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Ethylene plays an important role in regulating the development and ripening of fruits, and 1-aminocyclopropane carboxylic acid synthase is the key rate-limiting enzyme in ethylene synthesis pathway. In this study, eight PpACS genes were identified from the peach genome [Prunus persica (L.) Batsch], and their phylogeny, gene structures, promoter motifs and expression patterns were analyzed. The PpACS genes could be divided into four types, and the genes with similar structures and motif distribution clustered together. Identification of the cis-elements in the promoters revealed that the PpACS genes may respond to various hormones. Furthermore, expression analysis showed that five (PpACS1, PpACS5, PpACS6, PpACS7 and PpACS8) of the eight PpACS genes were expressed at different stages during peach fruit development. Among them, PpACS1 was highly expressed at the ripening stage and induced by ethylene. The expression peaks of PpACS5, PpACS6, PpACS7 and PpACS8 during the transition from first exponential growth to pit hardening (S1 to S2) indicated a potential function of ethylene during this important transition. Taken together, these results provide valuable information for future investigation into the functions of the PpACS genes during peach fruit development and ripening.

Laccases are the key enzymes responsible for plant lignin biosynthesis and responses to environment stress. However, the roles of LAC genes in plant disease resistance are still largely unknown, especially in grapevine, one of the most important horticultural crops in the world. Its quality and yield are very vulnerable to gray mold disease caused by Botrytis cinerea. In total, 30 VvLAC genes were identified and found to be unevenly distributed on seven chromosomes; they were classified into seven groups based on phylogenetic analysis according to the criteria applied in Arabidopsis thaliana. Collinearity and synteny analyses identified some orthologous gene pairs in Vitis vinifera and a few paralogous gene pairs among grape and peach. The VvLAC gene family has diverse gene structures and a highly conserved motif composition. The prominent presence of the MYB cis-elements in each VvLAC promoter highlighted MYB transcriptional factors as the main regulators of VvLAC genes. Furthermore, twenty-five VvLAC genes with functional redundancy are probably implicated in grape lignin biosynthesis. The expression patterns of the LAC genes in grape leaves of Chinese wild V. amurensis ‘Shuangyou’ (SY), a germplasm highly resistant to B. cinerea, were investigated through transcriptomic data and qRT-PCR verification. Combined with the phylogenetic analysis, with AtLACs participating in lignin metabolism, and the cis-element analysis, VaLAC14, VaLAC19, VaLAC24 and VaLAC30 were identified as key candidate genes for lignin biosynthesis in the grape response to B. cinerea. This study supplies a comprehensive understanding of the classification, evolution, structure and responses of the grape LAC genes against B. cinerea. It also provides valuable genetic resources for functional characterization towards enhancing grapevine disease resistance.

Jujube witches' broom (JWB) disease, referred to as 'Zaofeng' disease in Chinese, is associated with the JWB phytoplasma ('Candidatus Phytoplasma ziziphi') and causes heavy losses in the jujube industry. JWB disease has been recorded since the 1950s. Diseased trees have symptoms such as shoot proliferation and leaf-like flowers. The JWB phytoplasma is assigned to the 16SrV group, subgroup 16SrV-B, according to 16S rRNA gene sequence. The JWB phytoplasma is transmitted by leafhoppers and can infect a few plants other than jujube. Infection with phytoplasma affects biochemical and physiological process, altering the expression of genes encoding some transcription factors and functional genes, mainly involved in biotic stress response. The genome of JWB phytoplasma 'nky' strain has been sequenced and consists of 750,803 bp within one circular chromosome that encodes 694 protein-coding genes. The pathogenic mechanisms of a few JWB phytoplasma effectors have been investigated. The presence of JWB phytoplasma has been detected through symptoms observation, ELISA, DAPI staining and PCR, but new techniques, such as LAMP and CRISPR/Cas-12 based visual assay, have recently been developed. Some resistant jujube cultivars have been selected by infection screening. Although treatment with tetracycline antibiotics is effective, comprehensive control measures, including orchard management and sanitary measures, are needed for disease control. Further studies are needed in development of JWB phytoplasma culture method, expansion of genomic information, and phytoplasma effectors, resistance-related gene identification and resistant genotype developing.

Ethylene plays an important role in grape ripening, acting through ethylene response factors (ERFs) that are downstream of ethylene signaling pathways, yet little else is known about these molecular mechanisms. In our study, we demonstrate that ethylene treatment promotes grape ripening and that the transcription factor VvERF75 shows increased expression during grape ripening. VvERF75 was found to contain a conserved AP2/ERF domain, to be a member of the ERF IXa subfamily, and to localize to the nucleus and to have transactivation activity in yeast. Our in vitro assays showed that VvERF75 can positively regulate expression of pheophorbide a oxygenase (VvPAO1) and 1-aminocyclopropane-1-carboxylic acid synthase (VvACS5) by binding to DRE motifs in their promoters. Overexpression of VvERF75 in tomato increased ethylene production and resulted in dwarfed plants, early flowering and fruit ripening, increased SlPAO and SlACS transcript levels, and decreased chlorophyll content. In general, our study revealed that VvERF75 may regulate fruit ripening by promoting ethylene biosynthesis and chlorophyll degradation, a discovery that lays a foundation for deciphering the molecular mechanisms of grape ripening.

Abstract Background The rapid growth of annual shoots is detrimental to peach production. While gibberellin (GA) promotes the rapid growth of peach shoots, there is limited information on the identity and expression profiles of GA-metabolism genes for this species. Results All six GA biosynthetic gene families were identified in the peach genome, and the expression profiles of these family members were determined in peach shoots. The upstream biosynthetic gene families have only one or two members (1 CPS, 2 KSs, and 1 KO), while the downstream gene families have multiple members (7 KAOs, 6 GA20oxs, and 5 GA3oxs). Between the two KS genes, PpKS1 showed a relatively high transcript level in shoots, while PpKS2 was undetectable. Among the seven KAO genes, PpKAO2 was highly expressed in shoots, while PpKAO1 and − 6 were weakly expressed. For the six GA20ox genes, both PpGA20ox1 and − 2 were expressed in shoots, but PpGA20ox1 levels were higher than PpGA20ox2. For the five GA3ox genes, only PpGA3ox1 was highly expressed in shoots. Among these biosynthesis genes, PpGA20ox1 and PpGA3ox1 showed a gradual decrease in transcript level along shoots from top to bottom, and a similar trend was observed in bioactive GA1 and GA4 distribution. Among the GA-deactivation genes, PpGA2ox6 was highly expressed in peach shoots. PpGA2ox1 and − 5 transcripts were relatively lower and showed a similar pattern to PpGA20ox1 and PpGA3ox1 in peach shoots. Overexpression of PpGA20ox1, − 2, or PpGA2ox6 in Arabidopsis or tobacco promoted or depressed the plant growth, respectively, while PpGA3ox1 did not affect plant height. Transient expression of PpGA20ox1 in peach leaves significantly increased bioactive GA1 content. Conclusions Our results suggest that PpGA20ox and PpGA2ox expression are closely associated with the distribution of active GA1 and GA4 in peach annual shoots. Our research lays a foundation for future studies into ways to effectively repress the rapid growth of peach shoot.

Expansins (EXPs) loosen plant cell walls and are involved in diverse developmental processes through modifying cell-walls; however, little is known about the role of PpEXPs in peach fruit. In this study, 26 PpEXP genes were identified in the peach genome and grouped into four subfamilies, with 20 PpEXPAs, three PpEXPBs, one PpEXPLA and two PpEXPLBs. The 26 PpEXPs were mapped on eight chromosomes. The primary mode of gene duplication of the PpEXPs was dispersed gene duplication (DSD, 50%). Notably, cis-elements involved in light responsiveness and MeJA-responsiveness were detected in the promoter regions of all PpEXPs, while ethylene responsive elements were observed in 12 PpEXPs. Transcript profiling of PpEXPs in the peach fruit varieties of MF (melting), NMF (non-melting) and SH (stony hard) at different stages showed that PpEXPs displayed distinct expression patterns. Among the 26 PpEXPs, 15 PpEXPs were expressed in the fruit. Combining the expressing patterns of PpEXPs in fruits with different flesh textures, PpEXPA7, PpEXPA13 and PpEXPA15 were selected as candidate genes, as they were highly consistent with the patterns of previous reported key genes (PpPGM, PpPGF and PpYUC11) in regard to peach fruit texture. The genes with different expression patterns between MF and NMF were divided into 16 modules, of which one module, with pink and midnightblue, negatively correlated with the phenotype of fruit firmness and was identified as PpEXPA1 and PpEXPA7, while the other module was identified as PpERF in the pink module, which might potentially effect fruit texture development by regulating PpEXPs. These results provide a foundation for the functional characterization of PpEXPs in peach.

The interaction between miRNAs (microRNAs) and target genes plays an important role in plant pistil development. MiRNAs related to pistils were explored in pomegranate. The differentially expressed miRNAs were screened at different developmental stages of pomegranate pistils, and their target differentially expressed mRNAs were further identified to clarify the regulatory effect of miRNAs on pistil development. In our study, 61 conserved miRNAs were identified in 30 families, including miR395, miR394, miR393, miR161, miR162, and miR168. Among them, miR156, miR157, miR159, miR160, miR164, miR165, miR166, miR167, miR169, and miR172 were involved in the development of flower organs. Eight miRNAs were randomly selected and verified for qRT-PCR analysis. The result analysis indicated that miR160, miR164, and miR172 might be positive factors in the regulation of pomegranate pistil development. MiR156 and miR166 might be involved in regulation of pomegranate pistil development as negative factors.

Anthocyanins, a categories of metabolites derived from flavonoid biosynthesis pathway, are responsible for the red color of peach. Anthocyanin metabolism is modulated by a complicated regulatory network. In this study, our results demonstrated that PpNAC25 was a component of anthocyanin regulatory network in peach. PpNAC25 showed a closed relationship with the well-known ripening-related gene NOR (no-ripening) and was highly expressed in peach fruit. Overexpression of PpNAC25 in poplar resulted in a redder shoot tips compared with EV control. PpNAC25 overexpression upregulated the anthocyanin biosynthetic and transportation genes in transcript levels and then increased anthocyanin contents. In Y1H and Luc/Ren assay, PpNAC25 bound to the promoter of the anthocyanin-activator PpMYB10.1 and PpMYB10.2 and activated its transcript. Moreover, PpNAC25 formed a heterodimer with PpNAC1, an anthocyanin-regulator. Our researches provide evidence that PpNAC25 may be a positive regulator of anthocyanin biosynthesis in peach fruit.

Laccase (LAC) is the key enzyme responsible for lignin biosynthesis. Here, 57 PgLACs from pomegranate were identified and distributed on eight chromosomes and one unplaced scaffold. They were divided into six groups containing three typical Cu-oxidase domains. Totally, 51 cis-acting elements in the promoter region of PgLACs are involved in response to ABA, GA, light, stress, etc., indicating diverse functions of PgLACs. The expression profiles of 13 PgLACs during the seed development stage showed that most PgLACs expressed at a higher level earlier than at the later seed development stage in two pomegranate cultivars except PgLAC4. Also, PgLAC1/6/7/16 expressed at a significantly higher level in soft-seed ‘Tunisia’; on the contrary, PgLAC37 and PgLAC50 with a significantly higher expression in hard-seed ‘Taishanhong’. Combined with their distinguishing cis-acting elements, it was concluded that PgLAC1/6/7 may respond to GA via TATC-box and GARE-motif, and PgLAC16 repressed the promotor activity of embryo mid-maturation genes via RY-element so as to contribute to softer seed formation, whereas PgLAC37/50 may participate in seed formation and accelerate seed maturity via ABRE and G-box elements. Collectively, the dramatic gene expressions of PgLAC1/6/7/16/37/50 will provide valuable information to explore the formation of soft- and hard-seed in pomegranate.

Rootstocks play a vital role in regulating the environmental adaptability and controlling the growth and development of apple trees. M9T337, an excellent apple rootstock widely used in commercial orchards, could confer dwarf tree architectures, early fruiting and suitability for high-density planting. However, the rooting ability of M9T3337 is low when it is vegetatively propagated, and researchers have not yet established an efficient micropropagation system. The present study systematically evaluated the multiplication in adventitious shoots and the in vitro formation of adventitious roots to determine the effects of the culture media and plant growth regulators of M9T337 and a rapid micropropagation system was developed. For the shoot multiplication, the highest multiplication index of 3.93 was obtained on Murashige and Skoog (MS) media supplemented with 2.0 mg/L 6-BA, 0.1 mg/L NAA and 0.3 mg/L GA3 from 12 combinations of 6-BA and NAA. Stronger and taller adventitious shoots were grown on MS supplemented with 1.8 mg/L 6-BA and 0.5 mg/L NAA. The optimal media with 100% rooting was obtained using 1/2 MS supplemented with 0.3 mg/L IBA or MS supplemented with 0.6 mg/L IBA for the rooting induction, resulting in mean rooting numbers of 13.00 and 11.33, respectively. Additionally, the effect on rooting of adding 0.3 mg/L IBA or not on the 1/2 MS and MS media was compared; the results suggested that an appropriate IBA concentration was the key to successful rooting. The rooted plantlets were acclimatised in a shaded greenhouse with an 84% survival rate. The established micropropagation system could be used for the rapid propagation of M9T337 for commercial production.

Arginine is a natural preservative; however, its effects on the storage of different cultivars of pomegranates have not been investigated extensively. Therefore, the fruit quality of soft-seed Tunisia and hard-seed Yudazi pomegranates was investigated after treatment with arginine at four concentrations during cold storage for 80 days. Pomegranates treated with 1.0 mM arginine exhibited a relatively lower loss of vitamin C, soluble solid, total phenol, and anthocyanin contents in arils, together with a better fruit appearance. Combined with principal component analysis (PCA), the storage life of fruits treated with 1.0 mM arginine showed a higher correlation with antioxidant enzyme activity (e.g., superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)) during the first 40 days of cold storage, whereas after 40 days of cold storage, storage life was more dependent on the integrity of the cell membrane affected by malondialdehyde (MDA) content, electrolyte leakage (EL), and hydrogen peroxide (H2O2) accumulation. Arginine treatment contributed significantly to the appearance and inner quality of the hard-seed pomegranate cv. Yudazi fruit during cold storage compared to those of soft-seed Tunisia. Taken together, arginine application combined with cold storage enhanced the nutraceutical properties and marketability of pomegranate fruits.

The plant growth regulator ethylene influences rachis browning in grape (Vitis vinifera L.). Although the ethylene signaling pathway is well defined, there is limited knowledge on its mode of action during rachis browning. Here, we show that an ethylene response factor (VvERF111) positively regulates chlorophyll degradation in rachis by binding to a DRE motif in the promoter of VvCLH1. The expression of VvERF111 and VvCLH1 in rachis was induced by ethylene and inhibited by 1-methylcyclopropene (1-MCP). VvERF111 belongs to the ERF IX subfamily of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily, shows transcriptional activity in yeast, and is localized in the nucleus and membrane. The transient overexpression of VvERF111 or chlorophyllase (VvCLH1) in grape leaves accelerated chlorophyll degradation. In VvERF111-overexpressing leaves, transcript levels of VvCLH1 were also increased. Our findings offer a deeper understanding of the transcriptional regulation of chlorophyll degradation during the rachis browning of grape.

Fruit ripening includes several metabolic changes that lead to sweeter and softer fruit. Pectin depolymerization is one of major factors that softens developing grape berries. Pectin lyases (PLs) play important roles in pectin degradation in the grape berry. However, little is known about the temporal and spatial expression of grapevine (Vitis spp.) pectin lyase genes (VvPLs) or their function during fruit ripening and softening. In this study, 18 individual VvPL genes were identified in the grape genome. All VvPL genes were sorted into group I and group II, except VvPL12 which demonstrated higher and similar expression trends in different tissues and organs. In grape berry, VvPL1, 5, 7, 11 and 16 were highly expressed, whereas VvPL18, 15, 2, 13, 10, 14, 17, 6 and 8 showed lower expression levels at different berry developmental stages. Expression of VvPL11 firstly increased and then decreased, and the highest expression was shown at 6 weeks after full bloom (WAFB) during berry development. Over-expression of the VvPL11 gene in tomato caused higher ethylene production and lower firmness compared to wild-type fruit. Moreover, decreased propectin and increased water-soluble pectin (WSP) levels were observed in VvPL11 transgenic tomato fruit. Consistent with this result, the expression levels of SlPG2, SlEXP, and SlPME1, all of which are genes involved in fruit softening, were up-regulated in VvPL11-OE tomato fruit, which supported the idea that VvPL11 plays an important role in fruit ripening and softening. This study provided a comprehensive analysis of the grapevine PL family and advanced our knowledge of the functions of VvPLs during fruit softening.

As a predominant phenolic compound in apple fruits, chlorogenic acid (CGA) benefits human health due to its various antioxidant properties. However, little has been known regarding the molecular mechanism underlying the CGA accumulation in apple fruits. In this study, we measured the CGA content and relative enzymes’ activities during fruit development in two different flesh-colored cultivars ‘Huashuo’ and ‘Red Love’. The CGA content in both cultivars decreased sharply from 30 days after full bloom (DAFB) to 60 DAFB. Notably, the CGA content in fruit flesh was relatively higher than that in the peel. Further, the activities of C3H and HCT enzymes downstream of the CGA biosynthesis showed the similar changing trend as CGA content. Based on the transcriptome data of ‘Huashuo’ fruit at 30 DAFB and 60 DAFB, 23 differentially expressed CGA synthesis-related genes were screened. Gene expression analyses further showed that MdHCT1/2/4/5/6 and MdC3H1/2/3 were positively correlated to the variation of CGA content in two cultivars. These findings establish a theoretical foundation for further mechanism study on CGA biosynthesis and provide guidance for nutrient improvement in apple breeding programs.

Melatonin acts both as an antioxidant and as a growth regulatory substance in plants. Pseudomonas fluorescens endophytic bacterium has been shown to produce melatonin and increase plant resistance to abiotic stressors through increasing endogenous melatonin. However, in bacteria, genes are still not known to be melatonin-related. Here, we reported that the bacterial phenylalanine 4-hydroxylase (PAH) may be involved in the 5-hydroxytryptophan (5-HTP) biosynthesis and further influenced the subsequent production of melatonin in P. fluorescens. The purified PAH protein of P. fluorescens not only hydroxylated phenylalanine but also exhibited l-tryptophan (l-Trp) hydroxylase activity by converting l-Trp to 5-HTP in vitro. However, bacterial PAH displayed lower activity and affinity for l-Trp than l-phenylalanine. Notably, the PAH deletion of P. fluorescens blocked melatonin production by causing a significant decline in 5-HTP levels and thus decreased the resistance to abiotic stress. Overall, this study revealed a possible role for bacterial PAH in controlling 5-HTP and melatonin biosynthesis in bacteria, and expanded the current knowledge of melatonin production in microorganisms.

Abstract Background Ubiquitin ligases (E3) are the enzymes in the ubiquitin/26S proteasome pathway responsible for targeting proteins to the degradation pathway and play major roles in multiple biological activities. However, the E3 family and their functions are yet to be identified in the fruit of peach. Results In this study, genome-wide identification, classification and characterization of the E3 ligase genes within the genome of peach (Prunus persica) was carried out. In total, 765 E3 (PpE3) ligase genes were identified in the peach genome. The PpE3 ligase genes were divided into eight subfamilies according to the presence of known functional domains. The RBX subfamily was not detected in peach. The PpE3 ligase genes were not randomly distributed among the 8 chromosomes, with a greater concentration on the longer chromosomes. The primary mode of gene duplication of the PpE3 ligase genes was dispersed gene duplication (DSD). Four subgroups of the BTB subfamily never characterized before were newly identified in peach, namely BTBAND, BTBBL, BTBP and BTBAN. The expression patterns of the identified E3 ligase genes in two peach varieties that display different types of fruit softening (melting flesh, MF, and stony hard, SH) were analyzed at 4 different stages of ripening using Illumina technology. Among the 765 PpE3 ligase genes, 515 (67.3%) were expressed (FPKM > 1) in the fruit of either MF or SH during fruit ripening. In same-stage comparisons, 231 differentially expressed genes (DEGs) were identified between the two peach cultivars. The number of DEGs in each subfamily varied. Most DEGs were members of the BTB, F-box, U-box and RING subfamilies. PpE3 ligase genes predicted to be involved in ethylene, auxin, or ABA synthesis or signaling and DNA methylation were differentially regulated. Eight PpE3 ligase genes with possible roles in peach flesh texture and fruit ripening were discussed. Conclusions The results of this study provide useful information for further understanding the functional roles of the ubiquitin ligase genes in peach. The findings also provide the first clues that E3 ligase genes may function in the regulation of peach ripening.

Background Heat shock factors (HSFs) play important roles during normal plant growth and development and when plants respond to diverse stressors. Although most studies have focused on the involvement of HSFs in the response to abiotic stresses, especially in model plants, there is little research on their participation in plant growth and development or on the HSF (PpHSF) gene family in peach (Prunus persica). Methods DBD (PF00447), the HSF characteristic domain, was used to search the peach genome and identify PpHSFs. Phylogenetic, multiple alignment and motif analyses were conducted using MEGA 6.0, ClustalW and MEME, respectively. The function of PpHSF5 was confirmed by overexpression of PpHSF5 into Arabidopsis. Results Eighteen PpHSF genes were identified within the peach genome. The PpHSF genes were nonuniformly distributed on the peach chromosomes. Seventeen of the PpHSFs (94.4%) contained one or two introns, except PpHSF18, which contained three introns. The in silico-translated PpHSFs were classified into three classes (PpHSFA, PpHSFB and PpHSFC) based on multiple alignment, motif analysis and phylogenetic comparison with HSFs from Arabidopsis thaliana and Oryza sativa. Dispersed gene duplication (DSD at 67%) mainly contributed to HSF gene family expansion in peach. Promoter analysis showed that the most common cis-elements were the MYB (abiotic stress response), ABRE (ABA-responsive) and MYC (dehydration-responsive) elements. Transcript profiling of 18 PpHSFs showed that the expression trend of PpHSF5 was consistent with shoot length changes in the cultivar ‘Zhongyoutao 14’. Further analysis of the PpHSF5 was conducted in 5-year-old peach trees, Nicotiana benthamiana and Arabidopsis thaliana, respectively. Tissue-specific expression analysis showed that PpHSF5 was expressed predominantly in young vegetative organs (leaf and apex). Subcellular localization revealed that PpHSF5 was located in the nucleus in N. benthamiana cells. Two transgenic Arabidopsis lines were obtained that overexpressed PpHSF5. The root length and the number of lateral roots in the transgenic seedlings were significantly less than in WT seedlings and after cultivation for three weeks. The transgenic rosettes were smaller than those of the WT at 2–3 weeks. The two transgenic lines exhibited a dwarf phenotype three weeks after transplanting, although there was no significant difference in the number of internodes. Moreover, the PpHSF5-OE lines exhibited enhanced thermotolerance. These results indicated that PpHSF5 might be act as a suppresser of growth and development of root and aerial organs.

Peach (Prunus persica L. Batsch) trees grow vigorously and are subject to intense pruning during orchard cultivation. Reducing the levels of endogenous gibberellins (GAs) represents an effective method for controlling branch growth. Gibberellin 2-oxidases (GA2oxs) deactivate bioactive GAs, but little is known about the GA2ox gene family in peach. In this study, we identified seven PpGA2ox genes in the peach genome, which were clustered into three subgroups: C19-GA2ox-I, C19-GA2ox-II, and C20-GA2ox-I. Overexpressing representative genes from the three subgroups, PpGA2ox-1, PpGA2ox-5, and PpGA2ox-2, in tobacco resulted in dwarf plants with shorter stems and smaller leaves than the wild type. An analysis of the GA metabolic profiles of the transgenic plants showed that PpGA2ox-5 (a member of subgroup C19-GA2ox-II) is simultaneously active against both C19-GAs and C20-GAs,which implied that C19-GA2ox-II enzymes represent intermediates of C19-GA2oxs and C20-GA2oxs. Exogenous GA3 treatment of shoot tips activated the expression of all seven PpGA2ox genes, with different response times: the C19-GA2ox genes were transcriptionally activated more rapidly than the C20-GA2ox genes. GA metabolic profile analysis suggested that C20-GA2ox depletes GA levels more broadly than C19-GA2ox. These results suggest that the PpGA2ox gene family is responsible for fine-tuning endogenous GA levels in peach. Our findings provide a theoretical basis for appropriately controlling the vigorous growth of peach trees.

Apple (Malus domestica Borkh.), an economically important tree fruit worldwide, frequently suffers from temperature stress during growth and development, which strongly affects the yield and quality. Heat shock protein 20 (HSP20) genes play crucial roles in protecting plants against abiotic stresses. However, they have not been systematically investigated in apple. In this study, we identified 41 HSP20 genes in the apple ‘Golden Delicious’ genome. These genes were unequally distributed on 15 different chromosomes and were classified into 10 subfamilies based on phylogenetic analysis and predicted subcellular localization. Chromosome mapping and synteny analysis indicated that three pairs of apple HSP20 genes were tandemly duplicated. Sequence analysis revealed that all apple HSP20 proteins reflected high structure conservation and most apple HSP20 genes (92.6%) possessed no introns, or only one intron. Numerous apple HSP20 gene promoter sequences contained stress and hormone response cis-elements. Transcriptome analysis revealed that 35 of 41 apple HSP20 genes were nearly unchanged or downregulated under normal temperature and cold stress, whereas these genes exhibited high-expression levels under heat stress. Subsequent qRT-PCR results showed that 12 of 29 selected apple HSP20 genes were extremely up-regulated (more than 1,000-fold) after 4 h of heat stress. However, the heat-upregulated genes were barely expressed or downregulated in response to cold stress, which indicated their potential function in mediating the response of apple to heat stress. Taken together, these findings lay the foundation to functionally characterize HSP20 genes to unravel their exact role in heat defense response in apple.

Adventitious root (AR) formation is indispensable for vegetative asexual propagation. Indole-3-butyric acid (IBA) functioned indirectly as precursor of IAA in regulating AR formation. Ethylene affects auxin synthesis, transport, and/or signaling processes. However, the interactions between auxin and ethylene that control AR formation in apple have not been elucidated. In this study, we investigated the effects of IBA and its interaction with ethylene on AR development in apple. The results revealed that IBA stimulated the formation of root primordia, increased the number of ARs, and upregulated expression of genes (MdWOX11, MdLBD16, and MdLBD29) involved in AR formation. Comparison of different periods of IBA application indicated that IBA was necessary for root primordium formation, while long time IBA treatment obviously inhibited root elongation. RNA-seq analysis revealed that many plant hormone metabolism and signal transduction related genes were differentially expressed. IBA stimulated the production of ethylene during AR formation. Auxin inhibiting ARs elongation depended on ethylene. Together, our results suggest that the inhibitory role of auxin on AR elongation in apples is partially mediated by stimulated ethylene production.