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Melatonin pre-treatment significantly increases the tolerance of both drought-tolerant Malus prunifolia and drought-sensitive M. hupehensis plants. Its beneficial effects include better water conservation in leaves, less electrolyte leakage, steady chlorophyll contents, and greater photosynthetic performance under stress conditions. Melatonin selectively down-regulates MdNCED3, an abscisic acid (ABA) synthesis gene, and up-regulates its catabolic genes, MdCYP707A1 and MdCYP707A2, thereby reducing ABA contents in drought-stressed plants. Melatonin also directly scavenges H₂O₂ and enhances the activities of antioxidant enzymes to detoxify H₂O₂ indirectly. These two mechanisms work synergistically to improve the functions of stomata, i.e. causing them to re-open. Plants can effectively regulate their water balance under drought conditions by up-regulating the expression of melatonin synthesis genes MdTDC1, MdAANAT2, MdT5H4, and MdASMT1. Therefore, inducing melatonin production is an important mechanism by which plants can counteract the influence of this abiotic stressor.

MdMYB1 is a crucial regulator of light-induced anthocyanin biosynthesis and fruit coloration in apple (Malus domestica). In this study, it was found that MdMYB1 protein accumulated in the light but degraded via a ubiquitin-dependent pathway in the dark. Subsequently, the MdCOP1-1 and MdCOP1-2 genes were isolated from apple fruit peel and were functionally characterized in the Arabidopsis (Arabidopsis thaliana) cop1-4 mutant. Yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that MdMYBl interacts with the MdCOP1 proteins. Furthermore, in vitro and in vivo experiments indicated that MdCOP1s are necessary for the ubiquitination and degradation of MdMYB1 protein in the dark and are therefore involved in the light-controlled stability of the MdMYB1 protein. Finally, a viral vectorbased transformation approach demonstrated that MdCOP1s negatively regulate the peel coloration of apple fruits by modulating the degradation of the MdMYB1 protein. Our findings provide new insight into the mechanism by which light controls anthocyanin accumulation and red fruit coloration in apple and even other plant species.

Sugars play important roles in plant growth and development, crop yield and quality, as well as responses to abiotic stresses. Abscisic acid (ABA) is a multifunctional hormone. However, the exact mechanism by which ABA regulates sugar accumulation is largely unknown in plants. Here, we tested the expression profile of several sugar transporter and amylase genes in response to ABA treatment. MdSUT2 and MdAREB2 were isolated and genetically transformed into apple (Malus domestica) to investigate their roles in ABA-induced sugar accumulation. The MdAREB2 transcription factor was found to bind to the promoters of the sugar transporter and amylase genes and activate their expression. Both MdAREB2 and MdSUT2 transgenic plants produced more soluble sugars than controls. Furthermore, MdAREB2 promoted the accumulation of sucrose and soluble sugars in an MdSUT2-dependent manner. Our results demonstrate that the ABA-responsive transcription factor MdAREB2 directly activates the expression of amylase and sugar transporter genes to promote soluble sugar accumulation, suggesting a mechanism by which ABA regulates sugar accumulation in plants.

Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis.

SIZ1 (a SIZ/PIAS-type SUMO E3 ligase)-mediated small ubiquitin-like modifier (SUMO) modification of target proteins is important for various biological processes related to abiotic stress resistance in plants; however, little is known about its role in resistance toward iron (Fe) deficiency. Here, the SUMO E3 ligase MdSIZ1 was shown to be involved in the plasma membrane (PM) H⁺-ATPase-mediated response to Fe deficiency. Subsequently, a basic helix-loop-helix transcription factor, MdbHLH104 (a homolog of Arabidopsis bHLH104 in apple), which acts as a key component in regulating PM H⁺-ATPase-mediated rhizosphere acidification and Fe uptake in apples (Malus domestica), was identified as a direct target of MdSIZ1. MdSIZ1 directly sumoylated MdbHLH104 both in vitro and in vivo, especially under conditions of Fe deficiency, and this sumoylation was required for MdbHLH104 protein stability. Double substitution of K139R and K153R in MdbHLH104 blocked MdSIZ1-mediated sumoylation in vitro and in vivo, indicating that the K139 and K153 residues were the principal sites of SUMO conjugation. Moreover, the transcript level of the MdSIZ1 gene was substantially induced following Fe deficiency. MdSIZ1 overexpression exerted a positive influence on PM H⁺-ATPase-mediated rhizosphere acidification and Fe uptake. Our findings reveal an important role for sumoylation in the regulation of PM H⁺-ATPase-mediated rhizosphere acidification and Fe uptake during Fe deficiency in plants.

A plant pathway that initiates with the formation of citramalate from pyruvate and acetyl-CoA by citramalate synthase (CMS) is shown to contribute to the synthesis of α-ketoacids and important odor-active esters in apple (Malus × domestica) fruit. Microarray screening led to the discovery of a gene with high amino acid similarity to 2-isopropylmalate synthase (IPMS). However, functional analysis of recombinant protein revealed its substrate preference differed substantially from IPMS and was more typical of CMS. MdCMS also lacked the regulatory region present in MdIPMS and was not sensitive to feedback inhibition. 13C-acetate feeding of apple tissue labeled citramalate and α-ketoacids in a manner consistent with the presence of the citramalate pathway, labeling both straight- and branched-chain esters. Analysis of genomic DNA (gDNA) revealed the presence of two nearly identical alleles in “Jonagold” fruit (MdCMS_1 and MdCMS_2), differing by two nonsynonymous single-nucleotide polymorphisms (SNPs). The mature proteins differed only at amino acid 387, possessing either glutamine387 (MdCMS_1) or glutamate387 (MdCMS_2). Glutamate387 was associated with near complete loss of activity. MdCMS expression was fruit-specific, increasing severalfold during ripening. The translated protein product was detected in ripe fruit. Transient expression of MdCMS_1 in Nicotiana benthamiana induced the accumulation of high levels of citramalate, whereas MdCMS_2 did not. Domesticated apple lines with MdCMS isozymes containing only glutamate387 produced a very low proportion of 2-methylbutanol-and 2-methylbutanoate (2MB) and 1-propanol and propanoate (PROP) esters. The citramalate pathway, previously only described in microorganisms, is shown to function in ripening apple and contribute to isoleucine and 2MB and PROP ester biosynthesis without feedback regulation.

Summary In this study, we characterized the role of an apple cytosolic malate dehydrogenase gene (MdcyMDH) in the tolerance to salt and cold stresses and investigated its regulation mechanism in stress tolerance. The MdcyMDH transcript was induced by mild cold and salt treatments, and MdcyMDH‐overexpressing apple plants possessed improved cold and salt tolerance compared to wild‐type (WT) plants. A digital gene expression tag profiling analysis revealed that MdcyMDH overexpression largely altered some biological processes, including hormone signal transduction, photosynthesis, citrate cycle and oxidation–reduction. Further experiments verified that MdcyMDH overexpression modified the mitochondrial and chloroplast metabolisms and elevated the level of reducing power, primarily caused by increased ascorbate and glutathione, as well as the increased ratios of ascorbate/dehydroascorbate and glutathione/glutathione disulphide, under normal and especially stress conditions. Concurrently, the transgenic plants produced a high H2O2 content, but a low O2·− production rate was observed compared to the WT plants. On the other hand, the transgenic plants accumulated more free and total salicylic acid (SA) than the WT plants under normal and stress conditions. Taken together, MdcyMDH conferred the transgenic apple plants a higher stress tolerance by producing more reductive redox states and increasing the SA level; MdcyMDH could serve as a target gene to genetically engineer salt‐ and cold‐tolerant trees.

Malus hupehensis is an excellent Malus rootstock species, known for its strong adverse-resistance and apomixes. In the present study, stem cuttings of M. hupehensis were treated with three types of exogenous hormones, including indole acetic acid (IAA), naphthalene acetic acid (NAA), or green growth regulator (GGR). The effects and mechanisms of exogenous hormone treatment and antioxidant enzyme activity on adventitious root formation were investigated. The results showed that the apparent morphology of the adventitious root had four stages, including root pre-emergence stage (S0), early stage of root formation (S1), massive root formation stage (S2), and later stage of root formation (S3). The suitable concentrations of the three exogenous hormones, IAA, NAA and GGR, were 100 mg·L-1, 300 mg·L-1, and 300 mg·L-1, respectively. They shortened the rooting time by 25-47.4% and increased the rooting percentages of cuttings by 0.9-1.3 times, compared with that in the control. The dispersion in S0 stage was 3.6 times of that in the S1 stage after exogenous hormone application. The earlier the third critical point (P3) appeared, the shorter the rooting time and the greater the rooting percentage of the cuttings. During rhizogenesis, the activities of three antioxidant enzymes (POD, SOD, and PPO) showed an A-shaped trend. However, peak values of enzyme activity appeared at different points, which were 9 d before the P3, P3, and the fourth critical point (P4), respectively. Exogenous hormone treatment reduced the time to reach the peak value by 18 days, although the peak values of the enzymatic activities did not significantly changed. Our results suggested that exogenous hormone treatment mainly acted during the root pre-emergence stage, accelerated the synthesis of antioxidant enzymes, reduced the rooting time, and consequently promoted root formation. The three kinds of antioxidant enzymes acted on different stages of rooting.

Background While there is now a significant body of research correlating apple ( Malus x domestica ) fruit softening with the cell wall hydrolase ENDO -POLYGALACTURONASE1 (PG1), there is currently little knowledge of its physiological effects in planta . This study examined the effect of down regulation of PG1 expression in ‘Royal Gala’ apples, a cultivar that typically has high levels of PG1, and softens during fruit ripening. Results PG1 -suppressed ‘Royal Gala’ apples harvested from multiple seasons were firmer than controls after ripening, and intercellular adhesion was higher. Cell wall analyses indicated changes in yield and composition of pectin, and a higher molecular weight distribution of CDTA-soluble pectin. Structural analyses revealed more ruptured cells and free juice in pulled apart sections, suggesting improved integrity of intercellular connections and consequent cell rupture due to failure of the primary cell walls under stress. PG1 -suppressed lines also had reduced expansion of cells in the hypodermis of ripe apples, resulting in more densely packed cells in this layer. This change in morphology appears to be linked with reduced transpirational water loss in the fruit. Conclusions These findings confirm PG1’s role in apple fruit softening and suggests that this is achieved in part by reducing cellular adhesion. This is consistent with previous studies carried out in strawberry but not with those performed in tomato. In apple PG1 also appears to influence other fruit texture characters such as juiciness and water loss.

Background Iron (Fe) is an essential micronutrient for plants. Utilization of Fe deficiency-tolerant rootstock is an effective strategy to prevent Fe deficiency problems in fruit trees production. Malus halliana is an apple rootstock that is resistant to Fe deficiency; however, few molecular studies have been conducted on M. halliana . Results To evaluate short-term molecular response of M. halliana leaves under Fe deficiency condition, RNA sequencing (RNA-Seq) analyses were conducted at 0 (T1), 0.5 (T2) and 3 d (T3) after Fe-deficiency stress, and the timepoints were determined with a preliminary physiological experiment. In all, 6907, 5328, and 3593 differentially expressed genes (DEGs) were identified in pairs of T2 vs. T1, T3 vs. T1, and T3 vs. T2. Several of the enriched DEGs were related to heme binding, Fe ion binding, thylakoid membranes, photosystem II, photosynthesis-antenna protein, porphyrin and chlorophyll metabolism and carotenoid biosynthesis under Fe deficiency, which suggests that Fe deficiency mainly affects the photosynthesis of M. halliana . Additionally, we found that Fe deficiency induced significant down-regulation in genes involved in photosynthesis at T2 when seedlings were treated with Fe-deficient solution for 0.5 d, indicating that there was a rapid response of M. halliana to Fe deficiency. A strong up-regulation of photosynthesis genes was detected at T3, which suggested that M. halliana was able to recover photosynthesis after prolonged Fe starvation. A similar expression pattern was found in pigment regulation, including genes for coding chlorophyllide a oxygenase (CAO), β-carotene hydroxylase (β-OHase), zeaxanthin epoxidase (ZEP) and 9- cis -epoxycarotenoid dioxygenase (NCED). Our results suggest that pigment regulation plays an important role in the Fe deficiency response. In addition, we verified sixteen genes related to photosynthesis-antenna protein, porphyrin and chlorophyll metabolism and carotenoid biosynthesis pathways using quantitative real-time PCR (qRT-PCR) to ensure the accuracy of transcriptome data. Photosynthetic parameters, Chl fluorescence parameters and the activity of Chlase were also determined. Conclusions This study broadly characterizes a molecular mechanism in which pigment and photosynthesis-related regulations play indispensable roles in the response of M. halliana to short-term Fe deficiency and provides a basis for future analyses of the key genes involved in the tolerance of Fe deficiency.