Drought poses a significant global challenge to agriculture, substantially reducing crop yields. Abscisic acid (ABA) plays a crucial role in response to drought stress. Nevertheless, the molecular mechanism underlying the ABA-mediated drought stress response in apple remains poorly understood. We identified a drought- and ABA-induced AP2/ERF transcription factor (TF), MhSHINE2-like, which positively regulates drought stress tolerance in apple. Biochemical analysis showed that MhSHINE2-like directly binds to the GAGA-rich element in the promoter of the ABA biosynthesis gene MhNCED3, promoting its transcription under drought stress. Overexpression of MhNCED3 promotes ABA accumulation and enhances apple drought tolerance by regulating stomatal closure under drought stress. Further studies revealed that MhSHINE2-like physically interacts with 14-3-3 protein, MhGRF3, which also contributes positively to drought tolerance. Notably, MhSHINE2-like and MhGRF3 function cooperatively to modulate the expression of downstream genes, promoting ABA accumulation, and consequently enhancing drought tolerance in apple. These findings reveal a regulatory network mediated by the combined effects of TFs and chaperone proteins, offering valuable genetic resources for the development of drought-tolerant apple cultivars.

Drought poses a severe threat to the cultivation, yield, and quality of apple, which is an economically important fruit crop. DnaJ proteins, as members of the heat shock protein (HSP) family, play a crucial role in plant responses to abiotic stress. However, the functional characterization of apple DnaJ family genes in conferring drought tolerance remains largely unexplored. In this study, we have performed a genome-wide analysis for DnaJ genes in apple and a candidate gene functional role in drought tolerance was subsequently validated through genetic and physiological assays. A total of 111 DnaJ genes were identified in the apple genome and are distributed across 17 chromosomes. Gene structure analysis revealed notable variations in motifs and intron patterns among different DnaJ gene members in apple. Multiple stress-responsive cis-acting elements were identified within the 2kb promoter region upstream of the MdDnaJ genes. RNA-seq analysis revealed that the expression levels of most MdDnaJ genes were induced by drought and salt stress conditions. Subsequent RT-qPCR analysis demonstrated that MdDnaJ107 was significantly upregulated under drought stress. Overexpression of MdDnaJ107 enhanced drought tolerance in transgenic apple calli, as evidenced by reduced levels of electrolyte leakage, malondialdehyde (MDA), superoxide anion (O2−), and hydrogen peroxide (H2O2). In contrast, silencing MdDnaJ107 resulted in the opposite results. In addition, overexpression of MdDnaJ107 improved drought tolerance in apple plants, accompanied by enhanced expression of the stress-related genes. This study revealed that MdDnaJ107 plays a crucial role in conferring drought tolerance, suggesting its potential as a promising target gene for approaches aimed at enhancing plant tolerance to drought stress.

Protein lysine acetylation regulates diverse biological functions. This study investigated the effect of histone deacetylase inhibitor (HDACi) treatment on apple fruit quality. HDACi treatment markedly reprogrammed gene expression, upregulating pathways for cutin, suberin, and wax biosynthesis. In "Red Delicious", treatment altered aroma profiles, increasing alcohols and aldehydes while decreasing esters. Notably, HDACi enhanced peel wax content and strongly induced MdMYB96. Functional analyses revealed that MdMYB96 activates multiple wax biosynthetic genes; its silencing reduced the wax load, while overexpression in Arabidopsis increased wax accumulation and reduced water loss. Furthermore, across eight apple cultivars, denser epicuticular wax structures correlated with higher wax gene expression and better postharvest water retention. These findings demonstrate that inhibiting histone deacetylase reshapes the balance between wax metabolism and aroma production with MdMYB96 serving as a key regulator. Thus, manipulating histone acetylation offers a promising strategy to improve apple wax biosynthesis and modify volatile profiles.

The phytohormone abscisic acid (ABA) plays a pivotal role in regulating the ripening processes of both climacteric and non-climacteric fruits, including apple (Malus domestica). However, the underlying regulatory mechanisms remain elusive. In this study, exogenous ABA treatment markedly enhanced ethylene production, decreased fruit firmness, and accelerated ripening in 'Red Delicious' apples. Intriguingly, exogenous ethylene increased endogenous ABA levels and biosynthetic enzyme activities in 'Golden Delicious' fruit, whereas 1-MCP treatment suppressed ABA accumulation and delayed ripening. Subsequently, we identified two ABA-responsive transcription factors, MdERF2 and MdSCR11, that directly trans-repress the promoter of MdABA4-2, which shows a negative correlation with ABA accumulation. Notably, MdSCR11 physically interacts with MdERF2 and synergistically represses MdABA4-2 transcriptional activity. Furthermore, transient overexpression of MdABA4-2 in apple fruit significantly reduced ABA content and ethylene production, delaying ripening, whereas MdSCR11 overexpression suppressed MdABA4-2 expression and accelerated ripening. Finally, complementary evidence from transgenic calli confirmed that MdABA4-2 negatively regulates ABA biosynthesis while MdSCR11 positively modulates ABA accumulation through MdABA4-2 suppression. Our findings establish a novel transcriptional regulatory module wherein ABA promotes apple fruit ripening through MdERF2-MdSCR11-mediated repression of MdABA4-2, providing mechanistic insights into hormone crosstalk during climacteric fruit maturation.

Expansins are crucial cell wall-loosening proteins that play a vital role in various plant developmental processes, including fruit ripening and softening. However, a comprehensive genome-wide analysis of the expansin family in apple (Malus × domestica) and the specific functions of its members in postharvest fruit ripening remain to be explored. In this study, we identified 51 expansin genes in the apple genome and classified them into four subfamilies (EXPA, EXPB, EXLA, and EXLB). Cis-element analysis of the promoters of apple expansin genes showed that these promoters are rich in various hormone-responsive elements, including abscisic acid (ABA)-responsive elements (ABREs) and ethylene-responsive elements (EREs), suggesting potential hormonal regulation of expansin genes. Expression profiling identified six ripening-associated expansin genes. Among them, MdEXPA5, MdEXPA17, and MdEXPA23 were positively regulated by both ethylene and ABA, while being suppressed by the ethylene action inhibitor 1-MCP. Further functional characterization demonstrated that transient overexpression of MdEXPA17 accelerated fruit softening, skin yellowing, ethylene production, and increased total soluble solid (TSS) content. Conversely, silencing of MdEXPA17 significantly delayed these ripening processes. Our study provides a systematic overview of the apple expansin gene family and supports a role for MdEXPA17 in promoting postharvest fruit ripening and softening. These findings offer valuable insights into the molecular mechanisms of apple fruit ripening and provide potential targets for genetic improvement of fruit quality and shelf life.

MdNF-YB14 plays a critical role in enhancing drought stress tolerance by activating ROS- scavenging and stress-responsive genes. The nuclear factor Y (NF-Y) plays critical roles in maintaining plant growth, development, and stress tolerance. However, systematic research on the NF-Y gene family in apple remains insufficient. Here, a total of 47 MdNF-Y genes were identified in the apple genome, comprising 11 MdNF-YAs, 25 MdNF-YBs, and 11 MdNF-YCs. Analysis of cis-elements revealed that 30 out of 47 apple NF-Y gene promoters harbored one or more drought-responsive cis-elements, suggesting the potential involvement of NF-Y genes in plant drought tolerance. Among the 39 differentially expressed genes, 14 NF-Y genes were highly expressed in response to drought based on RNA-seq data. Notably, MdNF-YB14 showed significantly higher expression after 4 days of drought stress as confirmed by qRT-PCR analysis. Heterologous overexpression of MdNF-YB14 in transgenic Arabidopsis plants resulted in a higher germination rate, fresh weight, and root length under PEG treatment when compared with the wild-type plants. Similarly, overexpression of MdNF-YB14 positively enhanced drought tolerance in apple, which was associated with improved growth due to reduced electrolyte leakage, malondialdehyde (MDA), hydrogen peroxide (H2O2), and superoxide radicals (O2–). Furthermore, the transcript levels of genes related to reactive oxygen species (ROS) scavenging and stress response were also significantly up-regulated in MdNF-YB14 transgenic apple plants. Collectively, MdNF-YB14 plays a crucial role in enhancing drought tolerance and can serve as a promising candidate gene for improving plant drought tolerance through biotechnological approaches.

Drought is a major environmental stress that severely affects plant growth and poses a serious threat to crop productivity. Abscisic acid is a key plant hormone that plays a critical role in regulating plant responses to drought stress. However, the regulatory pathways controlling abscisic acid for drought tolerance in apple (Malus domestica Borkh) remain largely unclear. This study identified an NF-Y transcription factor, MhNF-YC6, from the drought-tolerant wild apple species (Malus hupehensis). Functional analysis revealed that MhNF-YC6 is a positive regulator of apple drought tolerance. Further research demonstrated that MhNF-YC6 directly binds to the promoters of the ABA biosynthesis genes MhNCED3 and MhAAO3 and activates their transcription. Silencing of MhNCED3 and MhAAO3 resulted in significantly reduced abscisic acid contents and drought tolerance. Furthermore, CONSTANS-like 4 (COL4) is identified as an MhNF-YC6-interacting protein that acts as a positive regulator of drought tolerance in apple. Notably, we discovered that the interaction between MhNF-YC6 and MhCOL4 substantially enhances the regulatory effect of MhNF-YC6 on MhNCED3 and MhAAO3, thereby promoting abscisic acid accumulation, inducing stomatal closure, and ultimately improving drought tolerance. Our findings offer valuable insights into the transcriptional regulation of abscisic acid biosynthesis and provide promising targets for the molecular breeding of drought-tolerant crops.

Drought stress severely impacts the growth, yield and quality of apple (Malus domestica). Abscisic acid (ABA) and basic helix-loop-helix (bHLH) transcription factors play crucial roles in regulating the drought response in many plants, but the potential interactions between bHLH and ABA in response to drought in apple still need to be discovered. Herein, we identified a bHLH transcription factor, ORG2 (OBP3-responsive gene 2), from M. hupehensis, and the expression of which is induced by drought and ABA. Apple plants that overexpressed MhORG2 were more sensitive to drought stress, while silencing MhORG2 caused the opposite phenotype. Specifically, we found that MhORG2 could directly bind to the DRE element in the MhAAO3 promoter and repress its expression, thereby ultimately reducing drought tolerance. Furthermore, MhORG2 represses the expression of antioxidant enzyme genes (MhSOD, MhAPX1 and MhCAT), leading to the accumulation of reactive oxygen species (ROS) and consequently reducing the drought tolerance of apple plants. Our findings uncover a novel mechanism by which MhORG2 negatively regulates drought tolerance in apple plants, offering a potential target for the development of drought-tolerant crops via biotechnological approaches.

Ascorbate oxidases (AAOs) are key regulators of extracellular redox homeostasis and plant stress responses, but their roles in grapevine defense remain unclear. Here, we performed a genome-wide analysis and characterization of the AAO gene family in grapevine Vitis amurensis, identifying 10 VaAAO genes that are unevenly distributed across six chromosomes, with notable clustering on chromosome 7. Promoter analysis revealed multiple phytohormone- and stress-responsive cis-elements (e.g., ARE, STRE, and TCA-element) and transcription factor binding sites (e.g., MYC/MYB, and WRKY), suggesting involvement in redox- and stress-related signaling pathways. Analysis of previously published transcriptomic data under Botrytis cinerea infection identified VaAAO7 as a key pathogen-responsive gene. VaAAO7 was rapidly induced by H2O2, and its transient ectopic overexpression in susceptible V. vinifera ‘Red Globe’ leaves significantly reduced lesion development. Together, these results demonstrate that VaAAO7 functions as a positive regulator of B. cinerea resistance and highlight its potential for genetic engineering to enhance systemic defense and develop disease-resistant grapevine cultivars.

Adventitious root (AR) formation is the key to asexual reproduction of horticultural crops. Auxin response factor (ARF) plays key role in mediating auxin signaling during AR formation and growth. However, the roles of apple ARF family genes in AR formation and growth remains limited. In this study, 29 MdARF genes were identified and were found to be unevenly distributed across 16 apple chromosomes. Gene structure and protein motif analysis revealed that the MdARF genes within the same subgroup exhibit similar intron phase patterns and motif compositions. The expression level of six of 29 MdARF genes was differentially regulated by indole-3-butyric acid (IBA). Among these genes, MdARF18-like was significantly upregulated in response to IBA treatment. Subcellular localization indicated that MdARF18-like was localized in the nucleus and cytoplasm. Overexpression of MdARF18-like promoted the elongation of apple AR and significantly increased AR biomass. Furthermore, the expression levels of genes associated with lateral root formation (MdLBD16, MdLBD29, and MdARF4), cell cycle regulation (MdCYCD3.2), and xylan biosynthesis (MdTCH4-1 and MdTCH4-2) were significantly up-regulated in MdARF18-like-OE apple plants compared to those in empty vector (EV) apple plants. Taken together, our findings indicate that MdARF18-like promotes the elongation of apple AR by positively regulating the expression of genes involved in AR formation and growth.

[Objective] Microtubule is composed of α-and β-tubulin heterodimers. It is known that α- and β-tubulins are encoded with large family genes and selected expression of α-and β-tubulin family genes plays key roles in regulating various biological processes, including plant growth and development, stress responses and signaling transduction. However, little is known about apple tubulin family genes and their potential functions. Therefore, the present study was to identify the apple β-tubulin family genes and to determine the candidate β-tubulin genes involved in regulating apple dwarfing. [Methods] Using Arabidopsis thaliana β-tubulin amino acid sequences as reference, apple β-tubulin family genes were identified from the apple reference genome GDDH13 v1.1. In the following, bioinformatic analysis was conducted to dissect the physicochemical properties, chromosome localization, phylogenetic relationships, gene structure, conserved motifs, collinearity, three-dimensional structure prediction and cis-acting elements in their promoter regions of the identified apple β-tubulin family genes. Different tissues including mature and young leaves, xylem, phloem and stem tips of column apple Runtai No.1 were collected as materials to analyze the tissue expression patterns of apple β-tubulin family genes through quantitative real-time PCR (RT-qPCR). To screen the candidate β-tubulin genes involved in regulating apple dwarfing, the relative expressions of apple β-tubulin family genes in the shoot apex of three apple dwarfing rootstocks (T337, Pamajul and JM7) and three common rootstocks (Malus prunifolia, M. micromalus and M. hupehensis) were compared by RT-qPCR. [Results] A total of 13 apple β-tubulin sequences were identified from the apple genome. The lengths of these 13 apple β-tubulins were from 444 aa to 450 aa, and the molecular weights ranged from 49.92 ku to 50.46 ku. The 13 apple β-tubulin genes were randomly distributed on 11 apple chromosomes, and fragment replication events were the main factor attributed to the expansion of apple β-tubulin family genes. Furthermore, phylogenetic tree analysis revealed that apple β-tubulin family can be divided into 5 subfamilies: ClassⅠhad 6 members with nomenclature of MdTUB1 to MdTUB6, and MdTUB10 and MdTUB11 were classified into ClassⅡ. Except for ClassⅢhaving only one member of MdTUB9, ClassⅣand ClassⅤboth had 2 members with nomenclatures of MdTUB7 and MdTUB8, MdTUB12 and MdTUB13 separately. Multiple sequence alignments showed that the amino acid sequences of the apple β-tubulins were highly conserved at the N-terminus, but only displayed slight differences at the C-terminus, which was also corroborated by three-dimensional structure prediction of apple β-tubulin family proteins, suggesting that the C-terminus differences might correlate with posttranslational modifications. Gene structure analysis showed that all 13 apple β-tubulin family genes contained 3 exons and 2 introns, and the amino acid sequences contained 10 conserved motifs, indicating that the functions of apple β-tubulin family members were probably highly conserved. The cis-acting element analysis of the promoter region showed that the abscisic acid responsive element, the methyl jasmonate responsive element and the anaerobic induction element were intensively distributed in the promoters of apple β-tubulin family genes. Furthermore, apple β-tubulin family genes displayed certain tissue expression specificities in the column apple Runtai No.1 variety, for instance, MdTUB2, MdTUB6, MdTUB9, MdTUB10 and Md-TUB11 were highly expressed in the phloem and MdTUB3 and MdTUB5 were mainly expressed in the young leaves. The relative expression of MdTUB1, MdTUB4 and MdTUB12 in xylems, young leaves and stem tips were significantly higher than in mature leaves and phloem. MdTUB8 was widely expressed in the detected tissues except for mature leaves, and MdTUB7 was highly expressed in young leaves and stem tips. In addition, the expression of MdTUB13 in stem tips were significantly higher than that in other detected tissues. This suggested that different tissues may selectively express different apple β-tubulin family genes to facilitate their growth and development. Furthermore, we found that the expression of MdTUB4 in the three common rootstocks (M. prunifolia, M. micromalus and M. hupehensis) was significantly higher than that in dwarfing rootstocks (T337, Pamajul and JM7), suggesting that MdTUB4 may positively regulate apple dwarfing. However, the expression pattern of MdTUB12 displayed opposite so that the relative expression of MdTUB12 was significantly higher in dwarfing rootstocks, suggesting MdTUB12 might negatively participate in regulating apple dwarfing. Apart from the MdTUB4 and MdTUB12, similar expression patterns were not observed with other apple β-tubulin family genes. These results indicated that MdTUB4 and MdTUB12 might be the candidate β-tubulin dwarfing genes involved in regulating apple dwarfing. [Conclusion] A total of 13 β-tubulin family genes were identified from the whole apple genome. Different apple tissues may selectively express specific β-tubulin genes to facilitate its growth and development. Furthermore, the selective expression of β-tubulin family genes is highly correlated with apple dwarfing and MdTUB4 and MdTUB12 might be the key genes involved in regulating apple dwarfing. The results will provide a theoretical basis for further research on the regulatory functions of apple dwarfing by β-tubulin genes.

Apple replant disease is a complex soil syndrome that occurs when the same fields are repeatedly utilized for apple orchard cultivation. It can be caused by various pathogens, and Fusarium solani is the main pathogen. Fusarium solani disrupts the structure and function of the orchard soil ecosystem and inhibits the growth and development of apple trees, significantly impacting the quality and yield of apples. In this study, we conducted a transcriptome comparison between uninoculated apple saplings and those inoculated with F. solani. The differentially expressed genes were mainly enriched in processes such as response to symbiotic fungus. Plant defensins are antimicrobial peptides, but their roles during F. solani infection remain unclear. We performed a genome-wide identification of apple defensin genes and identified 25 genes with the conserved motif of eight cysteine residues. In wild-type apple rootstock inoculated with F. solani, the root surface cells experienced severe damage, and showed significant differences in the total root length, total root projection area, root tips, root forks, and total root surface area compared to the control group. qRT-PCR analysis revealed that MdDEF3 and MdDEF25 were triggered in response to F. solani infection in apples. Subcellular localization showed specific expression of the MdDEF3-YFP and MdDEF25-YFP proteins on the cell membrane. Overexpressing the MdDEF25-YFP fusion gene enhanced resistance against F. solani in apple, providing a new strategy for the future prevention and biological control of apple replant disease.

Shoot branching in apple (Malus domestica) is primarily affected by the activation and outgrowth of axillary buds, which are regulated by a complex interplay of physiological and genetic factors. To gain deeper insight into the underlying regulatory mechanisms, we compared the physiological characteristics and transcriptomic profiles of two apple cultivars with different branching behaviours: 'Venus Gold' (VG, easy-to-branch) and 'Huashuo' (HS, hard-to-branch). Physiological assays revealed higher sucrose and cytokinin levels in 'VG', while 'HS' had higher abscisic acid, jasmonic acid, and strigolactones, which generally inhibit axillary bud outgrowth. Transcriptomic analysis identified 3022 differentially expressed genes, with 'VG' showing upregulation of pathways related to sugar metabolism, cytokinesis, and cell wall biogenesis, and 'HS' enriched in genes involved in meristem maintenance and stress responses. Our findings provide insights into the physiological and genetic regulation of shoot branching in apple and offer valuable targets for breeding programs of shoot architecture optimization.

Plant seed number depends on ovule number initiated within the carpels, and it serves as a primary factor shaping fruit yield. Pomegranate trees exhibit bisexual flowers and functional male flowers. Pomegranate have anatropous ovules which are bitegmic and crassinucellate. Bisexual flowers possess the fertile pistil, while functional male flowers have abnormally developed ovules, a small ovary with few chambers, and a short style. The formation of functional male flowers is due to abnormal and stagnant development of ovule integument. Ovule number directly determines the yield of pomegranate seeds. Recent studies have highlighted the molecular mechanisms through which ovule-related genes regulate pomegranate ovule development. Pomegranate PgCRC and PgINO genes positively regulate the increase in the number of ovules, and PgBEL1 to synergistically regulate seed development. PgAGL11 (the SEEDSTICK orthologous gene) promotes ovule development in transgenic Arabidopsis. PgSEP protein can bridge interactions among PgBEL1, PgSTK and PgAG, which regulate ovule development. At the level of post-transcriptional regulation, PgmiRNA167, PgmiRNA164 and PgmiRNA160 are differentially expressed during pomegranate flower development, and PgmiR166a interacts with its target genes to affect ovule development. This review summarizes the key regulators of ovule development and their molecular pathways, integrating these interactions into a model that describes pomegranate ovule development.

Drought is a major abiotic stress in agriculture that severely affects crop growth, yield, and quality. The APETALA2/ethylene responsive factor (AP2/ERF) plays a crucial role in maintaining plant growth, development, as well as stress tolerance. Herein, we cloned and characterized the MhERF113-like gene from Malus hupehensis. MhERF113-like is significantly induced by drought and highly expressed in leaves. Overexpression of MhERF113-like positively regulated the drought tolerance of apple calli and plants, as judged by less electrolyte leakage, lower malonaldehyde (MDA) and hydrogen peroxide (H2O2) contents in OE than those of the WT apple calli and plants under drought stress. In addition, ectopic expression of MhERF113-like gene in tomatoes improved the drought tolerance, accompanied by enhanced expression of antioxidant genes (SlAPX1 and SlSOD) and stress responsive genes (SlDREB and SlRD29), and reduced H2O2 and O2- contents in OE tomatoes. Taken together, our study demonstrated that MhERF113-like may play an important role in the regulation of plant drought tolerance, which may provide a key factor for future biotechnology applications to improve drought stress tolerance in plants.

Heat shock protein 70 (HSP70) is a class of important molecular chaperones that are involved in protein folding, stabilization, and maturation, and play a vital role in plant growth and response to environmental stress. Apple trees frequently suffer from different-degree salt stress, which seriously affects their growth, quality, and yield. However, whether HSP70 genes are involved in salt tolerance is unexplored in apple. In this study, 67 MdHSP70 genes were identified and unevenly distributed on 17 apple chromosomes. Gene structure and protein motif analysis revealed that MdHSP70 genes in the same subgroup have similar intron phase and motif organization, further supporting the phylogenetic results. RNA-seq analysis showed the expression level of nine of 67 MdHSP70 genes was induced by salt stress. Subsequent qRT-PCR analysis revealed that MdHSP70-38 was dramatically up-regulated in response to salt stress. The overexpression of MdHSP70-38 in transgenic tobacco and apple improved salt stress tolerance, which was associated with less electrolyte leakage and malondialdehyde (MDA), as well as diminished accumulation of hydrogen peroxide (H2O2) and superoxide radicals (O2-). Our findings demonstrated that MdHSP70-38 played a positive regulatory role in salt tolerance in tobacco and apple, and provided a promising candidate gene in genetic applications for improving salt tolerance.

Drought significantly impairs the growth, yield, and quality of apple (Malus domestica Borkh.). Nuclear factor-Y (NF-Y) transcription factors have well-documented functions in regulating stress tolerance in various plants. However, the specific functions and molecular mechanisms of NF-Ys in conferring drought tolerance in apples remain largely unexplored. Here, we identified and characterized a NF-Y transcription factor, MhNF-YA3-like, from Malus hupehensis, the expression of which is upregulated by drought stress and abscisic acid (ABA). Overexpressing MhNF-YA3-like enhanced tolerance to drought stress, while silencing MhNF-YA3-like increased susceptibility to drought stress. Further research demonstrated that MhNF-YA3-like directly binds to the promoter of MhAAO3 and activates its transcription. Notably, the interaction between MhNF-YA3-like and MhMSI4-like further amplified the regulatory effect of MhNF-YA3-like on MhAAO3, promoting the synthesis of ABA, which consequently improved drought stress tolerance. This study provides insights into how the MhNF-YA3-like-MhMSI4-like module finely tunes apple drought tolerance.

Graphene oxide (GO) is a versatile graphene derivative with excellent physicochemical properties and is extensively utilized across diverse fields. In this study, we explored the effect of GO on the regeneration and elongation of adventitious roots (AR) in apple rootstock M9-T337. The results showed that GO within the 0.1–1.0 mg/L concentration range promoted AR formation but inhibited root elongation. GO combined with indole-3-butyric acid (IBA) increased the rooting rate and the number of lateral roots of apple rootstock. The application of GO in conjunction with IBA treatments significantly enhanced the rooting rate and promoted the development of lateral roots in apple rootstock. Compared with the IBA treatment, the IBA + GO treatment resulted in a higher production of ethylene and enhanced the expression of ethylene synthesis genes MdACO1 and MdACS2, as well as the ethylene signal transduction gene MdEIN3. Both the IBA + GO treatment and the IBA + GO + silver nitrate (AgNO3) treatment significantly enhanced the capacity of root triphenyltetrazolium chloride (TTC) reduction. The IBA + GO treatment exhibited a significantly lower level of H2O2 compared to the other treatments, while exhibiting elevated levels of O2•– relative to other treatments. GO alters the expression of auxin transport genes. The IBA + GO treatment significantly increased the expression of the AR regeneration genes MdLBD29 and MdWOX11, as well as the cell cycle genes CYCD3;1 and CYCD3;2. These results indicate that GO and its interaction with auxin regulate AR formation and development in apple rootstock.

Drought, the most unfavorable and widespread environmental factor among abiotic stresses, has become a significant global issue that severely crop growth and yield. Nuclear factor-Y (NF-Y) transcription factors have well-documented functions in regulating stress tolerance in plants. However, the specific functions and molecular mechanisms of NF-YS in conferring drought tolerance in apples remain largely unexplored. Here, we reported that the NF-Y transcription factor MhNF-YA1-like positively modulates drought stress tolerance in apple (Malus domestica Borkh.). MhNF-YA1-like is transcriptionally upregulated in response to drought and abscisic acid (ABA) treatments. Overexpression of MhNF-YA1-like enhances drought tolerance in apples, whereas its silencing results in weakened drought tolerance. Further investigation reveals that MhNF-YA1-like directly binds to the promoter of MhAAO3 and activates its expression. Additionally, we identified MhDnaJC76 as an interacting protein of MhNF-YA1, and MhDnaJC76 positively modulates apple drought tolerance. Furthermore, the interaction between MhNF-YA1-like and MhDnaJC76 significantly amplifies the regulatory effect of MhNF-YA1-like on MhAAO3, thereby promoting ABA accumulation and consequently enhancing apple drought tolerance. Taken together, our findings elucidate a molecular framework by which the MhNF-YA1-like-MhDnaJC76 complex modulates drought tolerance in apples, thereby providing targets for developing drought-tolerant crops.

The plant-specific LIGHT-SENSITIVE SHORT HYPOCOTYL (LSH) genes are integral to the development of plant organs. However, the evolution and function of LSH genes in Rosaceae remain poorly understood. Here, 60 LSH genes were systematically identified across the genomes of six Rosaceae species. Phylogenetic analysis grouped these LSH genes into four distinct clades. The analysis of LSH gene family duplications revealed that whole-genome duplication (WGD) and transposed duplication (TRD) primarily drive the expansion of the LSH gene family in Rosaceae species. In apple, 18 MdLSH gene pairs generated through WGD have undergone purifying selection. Further transcriptome analysis demonstrated that MdLSH genes exhibit distinct expression patterns across different tissues. Among them, MdLSH10b was significantly induced at the induction stage of adventitious roots. The integration of transcriptome and promoter analyses indicated that MdLSH10b potentially regulates the expression of MdWOX4a. Yeast one-hybrid assays (Y1H) and β-glucuronidase (GUS) activity analyses demonstrated that MdLSH10b transcriptionally activates MdWOX4a. Notably, transient overexpression of MdLSH10b in apple resulted in an increased rate of adventitious root induction. In conclusion, this study suggests that MdLSH10b may promote adventitious root formation by transcriptionally activating MdWOX4a and provides new genetic resources for further functional investigations on MdLSH genes in apple development.