Cytokinin-responsive MdTCP17 interacts with MdWOX11 to repress adventitious root primordium formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and coexpression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple.

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX-LOOP-HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.

PpHY5 is involved in anthocyanin coloration in the peach flesh surrounding the stone.

Red coloration around the stone (Cs) is an important trait of canned peaches (Prunus persica). In this study, an elongated hypocotyl 5 gene in peach termed PpHY5 was identified to participate in the regulation of the Cs trait. The E3 ubiquitin ligase PpCOP1 was expressed in the flesh around the stone and could interact with PpHY5. Although HY5 is known to be degraded by COP1 in darkness, the PpHY5 gene was activated in the flesh tissue surrounding the stone at the ripening stages and its expression was consistent with anthocyanin accumulation. PpHY5 was able to promote the transcription of PpMYB10.1 through interacting with its partner PpBBX10. Silencing of PpHY5 in the flesh around the stone caused a reduction in anthocyanin pigmentation, while transient overexpression of PpHY5 and PpBBX10 resulted in anthocyanin accumulation in peach fruits. Moreover, transgenic Arabidopsis seedlings overexpressing PpHY5 showed increased anthocyanin accumulation in leaves. Our results improve our understanding of the mechanisms of anthocyanin coloration in plants.

Structured Psychological Nursing Combined with Group Health Education for Patients Receiving Blood Purification.

Context: Patients with ESRD undergoing blood purification still face various challenges, including psychological distress, lack of comprehensive understanding of their disease, and suboptimal health outcomes. Effective interventions for such patients are of paramount significance. Objective: The study intended to assess the benefits of structured psychological nursing combined with group health education for patients undergoing blood purification. Design: The research team conducted a randomized controlled trial. Setting: The study took place at Zhejiang Provincial People's Hospital in Xianyang, China. Participants: Participants were 96 patients at the hospital who were undergoing blood purification between May 2020 and March 2022. Interventions: The research team randomly assigned participants to one of two groups: (1) the study group, who received structured psychological nursing + group health education, and (2) the control group, who received routine nursing. Outcome Measures: At baseline and postintervention, the research team evaluated: (1) disease cognition, using the Mishel Uncertainty in Illness Scale (MUIS-A); (2) negative emotions, using the Self-rating Depression Scale (SDS) and Self-rating Anxiety Scale (SAS); (3) blood purification adequacy rate and nutritional status qualified rate; and (4) complication rate. Results: Postintervention, the intervention group's: (1) disease uncertainty, complexity, lack of disease information, and unpredictability were significantly lower than those of the control group (all P < .001); (2) SDS and SAS scores were significantly lower than those of the control group (both P < .001); (3) blood purification adequacy rate (P = .049) and nutritional status qualified rate (P = .037) were significantly higher than those of the control group; and (4) incidence of complications was significantly lower than that of the control group (P = .045). Conclusions: Group health education and structured psychological nursing for patients undergoing blood purification can effectively alleviate negative emotions, enhance disease cognition, improve blood purification adequacy and nutritional status, and reduce complication risk.

The East Asian wild apples, Malus baccata (L.) Borkh and Malus hupehensis (Pamp.) Rehder., are additional contributors to the genomes of cultivated European and Chinese varieties.

The domestication process in long-lived plant perennials differs dramatically from that of annuals, with a huge amount of genetic exchange between crop and wild populations. Though apple is a major fruit crop grown worldwide, the contribution of wild apple species to the genetic makeup of the cultivated apple genome remains a topic of intense study. We used population genomics approaches to investigate the contributions of several wild apple species to European and Chinese rootstock and dessert genomes, with a focus on the extent of wild-crop gene flow. Population genetic structure inferences revealed that the East Asian wild apples, Malus baccata (L.) Borkh and M. hupehensis (Pamp.), form a single panmictic group, and that the European dessert and rootstock apples form a specific gene pool whereas the Chinese dessert and rootstock apples were a mixture of three wild gene pools, suggesting different evolutionary histories of European and Chinese apple varieties. Coalescent-based inferences and gene flow estimates indicated that M. baccata - M. hupehensis contributed to the genome of both European and Chinese cultivated apples through wild-to-crop introgressions, and not as an initial contributor as previously supposed. We also confirmed the contribution through wild-to-crop introgressions of Malus sylvestris Mill. to the cultivated apple genome. Apple tree domestication is therefore one example in woody perennials that involved gene flow from several wild species from multiple geographical areas. This study provides an example of a complex protracted process of domestication in long-lived plant perennials, and is a starting point for apple breeding programmes.

MdNup62 interactions with MdHSFs involved in flowering and heat-stress tolerance in apple.

Because of global warming, the apple flowering period is occurring significantly earlier, increasing the probability and degree of freezing injury. Moreover, extreme hot weather has also seriously affected the development of apple industry. Nuclear pore complexes (NPCs) are main channels controlling nucleocytoplasmic transport, but their roles in regulating plant development and stress responses are still unknown. Here, we analysed the components of the apple NPC and found that MdNup62 interacts with MdNup54, forming the central NPC channel. MdNup62 was localized to the nuclear pore, and its expression was significantly up-regulated in 'Nagafu No. 2' tissue-cultured seedlings subjected to heat treatments. To determine MdNup62's function, we obtained MdNup62-overexpressed (OE) Arabidopsis and tomato lines that showed significantly reduced high-temperature resistance. Additionally, OE-MdNup62 Arabidopsis lines showed significantly earlier flowering compared with wild-type. Furthermore, we identified 62 putative MdNup62-interacting proteins and confirmed MdNup62 interactions with multiple MdHSFs. The OE-MdHSFA1d and OE-MdHSFA9b Arabidopsis lines also showed significantly earlier flowering phenotypes than wild-type, but had enhanced high-temperature resistance levels. Thus, MdNUP62 interacts with multiple MdHSFs during nucleocytoplasmic transport to regulate flowering and heat resistance in apple. The data provide a new theoretical reference for managing the impact of global warming on the apple industry.

Elucidation of the anti-inflammatory mechanism of Er Miao San by integrative approach of network pharmacology and experimental verification.

Traditional Chinese medicine (TCM) has been long time used in China and gains ever-increasing worldwide acceptance. Er Miao San (EMS), a TCM formula, has been extensively used to treat inflammatory diseases, while its bioactive components and therapeutic mechanisms remain unclear. In this study, we conducted an integrative approach of network pharmacology and experimental study to elucidate the underlying mechanisms of EMS in treating human rheumatoid arthritis (RA) and other inflammatory conditions. Quercetin, wogonin and rutaecarpine were probably the main active compounds of EMS in RA treatment as they affected the most RA-related targets, and TNF-α, IL-6 and IL-1ß were considered to be the core target proteins. The main compounds in EMS bound to these core proteins, which was further confirmed by molecular docking and bio-layer interferometry (BLI) analysis. Moreover, the potential molecular mechanisms of EMS predicted from network pharmacology analysis, were validated in vivo and in vitro experiments. EMS was found to inhibit the production of NO, TNF-α and IL-6 in lipopolysaccharide (LPS)-stimulated RAW264.7 cells; reduce xylene-induced mouse ear edema; and decrease the incidence of carrageenan-induced rat paw edema. The carrageenan-induced up-regulation of TNF-α, IL-6 and IL-1ß mRNA expression in rat paws was down-regulated by EMS, consistent with the network pharmacology results. This study provides evidence that EMS plays a critical role in anti-inflammation via suppressing inflammatory cytokines, indicating that EMS is a candidate herbal drug for further investigation in treating inflammatory and arthritic conditions.

Development of a Novel Analytical Method for Determining Trazodone in Human Plasma by Liquid Chromatography Coupled With Mass Spectrometry Coupled With Automatic 2-Dimensional Liquid Chromatograph-Mass Spectrometer Coupler 9500 and Its Application to Therapeutic Drug Monitoring.

BACKGROUND: Trazodone (TZD) is a tetracyclic serotonin antagonist and reuptake inhibitor that is used as a second-generation phenylpiperazine antidepressant. However, the plasma concentrations of TZD have shown individual variations in clinical practice. Quantification of TZD plasma concentrations may be an effective and valuable method to balance the clinical efficacy and adverse reactions. This study aimed to establish a novel liquid chromatography coupled with mass spectrometry (LC-MS) assay for measuring TZD concentrations in human plasma for therapeutic drug monitoring (TDM). METHODS: After protein precipitation with acetonitrile, LC-MS quantification of TZD was performed in the multiple reaction monitoring mode with chromatographic separation using a mobile phase of MeOH and 0.1% formic acid in water. This method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect, and stability according to United states food and drug administration guidelines. RESULTS: This method showed good selectivity because no interfering peaks were observed in the plasma samples during the 2-minute run time. The range of the calibration curve was 1-3000 ng/mL. The detection and quantification limits were 0.3 and 1 ng/mL, respectively. The intraday and interday accuracies were 96.5%-103.4%, with precision relative SD% values of <5%, except for the limit of quality. The mean TZD recovery from human plasma was 95.4%-104.5%. Finally, this method was successfully applied to TDM in 20 patients. The TZD plasma concentrations of the patients ranged between 21.5 and 2267.3 ng/mL. CONCLUSIONS: A novel analytical method was established to measure TZD by LC-MS coupled with an automatic 2-dimensional liquid chromatograph mass spectrometer coupler 9500 (LC-MS/MS-Mate 9500), which is superior to the ordinary LC-MS system in separation, transport, anti-interference, sensitivity, and quantitative analysis stability.

Genome-wide identification of the 14-3-3 gene family and its participation in floral transition by interacting with TFL1/FT in apple.

BACKGROUND: Apple (Malus domestica Borkh.) is a popular cultivated fruit crop with high economic value in China. Apple floral transition is an important process but liable to be affected by various environmental factors. The 14-3-3 proteins are involved in regulating diverse biological processes in plants, and some 14-3-3 members play vital roles in flowering. However, little information was available about the 14-3-3 members in apple. RESULTS: In the current study, we identified eighteen 14-3-3 gene family members from the apple genome database, designated MdGF14a to MdGF14r. The isoforms possess a conserved core region comprising nine antiparallel α-helices and divergent N and C termini. According to their structural and phylogenetic features, Md14-3-3 proteins could be classified into two major evolutionary branches, the epsilon (ɛ) group and the non-epsilon (non-ɛ) group. Moreover, expression profiles derived from transcriptome data and quantitative real-time reverse transcription PCR analysis showed diverse expression patterns of Md14-3-3 genes in various tissues and in response to different sugars and hormone treatments during the floral transition phase. Four Md14-3-3 isoforms (MdGF14a, MdGF14d, MdGF14i, and MdGF14j) exhibiting prominent transcriptional responses to sugars and hormones were selected for further investigation. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation experiments showed that the four Md14-3-3 proteins interact with key floral integrators, MdTFL1 (TERMINAL FLOWER1) and MdFT (FLOWERING LOCUS T). Subcellular localization of four selected Md14-3-3 proteins demonstrated their localization in both the cytoplasm and nucleus. CONCLUSION: We identified the Md14-3-3 s family in apple comprehensively. Certain Md14-3-3 genes are expressed predominantly during the apple floral transition stage, and may participate in the regulation of flowering through association with flower control genes. Our results provide a preliminary framework for further investigation into the roles of Md14-3-3 s in floral transition.

The downregulation of PpPG21 and PpPG22 influences peach fruit texture and softening.

MAIN CONCLUSION: The downregulation of PpPG21 and PpPG22 expression in melting-flesh peach delays fruit softening and hinders texture changes by influencing pectin solubilization and depolymerization. The polygalacturonase (PG)-catalyzed solubilization and depolymerization of pectin plays a central role in the softening and texture formation processes in peach fruit. In this study, the expression characteristics of 15 PpPG members in peach fruits belonging to the melting flesh (MF) and non-melting flesh (NMF) types were analyzed, and virus-induced gene silencing (VIGS) technology was used to identify the roles of PpPG21 (ppa006839m) and PpPG22 (ppa006857m) in peach fruit softening and texture changes. In both MF and NMF peaches, the expression of PpPG1, 10, 12, 23, and 25 was upregulated, whereas that of PpPG14, 24, 35, 38, and 39 was relatively stable or downregulated during shelf life. PpPG1 was highly expressed in NMF fruit, whereas PpPG21 and 22 were highly expressed in MF peaches. Suppressing the expression of PpPG21 and 22 by VIGS in MF peaches significantly reduced PG enzyme activity, maintained the firmness of the fruit during the late shelf life stage, and suppressed the occurrence of the "melting" stage compared with the control fruits. Moreover, the downregulation of PpPG21 and 22 expression also reduced the water-soluble pectin (WSP) content, increased the contents of ionic-soluble pectin (ISP) and covalent-soluble pectin (CSP) and affected the expression levels of ethylene synthesis- and pectin depolymerization-related genes in the late shelf life stage. These results indicate that PpPG21 and 22 play a major role in the development of the melting texture trait of peaches by depolymerizing cell wall pectin. Our results provide direct evidence showing that PG regulates peach fruit softening and texture changes.

Molecular mechanism of MdWUS2-MdTCP12 interaction in mediating cytokinin signaling to control axillary bud outgrowth.

Shoot branching is an important factor that influences the architecture of apple trees and cytokinin is known to promote axillary bud outgrowth. The cultivar 'Fuji', which is grown on ~75% of the apple-producing area in China, exhibits poor natural branching. The TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) family genes BRANCHED1/2 (BRC1/2) are involved in integrating diverse factors that function locally to inhibit shoot branching; however, the molecular mechanism underlying the cytokinin-mediated promotion of branching that involves the repression of BRC1/2 remains unclear. In this study, we found that apple WUSCHEL2 (MdWUS2), which interacts with the co-repressor TOPLESS-RELATED9 (MdTPR9), is activated by cytokinin and regulates branching by inhibiting the activity of MdTCP12 (a BRC2 homolog). Overexpressing MdWUS2 in Arabidopsis or Nicotiana benthamiana resulted in enhanced branching. Overexpression of MdTCP12 inhibited axillary bud outgrowth in Arabidopsis, indicating that it contributes to the regulation of branching. In addition, we found that MdWUS2 interacted with MdTCP12 in vivo and in vitro and suppressed the ability of MdTCP12 to activate the transcription of its target gene, HOMEOBOX PROTEIN 53b (MdHB53b). Our results therefore suggest that MdWUS2 is involved in the cytokinin-mediated inhibition of MdTCP12 that controls bud outgrowth, and hence provide new insights into the regulation of shoot branching by cytokinin.

Identification of apple TFL1-interacting proteins uncovers an expanded flowering network.

KEY MESSAGE: MdTFL1, a floral repressor, forms protein complexes with several proteins and could compete with MdFT1 to regulate reproductive development in apple. Floral transition is a key developmental stage in the annual growth cycle of perennial fruit trees that directly determines the fruit development in the subsequent stage. FLOWERING LOCUS T (FT)/TERMINAL FLOWER1 (TFL1) family is known to play a vital regulatory role in plant growth and flowering. In apple, the two TFL1-like genes (MdTFL1-1 and MdTFL1-2) function as floral inhibitors; however, their mechanism of action is still largely unclear. This study aimed to functionally validate MdTFL1 and probe into its mechanism of action in apple. MdTFL1-1 and MdTFL1-2 were expressed mainly in stem and apical buds of vegetative shoots, with little expression in flower buds and young fruit. Expression of MdTFL1-1 and MdTFL1-2 rapidly decreased during floral induction. On the other hand, transgenic Arabidopsis, which ectopically expressed MdTFL1-1 or MdTFL1-2, flowered later than wild-type plants; demonstrating their in planta capability to function redundantly as flower repressors. Furthermore, we identified hundreds of novel interaction proteins of the two apple MdTFL1 proteins using yeast two-hybrid screens. Independent experiments for several proteins confirmed the yeast two-hybrid interactions. Among them, the transcription factor Nuclear Factor-Y subunit C (MdNF-YC2) functions as a promoter of flowering in Arabidopsis by activating LEAFY (LFY) and APETALA1 (AP1) expression. MdFT1 showed a similar interaction pattern as MdTFL1, implying a possible antagonistic action in the regulation of flowering. These newly identified TFL1-interacting proteins (TIPs) not only expand the floral regulatory network, but may also introduce new roles for TFL1 in plant development.

Construction of a high-density SNP-based genetic map and identification of fruit-related QTLs and candidate genes in peach [Prunus persica (L.) Batsch].

BACKGROUND: High-density genetic mapping is a valuable tool for mapping loci that control specific traits for perennial fruit trees. Peach is an economically important fruit tree and a model Rosaceae species for genomic and genetic research. In peach, even though many molecular markers, genetic maps and QTL mappings have been reported, further research on the improvement of marker numbers, map densities, QTL accuracy and candidate gene identification is still warranted. RESULTS: A high-density single nucleotide polymorphism (SNP)-based peach linkage map was constructed using specific locus amplified fragment sequencing (SLAF-seq). This genetic map consisted of 7998 SLAF markers, spanning 1098.79 cM with an average distance of 0.17 cM between adjacent markers. A total of 40 QTLs and 885 annotated candidate genes were detected for 10 fruit-related traits, including fruit weight (FW), fruit diameter (FD), percentage of red skin colour (PSC), eating quality (EQ), fruit flavour (FV), red in flesh (RF), red around pit (RP), adherence to pit (AP), fruit development period (FDP) and fruit fibre content (FFC). Eighteen QTLs for soluble solid content (SSC) were identified along LGs 1, 4, 5, and 6 in 2015 and 2016, and 540 genes were annotated in QTL intervals. Thirty-two QTLs for fruit acidity content (FA) were detected on LG1, and 2, 4, 5, 6, and 1232 candidate genes were identified. The expression profiles of 2 candidate genes for SSC and 4 for FA were analysed in parents and their offspring. CONCLUSIONS: We constructed a high-density genetic map in peach based on SLAF-seq, which may contribute to the identification of important agronomic trait loci. Ninety QTLs for 12 fruit-related traits were identified, most of which overlapped with previous reports, and some new QTLs were obtained. A large number of candidate genes for fruit-related traits were screened and identified. These results may improve our understanding of the genetic control of fruit quality traits and provide useful information in marker-assisted selection for fruit quality in peach breeding programmes.

Transcription profiles reveal the regulatory mechanisms of spur bud changes and flower induction in response to shoot bending in apple (Malus domestica Borkh.).

KEY MESSAGE: Shoot bending, as an effective agronomic measure, has been widely used to promote flowering in 'Fuji' apple trees. Here, we examined the transcriptional responses of genes in 'Fuji' apple buds at different flowering stages under a shoot-bending treatment using RNA sequencing. A complex genetic crosstalk-regulated network, involving abscisic acid-related genes, starch metabolism and circadian rhythm-related genes, as well as stress response-related genes, was up-regulated by shoot bending, in which were contrbuted to apple flower bud formation in response to shoot-bending conditions. Flower induction plays an important role in the apple tree life cycle, but young trees produce fewer and inferior flower buds. Shoot bending, as an effective agronomic measure, has been widely used to promote flowering in 'Fuji' apple trees. However, little is known about the gene expression network patterns and molecular regulatory mechanisms caused by shoot bending during the induced flowering. Here, we examined the transcriptional responses of genes in 'Fuji' apple buds at different flowering stages under a shoot-bending treatment using RNA sequencing. A steady up-regulation of carbon metabolism-related genes led to relatively high levels of sucrose in early induced flowering stages and starch accumulation during shoot bending. Additionally, global gene expression profiling determined that cytokinin, indole-3-acetic acid, gibberellin synthesis and signalling-related genes were significantly regulated by shoot bending, contributing to cell division and differentiation, bud growth and flower induction. A complex genetic crosstalk-regulated network, involving abscisic acid-related genes, starch metabolism- and circadian rhythm-related genes, as well as stress response-related genes, was up-regulated by shoot bending. Additionally, some transcription factor family genes that were involved in sugar, abscisic acid and stress response signalling were significantly induced by shoot bending. These important flowering genes, which were mainly involved in photoperiod, age and autonomous pathways, were up-regulated by shoot bending. Thus, a complex genetic network of regulatory mechanisms involved in sugar, hormone and stress response signalling pathways may mediate the induction of apple tree flowering in response to shoot-bending conditions.

Comparative RNA-Sequencing and DNA Methylation Analyses of Apple (Malus domestica Borkh.) Buds with Diverse Flowering Capabilities Reveal Novel Insights into the Regulatory Mechanisms of Flower Bud Formation.

In plants, DNA methylation (i.e. chromatin modification) is important for various biological processes, including growth, development and flowering. Because 'Fuji' apple trees are alternate bearing and have a long ripening period and poor-quality flower buds, we used bud types with diverse flowering capabilities to investigate the epigenetic regulatory mechanisms influencing flower bud formation. We examined the DNA methylation changes and the transcriptional responses in the selected apple bud types. We observed that in the apple genome, approximately 79.5%, 67.4% and 23.7% of the CG, CHG and CHH sequences are methylated, respectively. For each sequence context, differentially methylated regions exhibited distinct methylation patterns among the analyzed apple bud types. Global methylation and transcriptional analyses revealed that nonexpressed genes or genes expressed at low levels were highly methylated in the gene-body regions, suggesting that gene-body methylation is negatively correlated with gene expression. Moreover, genes with methylated promoters were more highly expressed than genes with unmethylated promoters, implying promoter methylation and gene expression are positively correlated. Additionally, flowering-related genes (e.g. SOC1, AP1 and SPLs) and some transcription factor genes (e.g. GATA, bHLH, bZIP and WOX) were highly expressed in spur buds (highest flowering rate), but were associated with low methylation levels in the gene-body regions. Our findings indicate a potential correlation between DNA methylation and gene expression in apple buds with diverse flowering capabilities, suggesting an epigenetic regulatory mechanism influences apple flower bud formation.

Genome-wide identification of SERK genes in apple and analyses of their role in stress responses and growth.

BACKGROUND: Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases associated with various signaling pathways. These kinases have a relationship with stress signals, and they are also believed to be important for regulating plant growth. However, information about this protein family in apple is limited. RESULTS: Twelve apple SERK genes distributed across eight chromosomes were identified. These genes clustered into three distinct groups in a phylogenetic analysis. All of the encoded proteins contained typical SERK domains. The chromosomal locations, gene/protein structures, synteny, promoter sequences, protein-protein interactions, and physicochemical characteristics of MdSERK genes were analyzed. Bioinformatics analyses demonstrated that gene duplications have likely contributed to the expansion and evolution of SERK genes in the apple genome. Six homologs of SERK genes were identified between apple and Arabidopsis. Quantitative real-time PCR analyses revealed that the MdSERK genes showed different expression patterns in various tissues. Eight MdSERK genes were responsive to stress signals, such as methyl jasmonate, salicylic acid, abscisic acid, and salt (NaCl). The application of exogenous brassinosteroid and auxin increased the growth and endogenous hormone contents of Malus hupehensis seedlings. The expression levels of seven MdSERK genes were significantly upregulated by brassinosteroid and auxin. In addition, several MdSERK genes showed higher expression levels in standard trees of 'Nagafu 2' (CF)/CF than in dwarf trees of CF/'Malling 9' (M.9), and in CF than in the spur-type bud mutation "Yanfu 6" (YF). CONCLUSION: This study represents the first comprehensive investigation of the apple SERK gene family. These data indicate that apple SERKs may function in adaptation to adverse environmental conditions and may also play roles in controlling apple tree growth.

Molecular role of cytokinin in bud activation and outgrowth in apple branching based on transcriptomic analysis.

KEY MESSAGE: Axillary bud activation and outgrowth were dependent on local cytokinin, and that bud activation preceded the activation of cell cycle and cell growth genes in apple branching. Cytokinin is often applied to apple trees to produce more shoot branches in apple seedlings. The molecular response of apple to the application of cytokinin, and the relationship between bud activation and cell cycle in apple branching, however, are poorly understood. In this study, RNA sequencing was used to characterize differential expression genes in axillary buds of 1-year grafted "Fuji" apple at 4 and 96 h after cytokinin application. And comparative gene expression analyses were performed in buds of decapitated shoots and buds of the treatment of biosynthetic inhibitor of cytokinin (Lovastatin) on decapitated shoots. Results indicated that decapitation and cytokinin increased ZR content in buds and internodes at 4-8 h, and induced bud elongation at 96 h after treatment, relative to buds in shoots receiving the Lovastatin treatment. RNA-seq analysis indicated that differential expression genes in auxin and cytokinin signal transduction were significantly enriched at 4 h, and DNA replication was enriched at 96 h. Cytokinin-responsive type-A response regulator, auxin polar transport, and axillary meristem-related genes were up-regulated at 4 h in the cytokinin and decapitation treatments, while qRT-PCR analysis showed that cell cycle and cell growth genes were up-regulated after 8 h. Collectively, the data indicated that bud activation and outgrowth might be dependent on local cytokinin synthesis in axillary buds or stems, and that bud activation preceded the activation of cell cycle genes during the outgrowth of ABs in apple shoots.

Comparative RNA-sequencing-based transcriptome profiling of buds from profusely flowering 'Qinguan' and weakly flowering 'Nagafu no. 2' apple varieties reveals novel insights into the regulatory mechanisms underlying floral induction.

BACKGROUND: Floral induction is an important stage in the apple tree life cycle. In 'Nagafu No. 2', which was derived from a 'Fuji' bud sport, flower bud formation is associated with serious problems, such as fewer and inferior flower buds, a long juvenile phase, and an alternate bearing phenotype. Moreover, the molecular regulatory mechanisms underlying apple floral induction remain unknown. To characterize these mechanisms, we compared the RNA-sequencing-based transcriptome profiles of buds during floral induction in profusely flowering 'Qinguan' and weakly flowering 'Nagafu No. 2' apple varieties. RESULTS: Genes differentially expressed between the buds of the two varieties were mainly related to carbohydrate, fatty acid, and lipid pathways. Additionally, the steady up-regulated expression of genes related to the fatty acid and lipid pathways and the down-regulated expression of starch synthesis-related genes in the carbon metabolic pathway of 'Qinguan' relative to 'Nagafu No. 2' were observed to contribute to the higher flowering rate of 'Qinguan'. Additionally, global gene expression profiling revealed that genes related to cytokinin, indole-3-acetic acid, and gibberellin synthesis, signalling, and responses (i.e., factors contributing to cell division and differentiation and bud growth) were significantly differentially expressed between the two varieties. The up-regulated expression of genes involved in abscisic acid and salicylic acid biosynthesis via shikimate pathways as well as jasmonic acid production through fatty acid pathways in 'Qinguan' buds were also revealed to contribute to the floral induction and relatively high flowering rate of this variety. The differential expression of transcription factor genes (i.e., SPL, bZIP, IDD, and MYB genes) involved in multiple biological processes was also observed to play key roles in floral induction. Finally, important flowering genes (i.e., FT, FD, and AFL) were significantly more highly expressed in 'Qinguan' buds than in 'Nagafu No. 2' buds during floral induction. CONCLUSIONS: A complex genetic network of regulatory mechanisms involving carbohydrate, fatty acid, lipid, and hormone pathways may mediate the induction of apple tree flowering.

Exogenous application of GA3 inactively regulates axillary bud outgrowth by influencing of branching-inhibitors and bud-regulating hormones in apple (Malus domestica Borkh.).

Although gibberellin (GA) has been reported to control branching, little is known about how GA mediates signals regulating the outgrowth of axillary buds (ABs). In the current study, the effect of the exogenous application of 5.0 mM GA3 on ABs outgrowth on 1-year-old 'Nagafu No. 2'/T337/M. robusta Rehd. apple trees was investigated and compared to the bud-activating treatments, 5 mM BA or decapitation. Additionally, the expression of genes related to bud-regulating signals and sucrose levels in ABs was examined. Results indicated that GA3 did not promote ABs' outgrowth, nor down-regulate the expression of branching repressors [MdTCP40, MdTCP33, and MdTCP16 (homologs of BRANCHED1 and BRC2)], which were significantly inhibited by the BA and decapitation treatments. MdSBP12 and MdSBP18, the putative transcriptional activators of these genes, which are expressed at lower levels in BA-treated and decapitated buds, were up-regulated in the GA3 treatment in comparison to the BA treatment. Additionally, GA3 did not up-regulate the expression of CK response- and auxin transport-related genes, which were immediately induced by the BA treatment. In addition, GA3 also up-regulated the expression of several Tre6P biosynthesis genes and reduced sucrose levels in ABs. Sucrose levels, however, were still higher than what was observed in BA-treated buds, indicating that sucrose may not be limiting in GA3-controlled AB outgrowth. Although GA3 promoted cell division, it was not sufficient to induce AB outgrowth. Conclusively, some branching-inhibiting genes and bud-regulating hormones are associated with the inability of GA3 to activate AB outgrowth.

Identification and Characterization of miRNAs in Self-Rooted and Grafted Malus Reveals Critical Networks Associated with Flowering.

Grafting can improve the agricultural traits of crop plants, especially fruit trees. However, the regulatory networks and differentially expressed microRNAs (miRNAs) related to grafting in apple remain unclear. Herein, we conducted high-throughput sequencing and identified differentially expressed miRNAs among self-rooted Fuji, self-rooted M9, and grafted Fuji/M9. We analyzed the flowering rate, leaf morphology, and nutrient and carbohydrate contents in the three materials. The flowering rate, element and carbohydrate contents, and expression levels of flowering genes were higher in Fuji/M9 than in Fuji. We detected 206 known miRNAs and 976 novel miRNAs in the three materials, and identified those that were up- or downregulated in response to grafting. miR156 was most abundant in Fuji, followed by Fuji/M9, and then self-rooted M9, while miR172 was most abundant in M9, followed by Fuji/M9, and then Fuji. These expression patterns suggest that that these miRNAs were related to grafting. A Gene Ontology (GO) analysis showed that the differentially expressed miRNAs controlled genes involved in various biological processes, including cellular biosynthesis and metabolism. The expression of differentially expressed miRNAs and flowering-related genes was verified by qRT-PCR. Altogether, this comprehensive analysis of miRNAs related to grafting provides valuable information for breeding and grafting of apple and other fruit trees.