Iron (Fe) deficiency is a major limitation to apple (Malus domestica) growth in calcareous soils. Understanding the molecular mechanisms underlying Fe deficiency responses is crucial for improving Fe use efficiency in fruit trees. In this study, we identified the ETHYLENE RESPONSE FACTOR 4 (MxERF4) as a negative regulator of the Fe deficiency response in apple. Transgenic analysis revealed that overexpression of MxERF4 exacerbated leaf chlorosis and reduced root Fe content under Fe-deficient conditions, whereas RNA interference (RNAi) lines exhibited enhanced tolerance. We further identified the MITOGEN-ACTIVATED PROTEIN KINASE (MxMPK6-2) as an interactor of MxERF4. MxMPK6-2 phosphorylates MxERF4, reducing its protein stability and promoting its degradation. MxERF4 interacted with the key Fe uptake regulator FER-LIKE FE DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (MxFIT) and co-localized with it in root tissues. Phosphorylation weakened the MxERF4–MxFIT interaction, thereby relieving the inhibition of MxFIT–basic Helix-Loop-Helix 38/39 (MxbHLH38/39) complex formation. This in turn restored the activation of Fe uptake genes IRON TRANSPORTER1 (MxIRT1) and FERRIC REDUCTASE OXIDASE 2 (MxFRO2), enhanced ferric chelate reductase (FCR) activity, and promoted active Fe accumulation. Together, these findings reveal a previously uncharacterized MPK6-2–ERF4 signaling module that regulates Fe deficiency responses in apple by modulating ERF4 stability and its interaction with FIT, providing insights into the molecular basis of Fe efficiency and offering potential strategies for breeding Fe-efficient rootstocks.

The genus Zingiber possesses substantial economic and medicinal value, yet its mitochondrial genome (mitogenome) has remained entirely unexplored. Here, we present the first complete mitogenome assembly of ginger (Zingiber officinale), obtained through a hybrid sequencing (Illumina and PacBio) strategy. The genome features a complex, multi-branched structure totaling 8,977,507 bp with a GC content of 45.27%. It harbors 40 protein-coding genes (PCGs), 31 tRNAs, and three rRNAs. We identified abundant repetitive sequences, including 2954 SSRs and 54,887 dispersed repeats driving this genomic expansion, alongside widespread evidence of intracellular DNA transfer, with 273 plastid-derived fragments integrated into the mitogenome. Codon usage analysis revealed a pronounced bias toward A/U-ending synonymous codons. Importantly, Deepred-Mt predictions of extensive C-to-U RNA editing sites were experimentally corroborated via Sanger sequencing, confirming their critical role in maintaining the hydrophobicity of respiratory chain proteins. Comparative analyses revealed a striking evolutionary decoupling: highly conserved nucleotide substitution rates contrasting with extreme structural rearrangements relative to related Curcuma species, and phylogenetic reconstruction strongly supported the sister relationship between Z. officinale and the Curcuma clade. This study provides a foundational genomic resource for further research on cytoplasmic inheritance, genome gigantism, molecular breeding, and the links between mitogenomic variation and key agronomic or medicinal traits in Z. officinale.

The K-homology (KH) domain is a crucial RNA-binding motif central to post-transcriptional regulation. However, its corresponding gene family remains poorly characterized in tomato (Solanum lycopersicum), a key model species for studying fleshy fruit development. Here, we performed a genome-wide identification and comprehensive characterization of 47 SlKH genes in S. lycopersicum. Phylogenetic and synteny analyses indicated that the gene family expanded mainly through segmental duplications. While the core RNA-binding GXXG loop has evolved under strict purifying selection, specific orthologs, such as the SlKH3/AtKH6 pair (Ka/Ks = 1.78), exhibited putative signatures of positive selection. Haplotype variations in SlKH47, SlKH43, and SlKH35 are associated with significant differences in fruit weight, revealing their potential roles in crop domestication. Furthermore, expression profiling revealed distinct spatiotemporal patterns, highlighting several members that are significantly upregulated during fruit ripening. Structural modeling with AlphaFold 3 provided predictive insights into how the conserved GXXG motif mediates RNA recognition. This study provides a comprehensive genomic resource and foundational insights into the evolutionary and functional significance of KH proteins in S. lycopersicum development and breeding.

Apple Glomerella leaf spot (GLS), a major fungal disease, severely threatens the sustainable development of the apple industry and causes significant yield and quality losses. Enhancing host resistance through grafting is a promising strategy against GLS (caused by Colletotrichum fructicola); however, the underlying molecular mechanisms remain unclear. In this study, detached-leaf inoculation, grafting, and RT-PCR assays were integrated to confirm that rootstock-derived mobile mRNAs can significantly enhance scion resistance to GLS. Using six apple genotypes (‘Gala’, ‘Royal Gala’, Malus xiaojinensis, Malus hupehensis Rehd. var. pingyiensis Jiang, G935, M9-T337), we evaluated detached-leaf resistance and constructed grafting combinations. Resistant rootstocks were found to elevate resistance in susceptible scions by modulating leaf enzyme activities (POD, CAT, SOD) and lignin content. Importantly, we identified the CC-NBS-LRR (CNL) gene MdNRG1.1 as a mobile mRNA that translocates from rootstock to scion in specific grafting combinations (GL/G935 and GL/Mh), providing a mechanistic explanation for the enhanced resistance. This study establishes a theoretical basis for breeding resistant rootstocks and developing sustainable disease control strategies.

Salt stress is a major abiotic factor that severely restricts pepper (Capsicum annuum) production. Although abscisic acid (ABA) is vital for salt tolerance, the transcriptional regulatory networks governing ABA-mediated salt defense remain largely unknown. Here, we uncovered a negative feedback loop between CaMYB121 and CaABF2.1/2 that modulates the expression of CaNHX2.1/2/3, thereby enhancing salt tolerance in pepper plants. RNA-seq analysis revealed that CaMYB121 displayed an expression pattern consistent with that of CaNHX2 after salt treatment. Silencing CaMYB121 markedly reduced salt tolerance and inhibited root growth. Mechanistically, CaMYB121 directly binds to the CaNHX2 promoter to activate transcription, thereby promoting salt resilience. Salt stress also robustly triggered ABA signaling genes, with CaABF2.1/2 displaying expression patterns closely mirroring those of CaMYB121. Transient silencing of CaABF2.1/2 results in phenotypes similar to those observed with CaMYB121 suppression. Notably, CaMYB121 activates CaABF2.1/2 transcription by binding to its promoters, whereas CaABF2.1/2 represses CaMYB121 expression by directly targeting its promoter, forming a self-regulating feedback loop that prevents excessive defense activation. Collectively, our findings reveal a CaMYB121-CaABF2 feedback circuit that dynamically balances growth and defense to optimize salt tolerance in pepper plants.

High-throughput single-cell RNA sequencing (scRNA-seq) has substantially advanced plant transcriptional landscapes. However, decoding cell-type-specific transcriptional regulation in non-model crops like tomato (Solanum lycopersicum) remains challenging. An integrated computational pipeline was applied using high-dimensional weighted gene co-expression (hdWGCNA) and ensemble machine learning to analyze tomato leaf single-cell transcriptomes. Unsupervised clustering identified 19 cell subpopulations mapped to five major cell-types: mesophyll cells (50.6%), guard cells (31.0%), trichomes (8.3%), vascular cells (7.5%), and lamina epidermis (2.6%). hdWGCNA revealed eight cell-type-specific modules, linking mesophyll cells to photosynthesis and guard cells to redox homeostasis. Machine learning classifiers prioritized candidate transcription factors (TFs), with XGBoost achieving the highest accuracy (0.85) to define cell identity. A consensus of 33 core TFs was identified, from which four candidate TFs (SlWRKY-78, SlWRKY-75, SlERF-57, and SlGLK-49) were selected for in silico knockout (KO) analysis. The simulations predicted that these knockouts might dysregulate core functional pathways, such as serine-type endopeptidase inhibitor activity and protein binding. Furthermore, CellOracle simulations suggested that the virtual deletion of the guard-cell-associated SlWRKY-78 and SlWRKY-75 could induce a directional trajectory shift from the terminally differentiated guard cells back to the less differentiated mesophyll territory. These findings provide a promising computational framework for deciphering cell-type-specific regulatory programs in horticultural crops.

Objectives This study investigated the influence of xenia on disease resistance in apple fruits, with a specific focus on the impact of resistant pollen on the susceptibility of hybrid fruits to apple ring rot, a major threat to the apple industry in China.Materials and Methods Hybrid fruits were produced by pollinating susceptible apple cultivars with both resistant and susceptible pollen. Disease resistance was assessed through inoculation with the ring rot pathogen and subsequent evaluation of disease incidence, latent period, and lesion development. The expression of candidate mobile mRNAs associated with disease resistance was analyzed using quantitative reverse transcription polymerase chain reaction (qRT-RCR).Results The application of resistant pollen significantly reduced disease incidence (by up to 90% in the 'Fuji' x 'Jiguan' combination) and lesion development in hybrid fruits compared with those pollinated with susceptible cultivars. Fruits pollinated with resistant cultivars also exhibited longer latent periods and smaller lesions, with a 60%-86% reduction in lesion incidence observed between resistant and susceptible combinations. Two mobile mRNAs, MdRGA3 and MdMYC2, were identified as key signaling molecules, whose expression is significantly upregulated during late fruit maturation stages, suggesting their involvement in enhancing disease resistance.Conclusions The findings demonstrate that xenia can effectively improve disease resistance in apple fruits, highlighting its potential to enhance both fruit quality and resilience against diseases like apple ring rot. This research contributes valuable insights into the molecular mechanisms underlying xenia and supports the development of sustainable practices in apple cultivation.

Dissecting quantitative traits into Mendelian factors is a great challenge in genetics. Apple fruit storability is a complex trait controlled by multi-genes with unequal effects. We previously identified 62 quantitative trait loci (QTLs) associated with apple fruit storability and genomics-assisted prediction (GAP) models were trained using 56 QTL-based markers. Here, three candidate genes, MdNAC83, MdBPM2, and MdRGLG3, were screened from the regions of QTLs with large G' value and large genetic effects. Both a 216-bp deletion and an SNP934 T/C at the promoter of MdNAC83 were associated with higher MdNAC83 expression but an SNP388 G/A at the coding region significantly reduced the activity to activate the expression of the target genes MdACO1, MdMANA3, and MdXTH28. MdBPM2 and MdRGLG3 participated in the ubiquitination of MdNAC83. SNP657 T/A of MdBPM2 and SNP167 C/G of MdRGLG3 caused a reduction in the activity to ubiquitinate MdNAC83. By the addition of functional markers to the GenoBaits SNP array, the prediction accuracy of the updated GAP models increased to 0.7723/0.6231 and 0.5639/0.5345 for flesh firmness/crispness at harvest and flesh firmness/crispness retainability, respectively. The variation network involving eight simple Mendelian variations in six genes helps to gain insight into the molecular quantitative genetics, to improve breeding strategy, and to provide targets for future genome editing.

Environmental constraints such as drought and salt stress severely limit crop production worldwide. The WRKY TFs are very important in regulating plant growth and stress responses. Pineapple (Ananas comosus) is widely grown due to its unique flavor, high vitamins, and dietary fiber; however, the functional characterization of WRKY TFs in pineapple remains largely unexplored. In our study, we amplified and explored the molecular function of pineapple AcWRKY27, including its conserved domain, protein localization, transcriptional activity, and expression profiles in different tissues and stress treatments. Overexpression of AcWRKY27 in both rice and Arabidopsis thaliana (A. thaliana) resulted in growth inhibition, a decrease in primary root elongation, and a reduction in fresh weight under salt, drought, and ABA stress. RNA-Seq analysis and quantitative PCR (RT-qPCR) revealed that AcWRKY27 overexpression resulted in decreased expression levels of stress-responsive and ABA signaling pathway genes. These findings provide new perspectives on pineapple WRKY TFs and lay a foundation for improving pineapple stress tolerance through molecular breeding in the future.

Extreme heat driven by climate change poses a catastrophic threat to global vegetable production, undermining nutritional security because of the heightened physiological sensitivity and succulent tissues of these crops. This review synthesizes the multistage impacts of heat stress across critical developmental phases-from germination to reproduction-emphasizing morphological impairments (such as leaf wilting and floral abortion) and physiological disruptions (including photosynthetic inhibition and oxidative damage). We systematically dissect thermotolerance mechanisms in vegetables, highlighting transcriptional reprogramming by HSFs, WRKY, and NAC transcription factors; chaperone-mediated proteostasis via HSPs; epigenetic remodeling; Ca2+-ROS signaling pathways; and the role of phase separation dynamics. Importantly, we propose six strategic pathways to develop heat-resilient vegetables: harnessing natural variation through pan-genome-driven allele mining; employing biotechnological interventions such as CRISPR-mediated editing and synthetic promoters; engineering multistress tolerance by targeting conserved 'core response' pathways; exploiting epigenetic memory to achieve transgenerational resilience; optimizing source-sink dynamics with ''Climate-Responsive Carbon Optimization; and applying plant growth regulators and nanotechnology to enhance thermotolerance. Together, these strategies chart a clear roadmap for climate-smart vegetable breeding and call for interdisciplinary collaboration to translate molecular discoveries into practical breeding approaches for sustainable food systems under escalating thermal extremes.

Phosphorus in the soil is easily chelated into forms that are unavailable to plants, leading to phosphorus deficiency, which severely affects the growth, development, and fruit quality of apple trees. To address phosphorus deficiency, we used four different arbuscular mycorrhizal fungi (AMF) to investigate their effects on the growth and development of apple rootstocks and phosphorus uptake in the soil. We identified Glomus mosseae (Gm) fungi as the most effective AMF for promoting growth and found that under phosphorus-deficient conditions, inoculating with Gm fungi promoted the growth of the above-ground parts of the plants and phosphorus absorption, while it inhibited root growth. After inoculating with Gm fungi, we found phosphorus starvation response factors (PHRs) and auxin response factors (ARFs) were upregulated. Knockdown of MdPHR2 or MdARF6-4 resulted in decreased root arbuscular structures, total mycorrhizal colonization rate, and root phosphorus content, indicating that MdPHR2 and MdARF6-4 positively regulate the symbiosis of Gm fungi and phosphorus absorption. In contrast, overexpressing MdARF6-4 led to reduced root development but increased root phosphorus content under Gm fungi inoculation, suggesting that MdARF6-4 is involved in Gm-mediated phosphorus absorption and root development. Moreover, both MdPHR2 and MdARF6-4 directly bound to the promoter area of the downstream phosphorus transporter MdPHT1;13, and these two transcription factors interacted with each other in vivo and in vitro. In summary, our study demonstrates that the interaction between MdPHR2 and MdARF6-4 synergistically regulates the Gm symbiosis and the transcription of MdPHT1;13, thereby promoting phosphorus absorption in apple rootstocks.

With the ongoing rise in global temperatures, drought stress has become a significant threat to the normal growth and development of horticultural crops. Identifying the regulatory genes is the key to genetic improvement. Extensive research has highlighted the pivotal role of WRKY transcription factors in orchestrating plant responses to both biotic and abiotic stresses. However, their precise involvement in drought tolerance and the related molecular mechanisms have yet to be fully elucidated. In this study, we demonstrated that MdWRKY71 functioned as a positive regulator of drought tolerance in apple. Overexpressing MdWRKY71 in apple improved drought tolerance, while silencing it had the opposite effect. Additionally, under drought stress, compared with the control, chlorophyll fluorescence values, superoxide dismutase (SOD), and peroxidase levels were elevated in MdWRKY71-overexpressing apple and tobacco transgenic materials. Interaction analysis showed that MdWRKY71 directly binds to the W-box element of the MdFeSOD promoter and activates its transcription. We used yeast two-hybrid screening to identify potential interactors of MdWRKY71 and confirmed the interaction between MdWRKY71 and MdARF3 using Pull-down, bimolecular fluorescence complementation, and luciferase complementation imaging assays. Interestingly, MdARF3 enhanced MdWRKY71-mediated transcriptional activation of MdFeSOD through their interaction. In summary, our findings revealed that the MdWRKY71-MdARF3 module synergistically upregulates the expression of MdFeSOD and SOD enzyme activity in response to drought stress. This research uncovers a new mechanism of plant drought tolerance and presents a feasible strategy to enhance plant drought tolerance through stabilizing the biosynthesis of superoxide dismutase.

Phosphorus (P) mobilization via root-secreted organic acids, particularly citrate through metal chelation and proton-mediated acidification, is a key rhizosphere adaptation to P deficiency. Multidrug and Toxic Compound Extrusion (MATE) transporters emerge as critical regulators of citrate efflux. However, their roles in perennial fruit crops remain under explored. Here, we demonstrate root-specific induction of MdMATE3 in apple under low-P conditions. Overexpressing MdMATE3 enhanced citrate secretion and phosphorus uptake in both tobacco and apple systems. Transcriptomic and molecular analyses revealed that the low-P responsive transcription factor MdMYB1R directly activates MdMATE3 by binding to the conserved TTATC motif of the promoter. This regulatory cascade enhances rhizosphere P availability through citrate-mediated solubilization and chelation, ultimately improving P uptake. This study elucidates MdMATE3 pivotal role in low-P tolerance and provides molecular targets for developing P-efficient apple rootstocks.

This article is a Commentary on Liu et al . (2025), 246 : 2155–2173 .

In grafted combinations, the scion cultivar, representing the aboveground portion, possesses the potential to influence the rhizosphere bacterial community. However, the precise mechanisms by how the scion cultivar contributes to the recruitment and assembly of rhizosphere bacteria remain poorly understood. In this study, we conducted a comprehensive analysis of the root transcriptome and metabolomics coupled with the amplification of the rhizosphere bacterial 16S rRNA gene. We found that scion cultivars with different net photosynthesis rates significantly impacted root metabolites and the expression of root sugar metabolism genes. Moreover, rhizosphere-specific taxa (Rhizobium, Nitrospira and Ensifer) are associated with root sugar metabolites, particularly sucrose. The foliar application of 2% sucrose on leaves enhanced root sugar metabolism and shaped the rhizosphere microbiota, notably promoting the growth and colonization of the bacterial isolation in the rhizosphere of apple plantlets. This study has significant implications for future studies on plant-microbe interactions in grafted combinations.

During the long history of domestication and improvement of cultivated plants from their wild progenitors, unfavorable alleles of important traits, such as the small-fruit trait of wild apple (Malus) species, have undergone gradual negative selection. Utilizing wild species as breeding materials for elite lines requires removing these undesirable alleles, making it urgent to identify these allelic variations. We previously identified 90 quantitative trait loci (QTLs) for apple fruit weight via bulked segregant analysis-seq. In this study, we identified 37 genome regions associated with apple fruit weight by genome-wide association study (GWAS) using 253 Malus accessions. We then developed 147 GenoBaits markers within the QTL intervals or association regions, of which nine were major-effect markers for small-fruit traits and exhibited complementary epistasis. Nineteen candidate genes were predicted within the nine major-effect loci. Overexpressing three of these genes, MdPMEI51, MdTIP1-1, and MdARF9, inhibited cell proliferation in transgenic apple calli, and allelic variants in their coding sequences enhanced these inhibitory effects. We replaced the nearby linkage marker with MdTIP1-1 SNP700 A/G (Chr12_16 957 078 T/C, on the antisense strand) and identified seven markers as major-effect markers for the large-fruit trait. We added the joint effects of the nine and seven major-effect markers for the small- and large-fruit phenotypes, respectively, to genomics-assisted prediction (GAP) models as fixed effects. The prediction accuracy of the non-additive GAP model was 0.8436. The frequency of the small-fruit associated alleles of the nine major-effect markers is much higher in wild Malus species than in cultivated and semi-cultivated species, indicating that the alleles associated with the small-fruit trait underwent strong negative selection during domestication. These findings shed light on the genetic mechanism underlying the small-fruit trait in apple and could facilitate apple breeding.

Parapristipoma trilineatum is an edible and economically important marine fish with high farming potential; however, this fish has not been widely investigated. To understand the germplasm resources of this marine fish, we investigated the range of P. trilineatum from Maizuru, Japan, to Hainan, China. Using the mitochondrial control region as a molecular marker, we analyzed the population structure, genetic diversity, and population history dynamics of P. trilineatum. The control region exhibited high haplotype diversity and nucleotide diversity of 0.996 and 0.016, respectively. The genetic differentiation coefficients indicated low differentiation between Japanese and other populations. Additionally, based on the results of SAMOVA, AMOVA, and genetic differentiation coefficients, P. trilineatum can be divided into two management units in fishery resources management: China and Japan. Populations with high genetic diversity are useful as genetic breeding materials. This study provides a basis for the selection and breeding of P. trilineatum.

Anthocyanin generation in apples (Malus domestica) and the pigmentation that results from it may be caused by irradiation and through administration of methyl jasmonate (MeJA). However, their regulatory interrelationships associated with fruit coloration are not well defined. To determine whether MdERF109, a transcription factor (TF) involved in light-mediated coloration and anthocyanin biosynthesis, has synergistic effects with other proteins, we performed a yeast two-hybrid assessment and identified another TF, MdWER. MdWER was induced by MeJA treatment, and although overexpression of MdWER alone did not promote anthocyanin accumulation co-overexpression with MdERF109 resulted in significantly increase in anthocyanin biosynthesis. MdWER may form a protein complex with MdERF109 to promote anthocyanin accumulation by enhancing combinations between the proteins and their corresponding genes. In addition, MdWER, as a MeJA responsive protein, interacts with the anthocyanin repressor MdJAZ2. Transient co-expression in apple fruit and protein interaction assays allowed us to conclude that MdERF109 and MdJAZ2 interact with MdWER and take part in the production of anthocyanins upon MeJA treatment and irradiation. Our findings validate a role for the MdERF109-MdWER-MdJAZ2 module in anthocyanin biosynthesis and uncover a novel mechanism for how light and MeJA signals are coordinated anthocyanin biosynthesis in apple fruit.

Nitrogen is an essential nutrient for plant growth and serves as a signaling molecule to regulate gene expression inducing physiological, growth and developmental responses. An excess or deficiency of nitrogen may have adverse effects on plants. Studying nitrogen uptake will help us understand the molecular mechanisms of utilization for targeted molecular breeding. Here, we identified and functionally validated an NAC (NAM-ATAF1/2-CUC2) transcription factor based on the transcriptomes of two apple rootstocks with different nitrogen uptake efficiency. NAC1, a target gene of miR164, directly regulates the expression of the high-affinity nitrate transporter (MhNRT2.4) and citric acid transporter (MhMATE), affecting root nitrogen uptake. To examine the role of MhNAC1 in nitrogen uptake, we produced transgenic lines that overexpressed or silenced MhNAC1. Silencing MhNAC1 promoted nitrogen uptake and citric acid secretion in roots, and enhanced plant tolerance to low nitrogen conditions, while overexpression of MhNAC1 or silencing miR164 had the opposite effect. This study not only revealed the role of the miR164-MhNAC1 module in nitrogen uptake in apple rootstocks but also confirmed that citric acid secretion in roots affected nitrogen uptake, which provides a research basis for efficient nitrogen utilization and molecular breeding in apple.