concanamycin A; Co-IP: co-immunoprecipitation; DEPs: differentially expressed proteins; DMSO: dimethyl sulfoxide; GFP: green fluorescent protein; hpt: hours post-treatment; LCI: luciferase complementation imaging; MdATG8i: autophagy-related protein 8i in Malus domestica; MDC: monodansylcadaverine; MdEF-Tu: elongation factor Tu in Malus domestica; MdNBR1: neighbor of BRCA1 in Malus domestica; N. benthamiana: Nicotiana benthamiana; OE: overexpression; PAMP: pathogen-associated molecular pattern; PTI: pattern-triggered immunity; qRT-PCR: quantitative reverse transcription PCR; RFP: red fluorescent protein; RNAi: RNA interference; ROS: reactive oxygen species; Ub: ubiquitin; V. Mali: Valsa Mali; WT: wild-type plant; YFP: yellow fluorescent protein [ABSTRACT FROM AUTHOR]

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ‐aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one‐hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, β‐glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA. [ABSTRACT FROM AUTHOR]

Melatonin enhances plant tolerance to various environmental stressors. Although exogenous application of melatonin has been investigated, the role of endogenous melatonin metabolism in the response of apples to drought stress and nutrient utilization remains unclear. Here, we investigated the effects of ectopically expressing the human melatonin synthase gene HIOMT on transgenic apple plants under drought stress conditions. The tolerance of transgenic apple lines that ectopically expressed HIOMT improved significantly under drought conditions. After 10 days of natural drought stress treatment, the transgenic apple plants showed higher relative water content, chlorophyll levels and Fv/Fm , and lower relative electrolyte leakage and hydrogen peroxide accumulation, than wild-type plants. The activities of peroxidase, superoxide dismutase and catalase, as well as genes in the ascorbate–glutathione cycle, increased more in transgenic apple plants than in the wild-type. The ectopic expression of HIOMT also markedly alleviated the inhibitory effects of long-term drought stress on plant growth, photosynthetic rate and chlorophyll concentrations in apple plants. The uptake and utilization of 15N increased markedly in the transgenic lines under long-term moderate drought stress. Drought stress sharply reduced the activity of enzymes involved in nitrogen metabolism, but ectopic expression of HIOMT largely reversed that response. The expression levels of genes of nitrogen metabolism and uptake were more upregulated in transgenic apple plants than the wild-type. Overall, our study demonstrates that ectopic expression of HIOMT enhanced the tolerance of apple plants to drought stress, and transgenic apple plants showed improved growth due to higher nutrient utilization efficiency under drought conditions. [ABSTRACT FROM AUTHOR]

Summary: Drought limits apple yield and fruit quality. However, the molecular mechanism of apple in response to drought is not well known. Here, we report a Cys2/His2 (C2H2)‐type zinc‐finger protein, MdZAT5, that positively regulates apple drought tolerance by regulating drought‐responsive RNAs and microRNAs (miRNAs).DNA affinity purification and sequencing and yeast‐one hybrid analysis identified the binding motifs of MdZAT5, T/ACACT/AC/A/G. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP‐qPCR) and electrophoretic mobility shift assays (EMSAs) showed that MdZAT5 directly binds to the promoters of the drought‐responsive genes including MdRHA2a, MdLEA14, MdTPX1, and MdCAT3, and activates their expression under drought stress.MdZAT5 interacts with and directly targets HYPONASTIC LEAVES1 (MdHYL1). MdZAT5 may facilitate the interaction of MdHYL1 with pri‐miRNAs or MdDCL1 by activating MdHYL1 expression, thereby regulating the biogenesis of drought‐responsive miRNAs. Genetic dissection showed that MdHYL1 is essential for MdZAT5‐mediated drought tolerance and miRNA biogenesis. In addition, ChIP‐qPCR and EMSA revealed that MdZAT5 binds directly to the promoters of some MIR genes including Mdm‐miR171i and Mdm‐miR172c, and modulates their transcription.Taken together, our findings improve our understanding of the molecular mechanisms of drought response in apple and provide a candidate gene for the breeding of drought‐tolerant cultivars. [ABSTRACT FROM AUTHOR]

As a precursor of aromatic compounds, fatty acids play important roles in apple fruit quality; however, the genetic and molecular basis underlying fatty acid synthesis and metabolism is largely unknown. In this study, we conducted a genome-wide association study (GWAS) of seven fatty acids using genomic data of 149 Malus accessions and identified 232 significant signals (–log10 P >5) associated with 99 genes from GWAS of four fatty acids across 2 years. Among these, a significant GWAS signal associated with linoleic acid was identified in the transcriptional regulator SUPERMAN-like (SUP) MD13G1209600 at chromosome 13 of M. × domestica. Transient overexpression of MdSUP increased the contents of linoleic and linolenic acids and of three aromatic components in the fruit. Our study provides genetic and molecular information for improving the flavor and nutritional value of apple. [ABSTRACT FROM AUTHOR]

Used Random forest algorithm to construct a prediction model of aroma components based on the hybrid offspring of 'Honeycrisp' × 'Maodi', and different preprocessing methods were tried (Standardization (SS), First-order Derivative (D1) and Standard normal variate (SNV)). The aroma composition and content were determined by gas chromatography-mass spectrometry (GC-MS), and the main aroma components of apples were classified according to compound categories, including ester, aldehyde, ketone, alcohol. Taking the chemical groups as the research objects, the characteristic wavelengths were selected by grid search algorithm, and the characteristic wavelength-aroma chemical group model was established, and the same method was used to construct the model for single aroma components. The results show: SNV has the best noise removal effect among the five preprocessing methods. Under the SNV treatment, aroma chemical groups of apples showed a good correlation with the spectrum. The number of characteristic spectra of ester are 413, 493, 512, 551, 592, 600, 721, 727, 729, 733 nm, all in the visible light range. The determination coefficient ( R 2 ), the root mean square error (RMSE) and the ratio of the standard deviation values (RPD) of validation were 0.90, 4936.16 and 1.13. The characteristic spectrum of alcohols is 519, 562, 570, 571, 660, 676, 737, 738 nm, the range is close to that of ester. The R 2 and RMSE of alcohol validation are 0.92 and 83.21, and RPD is 1.30. The number of characteristic spectra of aldehyde is 20, and the most important band is 1000 nm, which is outside the visible light range. The number of characteristic spectra of ketone is 15, and also has some distribution outside the visible light range. The R 2 of aldehyde and ketone validation are 0.84 and 0.86. Except for cyclooctanol, the R 2 of single aroma compound prediction model performed poorly. Based on the models, we tried to visualize alcohol, which can roughly represent their distribution on apple. Their distributions all show significant differences in the center and edge of apple, but the results are still rough due to the accuracy of models. In conclusion, the study can preliminarily prove that hyperspectral imaging technology (HSI) can perform non-destructive detection of aroma in apple hybrid offspring. [ABSTRACT FROM AUTHOR]

Drought stress is an important environmental factor limiting apple yield and fruit quality. Previously, we identified GRETCHEN HAGEN3.6 (GH3.6) as a negative regulator of drought stress in apple trees. Using transgenic MdGH3 RNAi (knocking down MdGH3.6 and its five homologs) plants as rootstock can increase drought tolerance, water use efficiency, flowering, and fruit quality of the Fuji scion after drought stress. However, the molecular mechanism behind this phenomenon is still unknown. Here, we performed transcriptome sequencing of the grafted plants (Fuji/GL-3 where Fuji was used as the scion and non-transgenic GL-3 was used as the rootstock, and Fuji/MdGH3 RNAi where MdGH3 RNAi was used as the rootstock) under control and drought conditions. Under control conditions, 667 up-regulated genes and 176 down-regulated genes were identified in the scion of Fuji/MdGH3 RNAi, as compared to the scion of Fuji/GL-3. Moreover, 941 up-regulated genes and 2226 down-regulated genes were identified in the rootstock of MdGH3 RNAi plants relative to GL-3. GO terms of these differentially expressed genes (DEGs) in scion and rootstock showed associations with plant growth, fruit development, and stress responses. After drought stress, 220 up-regulated and 452 down-regulated genes were identified in MdGH3 RNAi rootstock, as compared to GL-3. Significantly enriched GO terms included response to abiotic stimulus, cell division, microtubule-based process, metabolic and biosynthetic process of flavonoid, pigment, and lignin. The comparison between the scion of Fuji/MdGH3 RNAi and Fuji/GL-3 yielded a smaller number of DEGs; however, all of them were significantly enriched in stress-related GO terms. Furthermore, 365 and 300 mRNAs could potentially move from MdGH3 RNAi rootstock to scion under control and drought conditions, respectively, including FIDDLEHEAD (FDH), RESPONSIVE TO DESICCATION 26 (RD26), ARS-binding factor 2 (ABF2), WRKY75, and ferritin (FER). Overall, our work demonstrates the effects of rootstock on scion at the transcriptional level after drought stress and provides theoretical support for further understanding and utilization of MdGH3 RNAi plants. [ABSTRACT FROM AUTHOR]

Apples are a rich source of polyphenols in the human diet. However, the distribution of polyphenols in different apple varieties and tissues is still largely unclear. In this study, a new liquid chromatography–tandem mass spectrometry (LC-MS/MS) strategy was developed to reveal the spatial distribution of polyphenols in different apple tissues and varieties. A method based on multiple reaction monitoring (MRM)-enhanced product ion (EPI) was established in the information-dependent acquisition (IDA) mode for pseudo-target screening of major apple polyphenols. A total of 39 apple polyphenolic metabolites were finally identified. Qualitative and quantitative results showed that the variety and content of polyphenols in apple peels were higher than those of other tissues. In apple roots, stems, and leaves, the highest polyphenol variety and content were found in wild species, followed by cultivars and elite varieties. Dihydrochalcone substances, one kind of major apple polyphenols, were more abundant in apple roots, stems, and leaves. This strategy can be applied as a model for other agricultural products, in addition to revealing the distribution of polyphenols in different tissues of apples, which provides a theoretical basis for the utilization of polyphenol resources and variety selection. [ABSTRACT FROM AUTHOR]

The photoprotective role of anthocyanin remains controversial. In this study, we explored the effects of anthocyanin on photosynthesis and photoprotection using transgenic 'Galaxy Gala' apple plants overexpressing MdMYB10 under high light stress. The overexpression of MdMYB10 dramatically enhanced leaf anthocyanin accumulation, allowing more visible light to be absorbed, particularly in the green region. However, through post-transcriptional regulation, anthocyanin accumulation lowered leaf photosynthesis in both photochemical reaction and CO2 fixation capacities. Anthocyanin accumulation also led to a decreased de-epoxidation state of the xanthophyll cycle and antioxidant capacities, but this is most likely a response to the light-shielding effect of anthocyanin, as indicated by a higher chlorophyll concentration and lower chlorophyll a/b ratio. Under laboratory conditions when detached leaves lost carbon fixation capacity due to the limitation of CO2 supply, the photoinhibition of detached transgenic red leaves was less severe under strong white, green, or blue light, but it became more severe in response to strong red light compared with that of the wild type. In field conditions when photosynthesis was performed normally in both green and transgenic red leaves, the degree of photoinhibition was comparable between transgenic red leaves and wild type leaves, but it was less severe in transgenic young shoot bark compared with the wild type. Taken together, these data show that anthocyanin protects plants from high light stress by absorbing excessive visible light despite reducing photosynthesis. [ABSTRACT FROM AUTHOR]

Drought resistance in plants is influenced by multiple signaling pathways that involve various transcription factors, many target genes, and multiple types of epigenetic modifications. Studies on epigenetic modifications of drought focus on DNA methylation and histone modifications, with fewer on chromatin remodeling. Changes in chromatin accessibility can play an important role in abiotic stress in plants by affecting RNA polymerase binding and various regulatory factors. However, the changes in chromatin accessibility during drought in apples are not well understood. In this study, the landscape of chromatin accessibility associated with the gene expression of apple (GL3) under drought conditions was analyzed by Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) and RNA-seq. Differential analysis between drought treatment and control identified 23,466 peaks of upregulated chromatin accessibility and 2447 peaks of downregulated accessibility. The drought-induced chromatin accessibility changed genes were mainly enriched in metabolism, stimulus, and binding pathways. By combining results from differential analysis of RNA-seq and ATAC-seq, we identified 240 genes with higher chromatin accessibility and increased gene expression under drought conditions that may play important functions in the drought response process. Among them, a total of nine transcription factor genes were identified, including ATHB7, HAT5, and WRKY26. These transcription factor genes are differentially expressed with different chromatin accessibility motif binding loci that may participate in apple response to drought by regulating downstream genes. Our study provides a reference for chromatin accessibility under drought stress in apples and the results will facilitate subsequent studies on chromatin remodelers and transcription factors. [ABSTRACT FROM AUTHOR]

The WRKY transcription factors are unique regulatory proteins in plants, which are important in the stress responses of plants. In this study, 113 WRKY genes were identified from the apple genome GDDH13 and a comprehensive analysis was performed, including chromosome mapping, and phylogenetic, motif and collinearity analysis. MdWRKYs are expressed in different tissues, such as seeds, flowers, stems and leaves. We analyzed seven WRKY proteins in different groups and found that all of them were localized in the nucleus. Among the 113 MdWRKYs, MdWRKY70L was induced by both drought and salt stresses. Overexpression of it in transgenic tobacco plants conferred enhanced stress tolerance to drought and salt. The malondialdehyde content and relative electrolyte leakage values were lower, while the chlorophyll content was higher in transgenic plants than in the wild-type under stressed conditions. In conclusion, this study identified the WRKY members in the apple genome GDDH13, and revealed the function of MdWRKY70L in the response to drought and salt stresses. [ABSTRACT FROM AUTHOR]

Summary: Although the N6‐methyladenosine (m6A) modification is the most prevalent RNA modification in eukaryotes, the global m6A modification landscape and its molecular regulatory mechanism in response to drought stress remain unclear.Transcriptome‐wide m6A methylome profiling revealed that m6A is mainly enriched in the coding sequence and 3′ untranslated region in response to drought stress in apple, by recognizing the plant‐specific sequence motif UGUAH (H=A, U or C). We identified a catalytically active component of the m6A methyltransferase complex, MdMTA. An in vitro methyl transfer assay, dot blot, LC‐MS/MS and m6A‐sequencing (m6A‐seq) suggested that MdMTA is an m6A writer and essential for m6A mRNA modification.Further studies revealed that MdMTA is required for apple drought tolerance. m6A‐seq and RNA‐seq analyses under drought conditions showed that MdMTA mediates m6A modification and transcripts of mRNAs involved in oxidative stress and lignin deposition. Moreover, m6A modification promotes mRNA stability and the translation efficiency of these genes in response to drought stress. Consistently, MdMTA enhances lignin deposition and scavenging of reactive oxygen species under drought conditions.Our results reveal the global involvement of m6A modification in the drought response of perennial apple trees and illustrate its molecular mechanisms, thereby providing candidate genes for the breeding of stress‐tolerant apple cultivars. [ABSTRACT FROM AUTHOR]

Ammonium is one of the prevalent nitrogen sources for growth and development of higher plants. Ammonium acquisition from soil is facilitated by ammonium transporters (AMTs), which are plasma membrane proteins that exclusively transport ammonium/ammonia. However, the functional characteristics and molecular mechanisms of AMTs in apple remain unclear. In this work, 15 putative AMT genes were identified and classified into four clusters (AMT1–AMT4) in apple. According to expression analysis, these AMTs had varying expressions in roots, leaves, stems, flowers and fruits. Some of them were strongly affected by diurnal cycles. AMT genes showed multiple transcript patterns to N regimes and were quite responsive to osmotic stress. In addition, phosphorylation analysis revealed that there were some conserved phosphorylation residues within the C-terminal of AMT proteins. Furthermore, detailed research was conducted on AMT1;2 functioning by heterologous expression in yeast. The present study is expected to provide basic bioinformatic information and expression profiles for the apple AMT family and to lay a basis for exploring the functional roles and regulation mechanisms of AMTs in apple. [ABSTRACT FROM AUTHOR]

Summary: SUMOylation is involved in various aspects of plant biology, including drought stress. However, the relationship between SUMOylation and drought stress tolerance is complex; whether SUMOylation has a crosstalk with ubiquitination in response to drought stress remains largely unclear. In this study, we found that both increased and decreased SUMOylation led to increased survival of apple (Malus × domestica) under drought stress: both transgenic MdSUMO2A overexpressing (OE) plants and MdSUMO2 RNAi plants exhibited enhanced drought tolerance. We further confirmed that MdDREB2A is one of the MdSUMO2 targets. Both transgenic MdDREB2A OE and MdDREB2AK192R OE plants (which lacked the key site of SUMOylation by MdSUMO2A) were more drought tolerant than wild‐type plants. However, MdDREB2AK192R OE plants had a much higher survival rate than MdDREB2A OE plants. We further showed SUMOylated MdDREB2A was conjugated with ubiquitin by MdRNF4 under drought stress, thereby triggering its protein degradation. In addition, MdRNF4 RNAi plants were more tolerant to drought stress. These results revealed the molecular mechanisms that underlie the relationship of SUMOylation with drought tolerance and provided evidence for the tight control of MdDREB2A accumulation under drought stress mediated by SUMOylation and ubiquitination. [ABSTRACT FROM AUTHOR]

Summary: N6‐methyladenosine (m6A) reader protein plays an important role in trichome morphology, developmental timing and morphogenesis in Arabidopsis. However, the function of m6A readers in plant‐microbe interaction remains unclear. Here, a Malus YTH‐domain family protein MhYTP2 was initially characterized as an m6A reader. MhYTP2 overexpression increased mRNA m6A modification level and translation efficiency. The m6A in the exon regions appeared to destabilize the mRNAs, whereas m6A in the untranslated regions positively correlated with the associated mRNA abundance. MhYTP2 overexpression enhanced apple powdery mildew resistance, possibly by rapidly degrading the bound mRNAs of MdMLO19 and MdMLO19‐X1 and improving the translation efficiency of the antioxidant genes. To conclude, the results shed light on the apple m6A profile, the effect of MhYTP2 on m6A profile, and the m6A roles in MdMLO19 and MdMLO19‐X1 mRNAs stability and glutamate dehydrogenase 1‐like MdGDH1L mRNA translation efficiency. [ABSTRACT FROM AUTHOR]

The high accumulation of phloridzin makes apple (Malus domestica) unique in the plant kingdom, which suggests a vital role of its biosynthesis in physiological processes. In our previous study, silencing MdUGT88F1 (a key UDP-GLUCOSE: PHLORETIN 2'-O-GLUCOSYLTRANSFERASE gene) revealed the importance of phloridzin biosynthesis in apple development and Valsa canker resistance. Here, results from MdUGT88F1 -silenced lines showed that phloridzin biosynthesis was indispensable for normal chloroplast development and photosynthetic carbon fixation by maintaining MdGLK1/2 (GOLDEN2-like1/2) expression. Interestingly, increased phloridzin biosynthesis did not affect plant (or chloroplast) development, but reduced nitrogen accumulation, leading to chlorophyll deficiency, light sensitivity, and sugar accumulation in MdUGT88F1- overexpressing apple lines. Further analysis revealed that MdUGT88F1-mediated phloridzin biosynthesis negatively regulated the cytosolic glutamine synthetase1-asparagine synthetase-asparaginase (GS1-AS-ASPG) pathway of ammonium assimilation and limited chlorophyll synthesis in apple shoots. The interference of phloridzin biosynthesis in the GS1-AS-ASPG pathway was also assumed to be associated with its limitation of the carbon skeleton of ammonium assimilation through metabolic competition with the tricarboxylic acid cycle. Taken together, our findings shed light on the role of MdUGT88F1-mediated phloridzin biosynthesis in the coordination between carbon and nitrogen accumulation in apple trees. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9 -cis -epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees. [ABSTRACT FROM AUTHOR]

Summary: Sugar transporters are necessary to transfer hexose from cell wall spaces into parenchyma cells to boost hexose accumulation to high concentrations in fruit. Here, we have identified an apple hexose transporter (HTs), MdHT2.2, located in the plasma membrane, which is highly expressed in mature fruit. In a yeast system, the MdHT2.2 protein exhibited high 14C‐fructose and 14C‐glucose transport activity. In transgenic tomato heterologously expressing MdHT2.2, the levels of both fructose and glucose increased significantly in mature fruit, with sugar being unloaded via the apoplastic pathway, but the level of sucrose decreased significantly. Analysis of enzyme activity and the expression of genes related to sugar metabolism and transport revealed greatly up‐regulated expression of SlLIN5, a key gene encoding cell wall invertase (CWINV), as well as increased CWINV activity in tomatoes transformed with MdHT2.2. Moreover, the levels of fructose, glucose and sucrose recovered nearly to those of the wild type in the sllin5‐edited mutant of the MdHT2.2‐expressing lines. However, the overexpression of MdHT2.2 decreased hexose levels and increased sucrose levels in mature leaves and young fruit, suggesting that the response pathway for the apoplastic hexose signal differs among tomato tissues. The present study identifies a new HTs in apple that is able to take up fructose and glucose into cells and confirms that the apoplastic hexose levels regulated by HT controls CWINV activity to alter carbohydrate partitioning and sugar content. [ABSTRACT FROM AUTHOR]

Drought and nitrogen (N) deficiency are common factors that limit apple production in the Loess Plateau region of China. Different apple cultivars respond to drought and low N differently; however, the mechanism that underlies the difference in nitrogen-use efficiency (NUE) under drought conditions is not well understood. In this study, by comparing the physiological responses of two apple (Malus domestica Borkh.) cultivars with contrasting NUE, 'Qinguan' (higher NUE) and 'Honeycrisp' (lower NUE), under low N and drought conditions, we discovered that, 'Qinguan' had larger stomatal apertures, higher chlorophyll fluorescence levels, more active N metabolism and antioxidant enzymes, higher abscisic acid and auxin concentrations, larger root size and more efficient N uptake mediated by higher expression of MdNRT2.4 in rootstock than that of 'Honeycrisp'. Additionally, we experimentally confirmed that MdNRT2.4 enhanced low N and osmotic stress tolerance in Arabidopsis when being overexpressed. Taken together, our findings shed light on the mechanism that underlies the difference in NUE of apple under drought and N-deficient conditionss and provide MdNRT2.4 as a candidate gene for future genetic engineering. [ABSTRACT FROM AUTHOR]

Melatonin has been reported to play roles in regulating carbohydrate levels and plant growth. However, little is known about the exact mechanism by which melatonin regulates sugar levels and growth in plants. In this study, it was found that high levels of melatonin inhibited the growth of wild-type (WT) apple plants and induced significant accumulations of fructose, glucose, and sucrose in apple leaves, while MdFRK2 expression was significantly downregulated. MdFRK2 promoter transiently expressed in tobacco leaves further supported that the expression of MdFRK2 could be inhibited by exogenous melatonin. After applying exogenous melatonin, the suppression of MdFRK2 expression was significantly rescued in transgenic apples overexpressing MdFRK2 via the 35S promoter. Fructose, glucose, and sucrose concentrations increased less as compared to WT apple plants. Wild-type plants showed a stunted phenotype 21 days after melatonin treatment, while MdFRK2 -overexpressing plants exhibited slightly inhibited growth, indicating that the downregulated MdFRK2 expression in response to melatonin was involved in melatonin-mediated growth inhibition. Taken together, these results demonstrate the involvement of MdFRK2 in melatonin-induced sugar accumulation and growth inhibition. Our findings shed light on the roles played by MdFRK2 in connecting melatonin action and plant growth. [ABSTRACT FROM AUTHOR]

Summary: Cytosine methylation is an essential feature of epigenetic regulation and is involved in various biological processes. Although cytosine methylation has been analysed at the genomic scale for several plant species, there is a general lack of understanding of the dynamics of global and genic DNA methylation in plants growing in environments challenged with biotic and abiotic stresses. In this study, we mapped cytosine methylation at single‐base resolution in the genome of commercial apple (Malus x domestica), and analysed changes in methylation patterns associated with water deficit in representative drought‐sensitive and drought‐tolerant cultivars. We found that the apple genome exhibits ~54%, ~38% and ~8.5% methylation at CG, CHG and CHH sequence contexts, respectively. We additionally documented changes in gene expression associated with water deficit in an attempt to link methylation and gene expression changes. Global methylation and transcription analysis revealed that promoter‐unmethylated genes showed higher expression levels than promoter‐methylated genes. Gene body methylation appears to be positively correlated with gene expression. Water deficit stress was associated with changes in methylation at a multitude of genes, including those encoding transcription factors (TFs) and transposable elements (TEs). These results present a methylome map of the apple genome and reveal widespread DNA methylation alterations in response to water deficit stress. These data will be helpful for understanding potential linkages between DNA methylation and gene expression in plants growing in natural environments and challenged with abiotic and biotic stresses. [ABSTRACT FROM AUTHOR]

Summary: Autophagy is a major and conserved pathway for delivering and recycling unwanted proteins or damaged organelles to be degraded in the vacuoles. AuTophaGy‐related (ATG) protein 18a has been established as one of the essential components for autophagy occurrence in Arabidopsis thaliana. We previously cloned the ATG18a homolog from Malus domestica (MdATG18a) and monitored its responsiveness to various abiotic stresses at the transcriptional level. However, it is still unclear what its function is under abiotic stress in apple. Here, we found that heterologous expression of MdATG18a in tomato plants markedly enhanced their tolerance to drought. Overexpression (OE) of that gene in apple plants improved their drought tolerance as well. Under drought conditions, the photosynthesis rate and antioxidant capacity were significantly elevated in OE lines when compared with the untransformed wild type (WT). Transcript levels of other important apple ATG genes were more strongly up‐regulated in transgenic MdATG18a OE lines than in the WT. The percentage of insoluble protein in proportion to total protein was lower and less oxidized protein accumulated in the OE lines than in the WT under drought stress. This was probably due to more autophagosomes being formed in the former. These results demonstrate that overexpression of MdATG18a in apple plants enhances their tolerance to drought stress, probably because of greater autophagosome production and a higher frequency of autophagy. Those processes help degrade protein aggregation and limit the oxidation damage, thereby suggesting that autophagy plays important roles in the drought response. [ABSTRACT FROM AUTHOR]

Abstract: Nitrogen (N) availability is an essential factor for plant growth. Recycling and remobilization of N have strong impacts on crop yield and quality under N deficiency. Autophagy is a critical nutrient‐recycling process that facilitates remobilization under starvation. We previously showed that an important AuTophaGy (ATG) protein from apple, MdATG18a, has a positive role in drought tolerance. In this study, we explored its biological role in response to low‐N. Overexpression of MdATG18a in both Arabidopsis and apple improved tolerance to N‐depletion and caused a greater accumulation of anthocyanin. The increased anthocyanin concentration in transgenic apple was possibly due to up‐regulating flavonoid biosynthetic and regulatory genes (MdCHI, MdCHS, MdANS, MdPAL, MdUFGT, and MdMYB1) and higher soluble sugars concentration. MdATG18a overexpression enhanced starch degradation with up‐regulating amylase gene (MdAM1) and up‐regulated sugar metabolism related genes (MdSS1, MdHXKs, MdFK1, and MdNINVs). Furthermore, MdATG18a functioned in nitrate uptake and assimilation by up‐regulating nitrate reductase MdNIA2 and 3 high‐affinity nitrate transporters MdNRT2.1/2.4/2.5. MdATG18a overexpression also elevated other important MdATG genes expression and autophagosomes formation under N‐depletion, which play key contributions to above changes. Together, these results demonstrate that overexpression of MdATG18a enhances tolerance to N‐deficiencies and plays positive roles in anthocyanin biosynthesis through greater autophagic activity. [ABSTRACT FROM AUTHOR]

Cadmium (Cd), a phytotoxic heavy metal accumulated in plants and fruits, has significant adverse effects on plant growth and development as well as human health. In particular, Cd pollution has become a serious agricultural issue in recent years. Apple is one of the most popular fruits consumed at the global scale. Improving apple Cd resistance via reductions in Cd absorption can benefit apple tree growth and ensure fruit safety. In this study, we determined that, under the 200 μM Cd treatment, 35S::MdIAA24 apple plants exhibited more biomass and less Cd accumulation in the tested tissues compared to wild type (WT). Furthermore, the 35S::MdIAA24 apple plants demonstrated more favorable photosynthesis characteristics, less reactive oxygen species (ROS) and a greater amount of active antioxidant enzymes under the Cd condition than WT. The expression levels of the Cd uptake genes were observed to be lower in the 35S::MdIAA24 apple plants compared with those of the WT under the Cd treatment. The results highlight the ability of the overexpression of MdIAA24 to enhance apple Cd resistance by improving antioxidant capacity and reducing Cd absorption. [Display omitted] ● Overexpression of MdIAA24 enhanced cadmium tolerance in apple. ● Photosynthetic activity, ROS scavengers and biomass of apple increased under Cd stress due to over expression of MdIAA24. ● Decreased uptake of Cd due to MdIAA24 ensures the quality produce of apple. [ABSTRACT FROM AUTHOR]

To examine the potential roles of melatonin in drought tolerance, we tested the effects of its long-term exogenous application on 'Hanfu' apple ( Malus domestica Borkh.). When 100 μ m melatonin was added to soils under drought conditions, the resultant oxidative stress was eased and leaf senescence was delayed. This molecule significantly reduced chlorophyll degradation and suppressed the up-regulation of senescence-associated gene 12 ( SAG12) and pheophorbide a oxygenase ( PAO). Such treatment also alleviated the inhibition of photosynthesis brought on by drought stress. We also investigated quenching and the efficiency of Photosystem II ( PSII) photochemistry under dark and light conditions and found that melatonin helped to maintain better function of PSII under drought. The addition of melatonin also controlled the burst of hydrogen peroxide, possibly through direct scavenging and by enhancing the activities of antioxidative enzymes and the capacity of the ascorbate-glutathione cycle. Thus, understanding this effect of melatonin on drought tolerance introduces new possibilities to use this compound for agricultural purposes. [ABSTRACT FROM AUTHOR]

The objectives of this study were to test the effects of exogenous melatonin on apple ( Malus domestica Borkh. cv. Golden Delicious) leaves and investigate its possible physiological role in delaying leaf senescence. Detached leaves treated with 10 m m melatonin solutions clearly showed a slowing in their process of dark-induced senescence, as evidenced by both biochemical and molecular parameters. Melatonin delayed the normal reduction in chlorophyll content and maximum potential photosystem II efficiency ( Fv/ Fm). It also suppressed the transcript levels of a key chlorophyll degradation gene, pheide a oxygenase ( PAO), and the senescence-associated gene 12 ( SAG12). This outcome was thought to be because of the enhanced antioxidant capabilities of melatonin. Indeed, H2O2 accumulation was inhibited by exogenous melatonin, which might have resulted from direct reactive oxygen species scavenging by melatonin and a great enhancement of ascorbate peroxidase (APX; ), which acted on both mRNA and protein activity levels. Melatonin treatment led to the maintenance of higher contents of ascorbic acid (AsA) and glutathione (GSH) but less dehydroascorbate (DHA) and oxidized glutathione (GSSG) compared with the control, possibly through its regulation of the AsA-GSH cycle. [ABSTRACT FROM AUTHOR]

Abstract: Effects of light irradiation on the photosynthetic electron transport chain between P680 and P700 in apple tree leaves was probed with chlorophyll a fluorescence transient and 820 nm transmission measurements during dehydration under different light intensities. The results showed that light accelerated the leaf water-loss rate during dehydration. Leaf dehydration lowered the maximum quantum yield of PSII and the far-red light induced maximal transmission change at 820 nm, but increased the relative variable fluorescence intensity at J-step, especially under increasing irradiation conditions. During leaf dehydration, irradiation lowered the relative variable fluorescence intensity at I-step. At the beginning of leaf dehydration, moderate light accelerated the leaf water-loss rate and then lowered the maximal light-trapping efficiency of P680. Upon further dehydration under moderate light or dehydration under high light, light accelerated the water-loss rate and also directly decreased the maximal light-trapping efficiency of P680. The more significant decrease in the exchange capacity of plastoquinones at the QB site was mainly attributed to the faster water-loss rate under moderate light than in the dark. Under high light, irradiation also directly lowered the capacity. The reoxidation of PQH2 in the dehydrated leaves was enhanced by the light irradiation. The rapidly decreased contents of P700 + plastocyanin were mainly attributed to the faster water-loss rate under light conditions in contrast with that in the dark. The different effects of light irradiations on the photosynthetic electron transport chain might be involved in the acclimation of apple tree leaves to dehydration. [Copyright &y& Elsevier]

Autophagy is a conserved degradation pathway for recycling damaged organelles and aberrant proteins, and its important roles in plant adaptation to nutrient starvation have been generally reported. Previous studies found that overexpression of autophagy-related (ATG) gene MdATG10 enhanced the autophagic activity in apple roots and promoted their salt tolerance. The MdATG10 expression was induced by nitrogen depletion condition in both leaves and roots of apple plants. This study aimed to investigate the differences in the growth and physiological status between wild type and MdATG10-overexpressing apple plants in response to nitrogen starvation. A hydroponic system containing different nitrogen levels was used. The study found that the reduction in growth and nitrogen concentrations in different tissues caused by nitrogen starvation was relieved by MdATG10 overexpression. Further studies demonstrated the increased root growth and the higher nitrogen absorption and assimilation ability of transgenic plants. These characteristics contributed to the increased uptake of limited nitrogen nutrients by transgenic plants, which also reduced the starvation damage to the chloroplasts. Therefore, the MdATG10-overexpressing apple plants could maintain higher photosynthetic ability and possess better growth under nitrogen starvation stress. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine how xanthophyll cycle-dependent thermal dissipation and the antioxidant system in the peel of apple fruit respond to the natural light exposure within the tree canopy. Fruit from exterior and interior canopies of both mature ‘Gala’ and ‘Smoothee’ apple trees were sampled at noon and/or predawn to measure chlorophyll fluorescence, xanthophyll cycle pool size and composition, and enzymatic and non-enzymatic antioxidants. Compared with the shaded side, the sun-exposed peel of the fruit had more excess absorbed photon flux density (PFD) as a result of a lower photosystem II operating efficiency and a higher incident PFD at noon. The efficiency of excitation transfer was lower in the sun-exposed peel than in the shaded peel, indicative of higher thermal dissipation. The sun-exposed peel had a larger xanthophyll cycle pool size and a higher conversion state. It also had higher activities of ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, and a larger size and a higher reduction state of the ascorbate pool and the glutathione pool. However, catalase activity was lower in the sun-exposed peel than in the shaded peel. Superoxide dismutase did not show significant trend with regard to fruit peel type or position in the canopy. We conclude that both the xanthophyll cycle and the ascorbate–glutathione pathway in the apple fruit peel are acclimated to the prevailing light exposure within the tree canopy to meet the respective needs for dissipating excess absorbed PFD and detoxifying reactive oxygen species. [Copyright &y& Elsevier]