A chromosome-scale genome assembly of Malus domestica, a multi-stress resistant apple variety.

Hanfu apple is the main cultivar grown in the cool areas of Northeast, Northwest, and North China. Here, we proposed a chromosome-level Hanfu genome assembly using PacBio, Illumina and Hi-C sequencing data. The total contig length was 628.99 Mb, with scaffold and contig N50 sizes of 36.18 Mb and 1.25 Mb, respectively. The Hanfu genome had a total of 39,617 genes, of which we predicted the function for 38,816. Evolutionary analysis showed that Hanfu may have undergone a γ-event, a recent whole-genome duplication. A comparative analysis was conducted on the genomes of Hanfu and homozygous triploid HFTH1, which were cultured using the anthers of diploid Hanfu apples. Three variants were identified, including 2,155,184 single nucleotide polymorphisms (SNPs), 413,108 insertions/deletions (indels), and 7,587 structural variants (SVs).This high-quality genome will provide a reference for the genetic improvement of apples and the breeding of more varieties with high resistance and high quality.

Preharvest application of prohydrojasmon affects color development, phenolic metabolism, and pigment-related gene expression in red pear (Pyrus ussuriensis).

BACKGROUND: Peel color is an economically relevant trait that influences the appearance and quality of red pear, whose red color is due to anthocyanin accumulation. Prohydrojasmon (PDJ), which has similar effects to endogenous jasmonates, was developed as a commercial bioregulator, particularly to improve fruits coloring. However, little information is available about the effect of PDJ on pears. This study investigated the effects of preharvest PDJ treatments on color development, phenolic compounds accumulation, and related gene expression in the red pear cultivar 'Nanhong'. The treatments were performed during the pre-color-change period by spraying 50 or 100 mg L-1 of PDJ on fruits. RESULTS: Preharvest PDJ treatments had a significant effect on color development, without affecting other quality parameters such as total soluble solids and fruit acidity. Liquid chromatography-mass spectrometry analysis showed that concentrations of anthocyanins and flavonols were enhanced in the peel after PDJ treatments, particularly when a concentration of 100 mg L-1 was used, whereas those of hydroxycinnamates and flavanols were decreased. After PDJ application, the transcription levels of anthocyanin biosynthesis genes PAL, CHS, CHI, ANS, F3H, and UFGT were enhanced, especially under the higher PDJ concentration tested. In addition, anthocyanin accumulation in the peels of PDJ-treated fruits was found to be positively correlated with the upregulation of the regulatory gene MYB114. CONCLUSION: Preharvest treatments with PDJ could be a useful tool to improve fruits coloring and increase phenolic content in pear. These findings also improve our understanding of the molecular mechanisms associated with PDJ-regulated anthocyanin accumulation in pear fruits.

Comparative proteomic analysis reveals the roots response to low root-zone temperature in Malus baccata.

Soil temperature is known to affect plant growth and productivity. In this study we found that low root-zone temperature (LRT) inhibited the growth of apple (Malus baccata Borkh.) seedlings. To elucidate the molecular mechanism of LRT response, we performed comparative proteome analysis of the apple roots under LRT for 6 days. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis (2-DE) and 29 differentially accumulated proteins were successfully identified by MALDI-TOF/TOF mass spectrometry. They were involved in protein transport/processing/degradation (21%), glycometabolism (20%), response to stress (14%), oxidoreductase activity (14%), protein binding (7%), RNA metabolism (7%), amino acid biosynthesis (3%) and others (14%). The results revealed that LRT inhibited glycometabolism and RNA metabolism. The up-regulated proteins which were associated with oxidoreductase activity, protein metabolism and defense response, might be involved in protection mechanisms against LRT stress in the apple seedlings. Subsequently, 8 proteins were selected for the mRNA quantification analysis, and we found 6 of them were consistently regulated between protein and mRNA levels. In addition, the enzyme activities in ascorbate-glutathione (AsA-GSH) cycle were determined, and APX activity was increased and GR activity was decreased under LRT, in consistent with the protein levels. This study provides new insights into the molecular mechanisms of M. baccata in responding to LRT.

Physiological and de novo transcriptome analysis of the fermentation mechanism of Cerasus sachalinensis roots in response to short-term waterlogging.

BACKGROUND: Cerasus sachalinensis is widely used in cool regions as a sweet cherry rootstock and is known for its sensitivity to soil waterlogging and waterlogging stress. However, the limited availability of Cerasus genomic resources has considerably restricted the exploration of its waterlogging response mechanism. To understand its reaction to short-term waterlogging, we analyzed the physiology and transcriptomes of C. sachalinensis roots in response to different waterlogging durations. RESULTS: In this study, 12,487 differentially expressed genes (DEGs) were identified from Cerasus sachalinensis roots under different waterlogging durations. Carbon metabolism and energy maintenance formed the first coping mechanism stage of C. sachalinensis in response to low oxygen conditions. Root energy processes, including root respiration and activities of the fermentation enzymes alcohol dehydrogenase, pyruvate decarboxylase, and lactate dehydrogenase, showed unique changes after 0 h, 3 h, 6 h, and 24 h of waterlogging exposure. Ribonucleic acid sequencing was used to analyze transcriptome changes in C. sachalinensis roots treated with 3 h, 6 h, and 24 h of waterlogging stress. After de novo assembly, 597,474 unigenes were recognized, of which 355,350 (59.47%) were annotated. To identify the most important pathways represented by DEGs, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases were used to compare these genes. The first stage of root reaction to waterlogging stress was activation of carbohydrate metabolism to produce more glucose and maintain energy levels. At 3 h, the glycolytic and fermentation pathways were activated to maintain adenosine triphosphate production. At 24 h, pathways involved in the translation of proteins were activated to further assist the plant in tolerating waterlogging stress. These findings will facilitate a further understanding of the potential mechanisms of plant responses to waterlogging at physiological and transcriptome levels. CONCLUSIONS: Carbon metabolism and energy maintenance formed the first coping mechanism C. sachalinensis in response to low oxygen conditions, and they may be responsible for its short-term waterlogging response. Our study not only provides the assessment of genomic resources of Cerasus but also paves the way for probing the metabolic and molecular mechanisms underlying the short-term waterlogging response in C. sachalinensis.

Differential Proteomic Analysis Reveals the Effect of Calcium on Malus baccata Borkh. Leaves under Temperature Stress.

In the cool apple-producing areas of northern China, air temperature during early spring changes in a rapid and dramatic manner, which affects the growth and development of apple trees at the early stage of the growing season. Previous studies have shown that the treatment of calcium can increase the cold tolerance of Malus baccata Borkh., a widely-used rootstock apple tree in northern China. To better understand the physiological function of calcium in the response of M. baccata to temperature stress, we analyzed the effect of calcium treatment (2% CaCl2) on M. baccata leaves under temperature stress. Physiological analysis showed that temperature stress aggravated membrane lipid peroxidation, reduced chlorophyll content and induced photo-inhibition in leaves, whereas these indicators of stress injuries were alleviated by the application of calcium. An isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics approach was used in this study. Among the 2114 proteins that were detected in M. baccata leaves, 41, 25, and 34 proteins were differentially regulated by the increasing, decreasing, and changing temperature treatments, respectively. Calcium treatment induced 9 and 15 proteins after increasing and decreasing temperature, respectively, in comparison with non-treated plants. These calcium-responsive proteins were mainly related to catalytic activity, binding, and structural molecule activity. Hierarchical cluster analysis indicated that the changes in abundance of the proteins under increasing temperature and changing temperature treatments were similar, and the changes in protein abundance under decreasing temperature and increasing temperature with calcium treatment were similar. The findings of this study will allow a better understanding of the mechanisms underlying the role of calcium in M. baccata leaves under temperature stress.

Exogenous Spermidine Alleviated Low-Temperature Damage by Affecting Polyamine Metabolism and Antioxidant Levels in Apples.

Low-temperature stress significantly limits the growth, development, and geographical distribution of apple cultivation. Spermidine (Spd), a known plant growth regulator, plays a vital role in the plant's response to abiotic stress. Yet, the mechanisms by which exogenous Spd enhances cold resistance in apples remain poorly understood. Therefore, the present study analyzed the effects of exogenous Spd on antioxidant enzyme activity, polyamine metabolism, and related gene expression levels of 1-year-old apple branches under low-temperature stress. Treatment with exogenous Spd was found to stabilize branch tissue biofilms and significantly reduce the levels of reactive oxygen species by elevating proline content and boosting the activity of antioxidants such as superoxide dismutase. It also upregulated the activities of arginine decarboxylase, S-adenosylmethionine decarboxylase, and spermidine synthase and the expression levels of MdADC1, MdSAMDC1, and MdSPDS1 under low-temperature stress and led to the accumulation of large amounts of Spd and spermine. Moreover, compared with the 2 mmol·L-1 Spd treatment, the 1 mmol·L-1 Spd treatment increased the expression levels of cold-responsive genes MdCBF1/2/3, MdCOR47, and MdKIN1, significantly. The findings suggest that exogenous Spd can enhance cold resistance in apple branches significantly. This enhancement is achieved by modulating polyamine metabolism and improving antioxidant defense mechanisms, which could be exploited to improve apple cultivation under cold stress conditions.

Responses of soil microorganisms, enzyme activities and nutrient contents to inter-row grass ploughing and returning to the field in a natural sod culture apple orchard.

Natural sod culture in orchard is an effective measure to improve the orchard productivity and promote the sustainable production. To explore the effects of inter-row grass ploughing and returning on soil biological cha-racteristics and nutrient contents, we examined the effects of different grass returning to the field on the amount of soil microorganisms, enzyme activities and nitrogen and potassium contents of 0-20 cm soil layer. There are three treatments, cleaning tillage as the control (CK), conventional mowing management (NG), and soil ploughing annually under natural sod culture with conventional mowing condition (NGR) treatments. The results showed that soil microorganisms were dominated by bacteria, followed by actinomycetes, with the least fungi. Compared with CK, both NG and NGR treatments significantly improved the abundance of soil bacteria and fungi, with the strongest effects in NGR treatment, and significantly increased the soil urease, sucrase and catalase activities by 59.0%, 20.7%, 38.3% and 73.5%, 45.9%, 67.8%, respectively. NGR treatment significantly increased soil nitrogen and potassium contents, with the contents of ammonium nitrogen, nitrate nitrogen, particulate organic nitrogen, microbial biomass nitrogen, available potassium and water-soluble potassium being 1.5, 1.8, 1.6, 2.0, 1.3 and 1.4 times of that in CK, respectively. NGR significantly increased soluble sugar content and sugar acid ratio and subsequently improved fruit quality. Overall, NGR increased soil microbial abundance, enzyme activities, nitrogen, potassium contents and fruit quality, which could be a feasible management of inter-row grasses in the natural sod culture apple orchard.

Natural Grass Cultivation Management Improves Apple Fruit Quality by Regulating Soil Mineral Nitrogen Content and Carbon-Nitrogen Metabolism.

Orchard grass cultivation management is an effective measure to safeguard the sustainable development of the fruit industry in China. However, details of the influence of natural sod culture management on carbon (C)-nitrogen (N) nutrition of trees and fruit quality in Hanfu apple orchards are lacking. Therefore, a field experiment was conducted, which consisted of two treatments: clean tillage (CT) and natural grass cultivation (NG). Results revealed that NG treatment contributed to the increases in soil organic matter (SOM), total N, and soil NH4+-N at depths of 0-20 cm and 20-40 cm, while the soil NO3--N concentration under NG treatment was significantly decreased at the same depth, within the range of 0-200 cm of the soil profile, compared with CT. NG treatment also significantly promoted leaf photosynthesis and enhanced leaf N and fruit sugar metabolism. The results of isotope labeling showed that NG treatment obviously elevated the 13C accumulation and distribution rate in fruits, as well as the 15N accumulation in the whole tree, whereas the 15N accumulation in fruits decreased. Furthermore, NG treatment significantly increased the fruit anthocyanin content. These results provide theoretical references for the feasibility of natural sod culture management to improve fruit quality in Hanfu apple orchards.

Rootstock-scion interaction affects Malus transcriptome profiles in response to cadmium.

Apple production is threatened by cadmium contamination in orchards. Cd accumulation and tolerance in grafted Malus plants is affected by rootstock, scion, and their interaction. This dataset is part of an experiment investigating the molecular mechanism of Cd bioaccumulation and tolerance in different apple rootstock-scion combinations. We exposed four rootstock-scion combinations to Cd treatment consisting of Hanfu and Fuji apple (Malus domestica) scions grafted onto apple rootstocks of M. baccata or M. micromalus "qingzhoulinqin". RNA sequencing was conducted in roots and leaves of grafting combinations under 0 or 50 µM CdCl2 conditions. A comprehensive transcriptional dataset of affected rootstock, scion, and their interaction among different graft combinations was obtained. This dataset provides new insights in the transcriptional control of Cd bioaccumulation and tolerance in grafting plants regulated by rootstock and scion. Herein, we discuss the molecular mechanism underlying Cd absorption and bioaccumulation.

Characteristics of cadmium accumulation and tolerance in apple plants grown in different soils.

Cadmium (Cd) is a nonessential element and highly toxic to apple tree. However, Cd accumulation, translocation and tolerance in apple trees planted in different soils remain unknown. To investigate soil Cd bioavailability, plant Cd accumulation, physiological changes as well as gene expression patterns in apple trees grown in five different soils, 'Hanfu' apple seedlings were planted in orchard soils collected from Maliangou village (ML), Desheng village (DS), Xishan village (XS), Kaoshantun village (KS) and Qianertaizi village (QT), and subjected to 500 µM CdCl2 for 70 d. Results showed that soils of ML and XS had higher content of organic matter (OM), clay and silt, and cation exchange capacity (CEC) but lower sand content than the other soils, thereby reduced Cd bioavailability, which could be reflected by lower concentrations and proportions of acid-soluble Cd but higher concentrations and proportions of reducible and oxidizable Cd. The plants grown in soils of ML and XS had relatively lower Cd accumulation levels and bio-concentration factors than those grown in the other soils. Excess Cd reduced plant biomass, root architecture, and chlorophyll content in all plants but to relatively lesser degree in those grown in soils of ML and XS. The plants grown in soils of ML, XS and QT had comparatively lower reactive oxygen species (ROS) content, less membrane lipid peroxidation, and higher antioxidant content and enzyme activity than those grown in soils of DS and KS. Transcript levels of genes regulating Cd uptake, transport and detoxification such as HA11, VHA4, ZIP6, IRT1, NAS1, MT2, MHX, MTP1, ABCC1, HMA4 and PCR2 displayed significant differences in roots of plants grown in different soils. These results indicate that soil types affect Cd accumulation and tolerance in apple plants, and plants grown in soils with higher OM content, CEC, clay and silt content and lower sand content suffer less Cd toxicity.

MdSWEET23, a sucrose transporter from apple (Malus × domestica Borkh.), influences sugar metabolism and enhances cold tolerance in tomato.

Photosynthetic products in most fleshy fruits are unloaded via the apoplasmic pathway. Sugar transporters play an important role in the apoplasmic unloading pathway and are involved in sugar transport for fruit development. The MdSWEET23, cloned from ''Hanfu'' apple (Malus × domestica Borkh.) fruits, belongs to Clade III of the SWEET family. Subcellular localization revealed that MdSWEET23 is localized on the plasma membrane. ß-glucuronidase activity assays showed that MdSWEET23 was primarily expressed in the sepal and carpel vascular bundle of apple fruits. Heterologous expression assays in yeast showed that MdSWEET23 functions in sucrose transport. The overexpression of MdSWEET23 in the ''Orin" calli increased the soluble sugar content. The silencing of MdSWEET23 significantly reduced the contents of sucrose and sorbitol in apple fruits. Ectopic overexpression of MdSWEET23 in tomato altered sugar metabolism and distribution in leaves and fruits, causing a reduction in photosynthetic rates and plant height, enhanced cold stress tolerance, and increased the content of sucrose, fructose, and glucose in breaking color fruits, but did not increase sugar sink potency of tomato fruits.

Alleviation effect of GR24, a strigolactone analogue, on low-nitrogen stress in Malus baccata seedlings.

To investigate the efficacy of foliar application of GR24, a strigolactone analogue, in alleviating low-nitrogen stress in Malus baccata, we applied GR24 with different concentrations (0, 1, 5, 10, and 20 µmol·L-1) to leaves of plants under low nitrogen stress. We evaluated the changes in photosynthetic characteristics of leaves, reactive oxygen metabolism, and nitrogen assimilation in roots. The results showed that shoot biomass of seedling significantly decreased and root-shoot ratio increased under low-nitrogen stress. The chlorophyll contents decreased, the carotenoid content increased, and the photosynthetic activity decreased. The activities of superoxide dismutase and catalase enzymes in roots changed little, while the activities of peroxidase and ascorbic acid peroxidase enzymes, along with the levels of soluble sugar, free proline, and reactive oxygen species showed a significant increase, and the soluble protein content decreased. The NO3- content in roots decreased, the NH4+ content increased, while activities of nitrate reductase and glutamine synthase decreased. Compared to the control group without GR24 application, foliar sprays of 10 and 20 µmol·L-1 GR24 under both normal and low-nitrogen increased biomass and root-shoot ratio to varying degrees. Additionally, GR24 application increased chlorophyll content, photosynthesis indices (net photosynthetic rate, transpiration rate and stomatal conductance), and fluorescence (maximum photochemical efficiency of PSⅡ and quantum yield of electron transfer per unit area) performance parameters, as well as the contents of osmotic regulation substances (soluble protein, soluble sugar, and free proline) and glutamine synthase activity. Application of 10 and 20 µmol·L-1 GR24 under low-nitrogen stress decreased carotenoid, reactive oxygen species, and NH4+ contents, while increased the activities of antioxidases and key enzymes in nitrogen metabolism (nitrate reductase and glutamine synthase) and NO3- content. The 10 µmol·L-1 GR24 treatment was the most effective in alleviating low nitrogen stress, which has potential for application in apple orchards with low nitrogen soil.

Glucose addition promotes C fixation and bacteria diversity in C-poor soils, improves root morphology, and enhances key N metabolism in apple roots.

The interaction between plant, soil and microorganism plays a crucial role in sustainable development of terrestrial ecosystem function and diversity. However, little information is known about how plant growth, soil organic carbon (C) fractions and microorganism respond to exogenous C addition in soils with low organic C content. Three levels of 13C-glucose (equal to 0, 100% and 500% of initial microbial biomass C) were added to non-sterilized (corresponding to treatment abbreviation of CK, Glu-1, Glu-2, respectively) and sterilized soils (corresponding to treatment abbreviation of SS, SS+Glu-1, SS+Glu-2, respectively) planted with apple rootstock (Malus baccata (L.) Borkh.) seedings. The objectives of this study were to analyse the dynamics of soil organic C (SOC) fractions and soil bacterial community diversity with glucose levels and soil sterilization, and to explore the morphology of roots and nitrogen (N) metabolism by plant after glucose addition to sterilized/non-sterilized soils. Results showed that the contents of labile organic C fractions were significantly varied (P<0.05) with the levels of glucose addition and soil sterilization. SS+Glu-2 and Glu-2 treatments increased the contents of labile organic C fractions, on average, by 48.47% and 35.33% compared with no glucose addition, respectively. About 21.42% and 16.17% of glucose-C remained in sterilized and non-sterilized soils, respectively at the end of experiment (day 45). Regardless of soil sterilized or not, the glucose addition increased the richness and diversity indices of soil bacterial community compared with no-glucose addition. The glucose addition optimized root zone conditions, and enhanced root vitality, morphology and biomass. Both SS+Glu-2 and Glu-2 treatments significantly enhanced (P<0.05) the contents of nitrate (NO3-N) and nitrite (NO2-N), but sharply decreased (P<0.05) the ammonium (NH4+-N) content compared with no glucose addition. Also, these two treatments significantly (P<0.05) increased the enzymic activities and gene transcript levels involved in root N metabolism, which demonstrated that the high level of glucose addition promoted N assimilation and transformation into free amino acids by root. Overall, the addition of exogenous C to not only promotes its fixation and bacterial community diversity in C-poor soils, but also improves root morphology and N absorption by plant.

Differential pulp cell wall structures lead to diverse fruit textures in apple (Malus domestica).

In this study, we aimed to elucidate the effect of pulp cell wall structure on fruit hardness and crispness in apples. To this end, we studied the cell wall polysaccharides in two apple varieties, "Hanfu" and "Honeycrisp," during fruit development. Compared with Hanfu, the crispness of Honeycrisp was higher, whereas its harness was lower. The intensity and distribution of immunofluorescence signals indicated that galactose and arabinose contributed to the higher hardness of Hanfu, whereas arabinose, egg-box structure, and fucosylated xyloglucans, distributed in the corners of tricellular junctions, enhanced the cell-cell adhesion and improved the crispness of Honeycrisp. Besides, fucosylated xyloglucan played an important role in promoting the formation and maintaining the strength of the cell wall skeleton and, consequently, retaining the fruit crispness. The esterification state of pectin had little effect on the fruit hardness and crispness in both varieties. Collectively, our findings provided information on the underlying mechanism of fruit texture formation in apples.

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To explore the effects of zinc levels on the synthesis and translocation of photosynthetic products from leaves to fruits, and to lay a theoretical foundation for improving fruit quality through zinc supplementation during the critical period of apple fruit development, a field experiment was carried out with a eight-year old 'Hanfu'/GM256/Malus baccata Borkh apple. We used the 13C tracer method to examine the effects of different zinc levels (ZnSO4·H2O 0, 0.1%, 0.2%, 0.3%, 0.4%, expressed by CK, Zn1, Zn2, Zn3, Zn4, respectively) on translocation of photosynthate to fruit during the stage of fruit expanding. The results showed that, with increasing zinc concentration, Rubisco enzyme activity, net photosynthetic rate, sorbitol and sucrose content, sorbitol 6-phosphate dehydrogenase, and sucrose phosphate synthase enzyme activities of leaves first increased and then decreased, with the highest values being observed in Zn3 treatment. Zn3 treatment significantly increased the 13C assimilation capability of leaves. Compared with other treatments, the 13C of self-retention (labeled leaves and labeled branches) was lowest in Zn3 treatment (61.2%) and the output of 13C photoassimilates was highest in Zn3 treatment (38.8%). 13C absorption of apple fruit showed a trend of Zn3 ＞ Zn2 ＞ Zn4 ＞ Zn1 ＞ CK. In summary, foliar zinc application under appropriate concentration (0.3% ZnSO4·H2O) enhanced photosynthesis, increased the assimilation capability of leaves, and promoted the directional transportation of photosynthate to fruit.

Malus rootstocks affect copper accumulation and tolerance in trees by regulating copper mobility, physiological responses, and gene expression patterns.

We investigated the roles of rootstocks in Cu accumulation and tolerance in Malus plants by grafting 'Hanfu' (HF) scions onto M. baccata (Mb) and M. prunifolia (Mp) rootstocks, which have different Cu tolerances. The grafts were exposed to basal or excess Cu for 20 d. Excess Cu-treated HF/Mb had less biomass, and pronounced root architecture deformation and leaf ultrastructure damage than excess Cu-challenged HF/Mp. Root Cu concentrations and bio-concentration factor (BCF) were higher in HF/Mp than HF/Mb, whereas HF/Mb had higher stem and leaf Cu concentrations than HF/Mp. Excess Cu lowered root and aerial tissue BCF and translocation factor (Tf) in all plants; however, Tf was markedly higher in HF/Mb than in HF/Mp. The subcellular distribution of Cu in the roots and leaves indicated that excess Cu treatments increased Cu fixation in the root cell walls, which decreased Cu mobility. Compared to HF/Mb, HF/Mp sequestered more Cu in its root cell walls and less Cu in leaf plastids, nuclei, and mitochondria. Moreover, HF/Mp roots and leaves had higher concentrations of water-insoluble Cu compounds than HF/Mb, which reduced Cu mobility and toxicity. Fourier transform infrared spectroscopy analysis showed that the carboxyl, hydroxyl and acylamino groups of the cellulose, hemicellulose, pectin and proteins were the main Cu binding sites in the root cell walls. Excess Cu-induced superoxide anion and malondialdehyde were 28.6% and 5.1% lower, but soluble phenolics, ascorbate and glutathione were 10.5%, 41.9% and 17.7% higher in HF/Mp than HF/Mb leaves. Compared with HF/Mb, certain genes involved in Cu transport were downregulated, while other genes involved in detoxification were upregulated in HF/Mp roots and leaves. Our results show that Mp inhibited Cu translocation and mitigated Cu toxicity in Malus scions by regulating Cu mobility, antioxidant defense mechanisms, and transcription of key genes involved in Cu translocation and detoxification.

RBOH-dependent hydrogen peroxide signaling mediates melatonin-induced anthocyanin biosynthesis in red pear fruit.

Although several studies have confirmed that exogenous melatonin promotes anthocyanin accumulation, the molecular mechanism of this remains elusive. Here, the signaling cross-talk between melatonin and NADPH oxidase (RBOH) -mediated ROS during anthocyanin biosynthesis were investigated. We found that application of exogenous melatonin not only induced anthocyanin biosynthesis, but also increased endogenous H2O2 and O2‾ content in pear fruits. The effect of melatonin on anthocyanin biosynthesis was abolished by inhibitors of RBOH. We also observed that genes encoding RBOH (PuRBOHF) were ubiquitously and highly expressed after melatonin treatment. Transient PuRBOHF overexpression significantly enhanced anthocyanin accumulation and activated transcription of anthocyanin biosynthesis genes, whereas PuRBOHF silencing repressed melatonin-promoted anthocyanin accumulation and H2O2 production. Moreover, RBOH-derived H2O2 induced PuMYB10 transcription, and PuRBOHF enhanced the PuMYB10-induced activation of the PuUFGT promoter. PuMYB10, in turn, activated PuRBOHF transcription, revealing a positive feedback loop. These results provide molecular evidence supporting the essential roles of PuRBOHF-dependent H2O2 in melatonin-induced anthocyanin accumulation in pears.

Rootstock-Scion Interaction Affects Cadmium Accumulation and Tolerance of Malus.

To understand the roles of Malus rootstock, scion, and their interaction in Cd accumulation and tolerance, four scion/rootstock combinations consisting of the apple cultivars "Hanfu" (HF) and "Fuji" (FJ) grafted onto M. baccata (Mb) or M. micromalus "qingzhoulinqin" (Mm) rootstocks differing in relative Cd tolerance were exposed either to 0 µM or 50 µM CdCl2 for 18 d. Cd accumulation and tolerance in grafted Malus plants varied within rootstock, scion, and rootstock-scion interaction. Cd-induced decreases in photosynthesis, photosynthetic pigment level, and biomass were lower for HF grafted onto Mb than those for HF grafted onto Mm. Reductions in growth and photosynthetic rate were always the lowest for HF/Mb. Cd concentration, bioconcentration factor (BCF), and translocation factor (Tf ) were always comparatively higher in HF and FJ grafted onto rootstock Mm than in HF and FJ grafted on Mb, respectively. When HF and FJ were grafted onto the same rootstock, the root Cd concentrations were always higher in HF than FJ, whereas the shoot Cd concentrations displayed the opposite trend. The shoot Cd concentrations and Tf were lower for HF/Mb than the other scion/rootstock combinations. Rootstock, scion, and rootstock-scion interaction also affected subcellular Cd distribution. Immobilization of Cd in the root cell walls may be a primary Cd mobility and toxicity reduction strategy in Malus. The rootstock and scion also had statistically significant influences on ROS level and antioxidant activity. Cd induced more severe oxidative stress in HF and FJ grafted onto Mm than it did in HF and FJ grafted onto Mb. Compared with FJ, HF had lower foliar O2 -, root H2O2, and root and leaf MDA levels, but higher ROS-scavenging capacity. The rootstock, scion, and rootstock-scion interaction affected the mRNA transcript levels of several genes involved in Cd uptake, transport, and detoxification including HA7, FRO2-like, NRAMP1, NRAMP3, HMA4, MT2, NAS1, and ABCC1. Hence, the responses of grafted Malus plants to Cd toxicity vary with rootstock, scion, and rootstock-scion interaction.

Exogenous melatonin alleviates cadmium uptake and toxicity in apple rootstocks.

To examine the potential roles of melatonin in cadmium (Cd) uptake, accumulation and detoxification in Malus plants, we exposed two different apple rootstocks varying greatly in Cd uptake and accumulation to either 0 or 30 µM Cd together with 0 or 100 µM melatonin. Cadmium stress stimulated endogenous melatonin production to a greater extent in the Cd-tolerant Malus baccata Borkh. than in the Cd-susceptible Malus micromalus 'qingzhoulinqin'. Melatonin application attenuated Cd-induced reductions in growth, photosynthesis and enzyme activity, as well as reactive oxygen species (ROS) and malondialdehyde accumulation. Melatonin treatment more effectively restored photosynthesis, photosynthetic pigments and biomass in Cd-challenged M. micromalus 'qingzhoulinqin' than in Cd-stressed M. baccata. Exogenous melatonin lowered root Cd2+ uptake, reduced leaf Cd accumulation, decreased Cd translocation factors and increased root, stem and leaf melatonin contents in both Cd-exposed rootstocks. Melatonin application increased both antioxidant concentrations and enzyme activities to scavenge Cd-induced ROS. Exogenous melatonin treatment altered the mRNA levels of several genes regulating Cd uptake, transport and detoxification including HA7, NRAMP1, NRAMP3, HMA4, PCR2, NAS1, MT2, ABCC1 and MHX. Taken together, these results suggest that exogenous melatonin reduced aerial parts Cd accumulation and mitigated Cd toxicity in Malus plants, probably due to the melatonin-mediated Cd allocation in tissues, and induction of antioxidant defense system and transcriptionally regulated key genes involved in detoxification.

Biochemical characterization and expression analysis of lignification in two pear (Pyrus ussuriensis Maxim.) varieties with contrasting stone cell content.

As lignified stone cells reduce fruit quality, we investigated lignin deposition, phenolic metabolites, and expression of lignin biosynthetic genes during fruit development to elucidate the molecular mechanism of stone cell lignification using histological, biochemical, and transcriptional data from two Ussurian pear varieties (Jianba and Nanguo) with contrasting stone cell content. Lignin content and distribution coincided with stone cell accumulation. As per LC-MS analysis, Jianba exhibited higher levels of lignin monomers and hydroxycinnamates than Nanguo, consistently with lignin amount in each case. However, flavonoid content was much higher in Nanguo. Transcriptional data showed that most monolignol biosynthesis-related genes were particularly upregulated in Jianba during lignin accumulation; especially CCR and LAC, two monolignol biosynthesis-specific genes, were substantially upregulated in Jianba fruits at critical stages. Therefore, differences in stone cell content between "Jianba" and "Nanguo" may result from differential expression of lignin synthase genes located downstream of the lignin biosynthesis pathway. Taken together, our data may provide a deeper understanding of the molecular mechanism for stone cell lignification in pear fruit.