The transcription factor MdERF023 negatively regulates salt tolerance by modulating ABA signaling and Na+/H+ transport in apple.

KEY MESSAGE: MdERF023 is a transcription factor that can reduce salt tolerance by inhibiting ABA signaling and Na+/H+ homeostasis. Salt stress is one of the principal environmental stresses limiting the growth and productivity of apple (Malus × domestica). The APETALA2/ethylene response factor (AP2/ERF) family plays key roles in plant growth and various stress responses; however, the regulatory mechanism involved has not been fully elucidated. In the present study, we identified an AP2/ERF transcription factor (TF), MdERF023, which plays a negative role in apple salt tolerance. Stable overexpression of MdERF023 in apple plants and calli significantly decreased salt tolerance. Biochemical and molecular analyses revealed that MdERF023 directly binds to the promoter of MdMYB44-like, a positive modulator of ABA signaling-mediated salt tolerance, and suppresses its transcription. In addition, MdERF023 downregulated the transcription of MdSOS2 and MdAKT1, thereby reducing the Na+ expulsion, K+ absorption, and salt tolerance of apple plants. Taken together, these results suggest that MdERF023 reduces apple salt tolerance by inhibiting ABA signaling and ion transport, and that it could be used as a potential target for breeding new varieties of salt-tolerant apple plants via genetic engineering.

The HD-Zip I transcription factor MdHB-7 negatively regulates resistance to Glomerella leaf spot in apple.

Glomerella leaf spot (GLS), caused by Colletotrichum fructicola (Cf), has been one of the main fungal diseases afflicting apple-producing areas across the world for many years, and it has led to substantial reductions in apple output and quality. HD-Zip transcription factors have been identified in several species, and they are involved in the immune response of plants to various types of biotic stress. In this study, inoculation of MdHB-7 overexpressing (MdHB-7-OE) and interference (MdHB-7-RNAi) transgenic plants with Cf revealed that MdHB-7, which encodes an HD-Zip transcription factor, adversely affects GLS resistance. The SA content and the expression of SA pathway-related genes were lower in MdHB-7-OE plants than in 'GL-3' plants; the content of ABA and the expression of ABA biosynthesis genes were higher in MdHB-7-OE plants than in 'GL-3' plants. Further analysis indicated that the content of phenolics and chitinase and ß-1, 3 glucanase activities were lower and H2O2 accumulation was higher in MdHB-7-OE plants than in 'GL-3' plants. The opposite patterns were observed in MdHB-7-RNAi apple plants. Overall, our results indicate that MdHB-7 plays a negative role in regulating defense against GLS in apple, which is likely achieved by altering the content of SA, ABA, polyphenols, the activities of defense-related enzymes, and the content of H2O2.

Apple Glycosyltransferase MdUGT73AR4 Glycosylates ABA to Regulate Stomatal Movement Involved in Drought Stress.

Abscisic acid (ABA) is a drought-stress-responsive hormone that plays an important role in the stomatal activity of plant leaves. Currently, ABA glycosides have been identified in apples, but their glycosyltransferases for glycosylation modification of ABA are still unidentified. In this study, the mRNA expression of glycosyltransferase gene MdUGT73AR4 was significantly up-regulated in mature apple leaves which were treated in drought stress by Real-Time PCR. It was hypothesised that MdUGT73AR4 might play an important role in drought stress. In order to further characterise the glycosylation modification substrate of glycosyltransferase MdUGT73AR4, we demonstrated through in vitro and in vivo functional validation that MdUGT73AR4 can glycosylate ABA. Moreover, the overexpression lines of MdUGT73AR4 significantly enhance its drought stress resistance function. We also found that the adversity stress transcription factor AREB1B might be an upstream transcription factor of MdUGT73AR4 by bioinformatics, EMSA, and ChIP experiments. In conclusion, this study found that the adversity stress transcription factor AREB1B was significantly up-regulated at the onset of drought stress, which in turn positively regulated the downstream glycosyltransferase MdUGT73AR4, causing it to modify ABA by mass glycosylation and promoting the ABA synthesis pathway, resulting in the accumulation of ABA content, and displaying a stress-resistant phenotype.

The MdNAC72-MdABI5 module acts as an interface integrating jasmonic acid and gibberellin signals and undergoes ubiquitination-dependent degradation regulated by MdSINA2 in apple.

Jasmonic acid (JA) and gibberellin (GA) coordinately regulate plant developmental programs and environmental cue responses. However, the fine regulatory network of the cross-interaction between JA and GA remains largely elusive. In this study, we demonstrate that MdNAC72 together with MdABI5 positively regulates anthocyanin biosynthesis through an exquisite MdNAC72-MdABI5-MdbHLH3 transcriptional cascade in apple. MdNAC72 interacts with MdABI5 to promote the transcriptional activation of MdABI5 on its target gene MdbHLH3 and directly activates the transcription of MdABI5. The MdNAC72-MdABI5 module regulates the integration of JA and GA signals in anthocyanin biosynthesis by combining with JA repressor MdJAZ2 and GA repressor MdRGL2a. MdJAZ2 disrupts the MdNAC72-MdABI5 interaction and attenuates the transcriptional activation of MdABI5 by MdNAC72. MdRGL2a sequesters MdJAZ2 from the MdJAZ2-MdNAC72 protein complex, leading to the release of MdNAC72. The E3 ubiquitin ligase MdSINA2 is responsive to JA and GA signals and promotes ubiquitination-dependent degradation of MdNAC72. The MdNAC72-MdABI5 interface fine-regulates the integration of JA and GA signals at the transcriptional and posttranslational levels by combining MdJAZ2, MdRGL2a, and MdSINA2. In summary, our findings elucidate the fine regulatory network connecting JA and GA signals with MdNAC72-MdABI5 as the core in apple.

VmRDR2 of Valsa mali mediates the generation of VmR2-siR1 that suppresses apple resistance by RNA interference.

Cross-kingdom RNA interference (RNAi) is a crucial mechanism in host-pathogen interactions, with RNA-dependent RNA polymerase (RdRP) playing a vital role in signal amplification during RNAi. However, the role of pathogenic fungal RdRP in siRNAs generation and the regulation of plant-pathogen interactions remains elusive. Using deep sequencing, molecular, genetic, and biochemical approaches, this study revealed that VmRDR2 of Valsa mali regulates VmR2-siR1 to suppress the disease resistance-related gene MdLRP14 in apple. Both VmRDR1 and VmRDR2 are essential for the pathogenicity of V. mali in apple, with VmRDR2 mediating the generation of endogenous siRNAs, including an infection-related siRNA, VmR2-siR1. This siRNA specifically degrades the apple intracellular LRR-RI protein gene MdLRP14 in a sequence-specific manner, and overexpression of MdLRP14 enhances apple resistance against V. mali, which can be suppressed by VmR2-siR1. Conversely, MdLRP14 knockdown reduces resistance. In summary, this study demonstrates that VmRDR2 contributes to the generation of VmR2-siR1, which silences the host's intracellular LRR protein gene, thereby inhibiting host resistance. These findings offer novel insights into the fungi-mediated pathogenicity mechanism through RNAi.

Apple MdZAT5 mediates root development under drought stress.

Root plays an important role in plant drought tolerance, especially in horticultural crops like apples. However, the crucial regulator and molecular mechanism in root development of apple trees under drought are not well unknown. Cys2/His2-type Zinc-finger proteins are essential for plant response to drought, while the members of C2H2 Zinc-finger proteins in apple are largely unknown. In this study, we identified the members of the C1-2i subclass family of C2H2 Zinc-finger proteins in apple (Malus × domestica). Among them, MdZAT5 is significantly induced in apple roots under drought conditions and positively regulates apple root development under drought. Further investigation revealed that MdZAT5 positively regulates root development and root hydraulic conductivity by mediating the transcription level of MdMYB88 under drought stress. Taken together, our results demonstrate the importance of MdZAT5 in root development under drought in apple trees. This finding provides a new candidate direction for apple breeding for drought resistance.

A non-invasive method for phenotyping scab-tolerant apple plants using volatile organic compounds.

One of the most devastating diseases of apples is scab, caused by the fungus Venturia inaequalis. Most commercial apple varieties are susceptible to this disease; only a few are resistant. Breeding approaches are being used to develop better apple varieties that are resistant to scab. Volatile organic compounds (VOCs) contribute greatly to a plant's phenotype, and their emission profile largely depends on the genotype. In the non-destructive phenotyping of plants, VOCs can be used as biomarkers. In this study, we assessed non-destructively the scab tolerance potential of resistant (cv. 'Prima') and susceptible (cv. 'Oregon Spur') apple cultivars by comparing their major leaf VOC compositions and relative proportions. A comparison of the leaf VOC profiles of the two cultivars revealed 16 different VOCs, with cis-3-hexenyl acetate (3HA) emerging as a biomarker of cultivar differences. V. inaequalis growth was significantly inhibited in vitro by 3HA treatment. 3HA was significantly effective in reducing scab symptoms on V. inaequalis-inoculated leaves of 'Oregon Spur.' The resistant cultivar 'Prima' also exhibited higher lipoxygenase (LOX) activity and α-linolenic acid (ALA) levels, suggesting that V. inaequalis resistance is linked to LOX activity and 3HA biosynthesis. This study proposes 3HA as a potential biomarker for rapid non-destructive screening of scab-resistant apple germplasm of 'Prima' based on leaf VOCs.

A haplotype-resolved genome assembly of Malus domestica 'Red Fuji'.

The 'Red Fuji' apple (Malus domestica), is one of the most important and popular economic crops worldwide in the fruit industry. Using PacBio HiFi long reads and Hi-C reads, we assembled a high-quality haplotype-resolved genome of 'Red Fuji', with sizes of 668.7 and 668.8 Mb, and N50 sizes of 34.1 and 31.4 Mb. About 97.2% of sequences were anchored in 34 chromosomes. We annotated both haploid genomes, identifying a total of 95,439 protein-coding genes in the two haplotype genomes, with 98% functional annotation. The haplotype-resolved genome of 'Red Fuji' apple stands as a precise benchmark for an array of analyses, such as comparative genomics, transcriptomics, and allelic expression studies. This comprehensive resource is paramount in unraveling variations in allelic expression, advancing quality improvements, and refining breeding efforts.

The apple MdGA2ox7 modulates the balance between growth and stress tolerance in an anthocyanin-dependent manner.

Apple (Malus domestica Borkh.) is a widely cultivated fruit crop worldwide but often suffers from abiotic stresses such as salt and cold. Gibberellic acid (GA) plays a pivotal in controlling plant development, environmental adaptability, and secondary metabolism. The GA2-oxidase (GA2ox) is responsible for the deactivation of bioactive GA. In this study, seventeen GA2-oxidase genes were identified in the apple genome, and these members could be clustered into four clades based on phylogenetic relationships and conserved domain structures. MdGA2ox7 exhibited robust expression across various tissues, responded to cold and salt treatments, and was triggered in apple fruit peels via light-induced anthocyanin accumulation. Subcellular localization prediction and experiments confirmed that MdGA2ox7 was located in the cytoplasm. Overexpression of MdGA2ox7 in Arabidopsis caused a lower level of active GA and led to GA-deficient phenotypes, such as dwarfism and delayed flowering. MdGA2ox7 alleviated cold and salt stress damage in both Arabidopsis and apple in concert with melatonin (MT). Additionally, MdGA2ox7 enhanced anthocyanin biosynthesis in apple calli and activated genes involved in anthocyanin synthesis. These findings provide new insights into the functions of apple GA2ox in regulating development, stress tolerance, and secondary metabolism.

Molecular evolution of Phytocyanin gene and analysis of expression at different coloring periods in apple (Malus domestica).

BACKGROUND: PC (phytocyanin) is a class of copper-containing electron transfer proteins closely related to plant photosynthesis, abiotic stress responses growth and development in plants, and regulation of the expression of some flavonoids and phenylpropanoids, etc., however, compared with other plants, the PC gene family has not been systematically characterized in apple. RESULTS: A total of 59 MdPC gene members unevenly distributed across 12 chromosomes were identified at the genome-wide level. The proteins of the MdPC family were classified into four subfamilies based on differences in copper binding sites and glycosylation sites: Apple Early nodulin-like proteins (MdENODLs), Apple Uclacyanin-like proteins (MdUCLs), Apple Stellacyanin-like proteins (MdSCLs), and Apple Plantacyanin-like proteins (MdPLCLs). Some MdPC members with similar gene structures and conserved motifs belong to the same group or subfamily. The internal collinearity analysis revealed 14 collinearity gene pairs among members of the apple MdPC gene. Interspecific collinearity analysis showed that apple had 31 and 35 homologous gene pairs with strawberry and grape, respectively. Selection pressure analysis indicated that the MdPC gene was under purifying selection. Prediction of protein interactions showed that MdPC family members interacted strongly with the Nad3 protein. GO annotation results indicated that the MdPC gene also regulated the biosynthesis of phenylpropanoids. Chip data analysis showed that (MdSCL3, MdSCL7 and MdENODL27) were highly expressed in mature fruits and peels. Many cis-regulatory elements related to light response, phytohormones, abiotic stresses and flavonoid biosynthetic genes regulation were identified 2000 bp upstream of the promoter of the MdPC gene, and qRT-PCR results showed that gene members in Group IV (MdSCL1/3, MdENODL27) were up-regulated at all five stages of apple coloring, but the highest expression was observed at the DAF13 (day after fruit bag removal) stage. The gene members in Group II (MdUCL9, MdPLCL3) showed down-regulated or lower expression in the first four stages of apple coloring but up-regulated and highest expression in the DAF 21 stage. CONCLUSION: Herein, one objective of these findings is to provide valuable information for understanding the structure, molecular evolution, and expression pattern of the MdPC gene, another major objective in this study was designed to lay the groundwork for further research on the molecular mechanism of PC gene regulation of apple fruit coloration.

Brassinosteroids biosynthetic gene MdBR6OX2 regulates salt stress tolerance in both apple and Arabidopsis.

Salt stress is a critical limiting factor for fruit yield and quality of apples. Brassinosteroids (BRs) play an important role in response to abiotic stresses. In the present study, application of 2,4- Epicastasterone on seedlings of Malus 'M9T337' and Malus domestica 'Gala3' alleviated the physiological effects, such as growth inhibition and leaf yellowing, induced by salt stress. Further analysis revealed that treatment with NaCl induced expression of genes involved in BR biosynthesis in 'M9T337' and 'Gala3'. Among which, the expression of BR biosynthetic gene MdBR6OX2 showed a three-fold upregulation upon salt treatment, suggesting its potential role in response to salt stress in apple. MdBR6OX2, belonging to the CYP450 family, contains a signal peptide region and a P450 domain. Expression patterns analysis showed that the expression of MdBR6OX2 can be significantly induced by different abiotic stresses. Overexpressing MdBR6OX2 enhanced the tolerance of apple callis to salt stress, and the contents of endogenous BR-related compounds, such as Typhastero (TY), Castasterone (CS) and Brassinolide (BL) were significantly increased in transgenic calli compared with that of wild-type. Extopic expression of MdBR6OX2 enhanced tolerance to salt stress in Arabidopsis. Genes associated with salt stress were significantly up-regulated, and the contents of BR-related compounds were significantly elevated under salt stress. Our data revealed that BR-biosynthetic gene MdBR6OX2 positively regulates salt stress tolerance in both apple calli and Arabidopsis.

Melatonin promotes the recovery of apple plants after waterlogging by shaping the structure and function of the rhizosphere microbiome.

The dynamics of the physiological adaptability of plants and the rhizosphere soil environment after waterlogging remain unclear. Here we investigated the mechanisms regulating plant condition and shaping of the rhizosphere microbiome in a pot experiment. In the experiment, we added melatonin to waterlogged plants, which promoted waterlogging relief. The treatment significantly enhanced photosynthesis and the antioxidant capacity of apple plants, and significantly promoted nitrogen (N) utilization efficiency by upregulating genes related to N transport and metabolism. Multiperiod soil microbiome analysis showed the dynamic effects of melatonin on the diversity of the microbial community during waterlogging recovery. Random forest and linear regression analyses were used to screen for potential beneficial bacteria (e.g., Azoarcus, Pseudomonas and Nocardioides) specifically regulated by melatonin and revealed a positive correlation with soil nutrient levels and plant growth. Furthermore, metagenomic analyses revealed the regulatory effects of melatonin on genes involved in N cycling in soil. Melatonin positively contributed to the accumulation of plant dry weight by upregulating the expression of nifD and nifK (N fixation). In summary, melatonin positively regulates physiological functions in plants and the structure and function of the microbial community; it promoted the recovery of apple plants after waterlogging stress.

The potential range of west Asian apple species Malus orientalis Uglitzk. under climate change.

The wild relatives of cultivated apples would be an ideal source of diversity for breeding new varieties, which could potentially grow in diverse habitats shaped by climate change. However, there is still a lack of knowledge about the potential distribution of these species. The aim of the presented work was the understand the impacts of climate change on the potential distribution and habitat fragmentation of Caucasian crab apple (Malus orientalis Uglitzk.) and the designation of areas of high interest according to climatic conditions. We used the MaxEnt models and Morphological-Spatial Analysis (MSPA) to evaluate the potential distribution, suitability changes, habitat fragmentation, and connectivity throughout the species range in Turkey, Armenia, Georgia, Russia, and Iran. The results revealed that the potentially suitable range of M. orientalis encompasses 858,877 km², 635,279 km² and 456,795 km² under the present, RCP4.5 and RCP8.5 scenario, respectively. The range fragmentation analysis demonstrated a notable shift in the edge/core ratio, which increased from 50.95% in the current scenario to even 67.70% in the future. The northern part of the range (Armenia, northern Georgia, southern Russia), as well as the central and western parts of Hyrcania will be a core of the species range with suitable habitats and a high connectivity between M. orientalis populations and could work as major refugia for the studied species. However, in the Zagros and central Turkey, the potential range will shrink due to the lack of suitable climatic conditions, and the edge/core ratio will grow. In the southern part of the range, a decline of M. orientalis habitats is expected due to changing climatic conditions. The future outlook suggests that the Hyrcanian forest and the Caucasus region could serve as important refuges for M. orientalis. This study helps to understand spatial changes in species' range in response to climate change and can help develop conservation strategies. This is all the more important given the species' potential use in future breeding programs aimed at enriching the gene pool of cultivated apple varieties.

Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by Wall-associated kinase gene MbWAK1.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.

Assessment and Partial Characterization of Candidate Genes in Dihydrochalcone and Arbutin Biosynthesis in an Apple-Pear Hybrid by De Novo Transcriptome Assembly.

Apples (Malus × domestica Borkh.) and pears (Pyrus communis L.) are valuable crops closely related within the Rosaceae family with reported nutraceutical properties derived from secondary metabolites including phloridzin and arbutin, which are distinctive phenolic metabolites characterizing apples and pears, respectively. Here, we generated a de novo transcriptome assembly of an intergeneric hybrid between apple and pear, accumulating intermediate levels of phloridzin and arbutin. Combining RNA-seq, in silico functional annotation prediction, targeted gene expression analysis, and expression-metabolite correlations, we identified candidate genes for functional characterization, resulting in the identification of active arbutin synthases in the hybrid and parental genotypes. Despite exhibiting an active arbutin synthase in vitro, the natural lack of arbutin in apples is reasoned by the absence of the substrate and broad substrate specificity. Altogether, our study serves as the basis for future assessment of potential physiological roles of identified genes by genome editing of hybrids and pears.

Evolution of the SPX gene family and its role in the response mechanism to low phosphorus stress in self-rooted apple stock.

BACKGROUND: Phosphorus plays a key role in plant adaptation to adversity and plays a positive role in the yield and quality formation of apples. Genes of the SPX domain-containing family are widely involved in the regulation of phosphorus signalling networks. However, the mechanisms controlling phosphorus deficiency are not completely understood in self-rooted apple stock. RESULTS: In this study, 26 members of the apple SPX gene family were identified by genome-wide analysis, and further divided into four subfamilies (SPX, SPX-MFS, SPX-EXS, and SPX-RING) based on their structural features. The chromosome distribution and gene duplications of MdSPXs were also examined. The promoter regions of MdSPXs were enriched for multiple biotic/abiotic stresses, hormone responses and typical P1BS-related elements. Analysis of the expression levels of 26 MdSPXs showed that some members were remarkably induced when subjected to low phosphate (Pi) stress, and in particular MdSPX2, MdSPX3, and MdPHO1.5 exhibited an intense response to low Pi stress. MdSPX2 and MdSPX3 showed significantly divergent expression levels in low Pi sensitive and insensitive apple species. Protein interaction networks were predicted for 26 MdSPX proteins. The interaction of MdPHR1 with MdSPX2, MdSPX3, MdSPX4, and MdSPX6 was demonstrated by yeast two-hybrid assay, suggesting that these proteins might be involved in the Pi-signaling pathway by interacting with MdPHR1. CONCLUSION: This research improved the understanding of the apple SPX gene family and contribute to future biological studies of MdSPX genes in self-rooted apple stock.

Apple-arbuscular mycorrhizal symbiosis confers resistance to Fusarium solani by inducing defense response and elevating nitrogen absorption.

Fusarium solani exerts detrimental effects on plant growth, which is one of the reasons for the incidence of apple replant disease. Arbuscular mycorrhizal fungi (AMF) enhance plant resistance to Fusarium wilt; however, the mechanism remains poorly understood. Therefore, the present study investigated the symbiosis between apple and AMF and explored the physiology, especially nitrate metabolism, antioxidant defense, and photosynthetic performance, when infected by F. solani. The experiment was carried out with four treatments, namely -AMF - F. solani, -AMF + F. solani, -AMF + F. solani, and + AMF + F. solani. In this study, the -AMF + F. solani treatment increased the activity of enzymes associated with nitrogen metabolism, such as the nitrate and nitrite reductases, in the apple root system. The +AMF + F. solani treatment showed higher antioxidant enzyme activities than the -AMF + F. solani by F. solani infection. The apple seedlings of the +AMF + F. solani treatment decreased reactive oxygen accumulation and reduced the oxidative damages triggered by F. solani infection. The improvement in antioxidant capacity due to the +AMF + F. solani treatment was closely associated with the upregulation of genes related to the antioxidant system. The F. solani infection greatly damaged the photosynthetic process, while the +AMF + F. solani treatment significantly improved it compared to the -AMF + F. solani treatment. In conclusion, the study demonstrated that the apple-AMF symbiosis plays an active role in regulating the resistance against F. solani infection by enhancing defense response and nitrogen metabolism.

Transcriptome and metabolome analyses reveal the regulatory role of MdPYL9 in drought resistance in apple.

BACKGROUND: The mechanisms by which the apple MdPYL9 gene mediates the response to drought stress remain unclear. Here, transcriptome and metabolome analyses of apple plants under drought were used to investigate the mechanisms by which MdPYL9 regulates the response to drought stress in apple. MdPYL9-overexpressed transgenic and non-transgenic apple histoculture seedlings were rooted, transplanted, and subjected to drought treatments to clarify the mechanisms underlying the responses of apples to drought stress through phenotypic observations, physiological and biochemical index measurements, and transcriptomic and metabolomic analyses. RESULTS: Under drought stress treatment, transgenic plants were less affected by drought stress than non-transgenic plants. Decreases in the net photosynthetic rate, stomatal conductance, and transpiration rate of transgenic apple plants were less pronounced in transgenic plants than in non-transgenic plants, and increases in the intercellular CO2 concentration were less pronounced in transgenic plants than in non-transgenic plants. The relative electrical conductivity and content of malondialdehyde, superoxide anion, and hydrogen peroxide were significantly lower in transgenic plants than in non-transgenic plants, and the chlorophyll content and activities of antioxidant enzymes (superoxide dismutase, peroxidase, and catalase) were significantly higher in transgenic plants than in non-transgenic plants. The number of differentially expressed genes (DEGs) involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and the most significant and highly annotated DEGs in the transgenic plants were involved in the flavonoid biosynthesis pathway, and the most significant and highly annotated DEGs in control plants were involved in the phytohormone signal transduction pathway. The number of differentially accumulated metabolites involved in the response to drought stress was lower in transgenic plants than in non-transgenic plants, and up-regulated metabolites were significantly enriched in apigenin-7-O-glucoside in transgenic plants and in abscisic acid in non-transgenic plants. In the flavonoid biosynthetic pathway, the expression of genes encoding chalcone synthase (CHS) and chalcone isomerase (CHI) was more significantly down-regulated in non-transgenic plants than in transgenic plants, and the expression of the gene encoding 4-coumarate-CoA ligase (4CL) was more significantly up-regulated in transgenic plants than in non-transgenic plants, which resulted in the significant up-regulation of apigenin-7-O-glucoside in transgenic plants. CONCLUSIONS: The above results indicated that the over-expression of MdPYL9 increased the drought resistance of plants under drought stress by attenuating the down-regulation of the expression of genes encoding CHS and CHI and enhancing the up-regulated expression of the gene encoding 4CL, which enhanced the content of apigenin-7-O-glucoside.

The chromosome-level genome assembly of the dwarfing apple interstock Malus hybrid 'SH6'.

Malus hybrid 'SH6' (M. honanensis × M. domestica)is a commonly used apple interstock in China, known for its excellent dwarfing characteristics and cold tolerance. In this study, a combined strategy utilizing PacBio HiFi, Hi-C and parental resequencing data were employed to assemble two haploid genomes for 'SH6'. After chromosome anchoring, the final hapH genome size was 596.63 Mb, with a contig N50 of 34.38 Mb. The hapR genome was 649.37 Mb, with a contig N50 of 36.84 Mb. Further analysis predicted that repeated sequences made up 59.69% and 62.52% of the entire genome, respectively. Gene annotations revealed 45,435 genes for hapH and 48,261 genes for hapR. Combined with genomic synteny we suggest that the hapR genome originates from its maternal parent M. domestica cv. Ralls Janet, while the hapH genome comes from its paternal parent, M. honanensis. The assembled genome significantly contributes to the discovery of genes associated with apple dwarfing and the molecular mechanisms governing them.

Strigolactones alleviate AlCl3 stress by vacuolar compartmentalization and cell wall blocking in apple.

Poor management and excess fertilization of apple (Malus domestica Borkh.) orchards are causing increasingly serious soil acidification, resulting in Al toxicity and direct poisoning of roots. Strigolactones (SLs) are reported to be involved in plant responses to abiotic stress, but their role and mechanism under AlCl3 stress remain unknown. Here, we found that applying 1 µm GR24 (an SL analoge) significantly alleviated AlCl3 stress of M26 apple rootstock, mainly by blocking the movement of Al through cell wall and by vacuolar compartmentalization of Al. RNA-seq analysis identified the core transcription factor gene MdWRKY53, and overexpressing MdWRKY53 enhanced AlCl3 tolerance in transgenic apple plants through the same mechanism as GR24. Subsequently, we identified MdPMEI45 (encoding pectin methylesterase inhibitor) and MdALS3 (encoding an Al transporter) as downstream target genes of MdWRKY53 using chromatin immunoprecipitation followed by sequencing (ChIP-seq). GR24 enhanced the interaction between MdWRKY53 and the transcription factor MdTCP15, further increasing the binding of MdWRKY53 to the MdPMEI45 promoter and inducing MdPMEI45 expression to prevent Al from crossing cell wall. MdWRKY53 also bound to the promoter of MdALS3 and enhanced its transcription to compartmentalize Al in vacuoles under AlCl3 stress. We therefore identified two modules involved in alleviating AlCl3 stress in woody plant apple: the SL-WRKY+TCP-PMEI module required for excluding external Al by blocking the entry of Al3+ into cells and the SL-WRKY-ALS module allowing internal detoxification of Al through vacuolar compartmentalization. These findings lay a foundation for the practical application of SLs in agriculture.