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Melatonin enhances plant tolerance to various environmental stressors. Although exogenous application of melatonin has been investigated, the role of endogenous melatonin metabolism in the response of apples to drought stress and nutrient utilization remains unclear. Here, we investigated the effects of ectopically expressing the human melatonin synthase gene HIOMT on transgenic apple plants under drought stress conditions. The tolerance of transgenic apple lines that ectopically expressed HIOMT improved significantly under drought conditions. After 10 days of natural drought stress treatment, the transgenic apple plants showed higher relative water content, chlorophyll levels and Fv/Fm , and lower relative electrolyte leakage and hydrogen peroxide accumulation, than wild-type plants. The activities of peroxidase, superoxide dismutase and catalase, as well as genes in the ascorbate–glutathione cycle, increased more in transgenic apple plants than in the wild-type. The ectopic expression of HIOMT also markedly alleviated the inhibitory effects of long-term drought stress on plant growth, photosynthetic rate and chlorophyll concentrations in apple plants. The uptake and utilization of 15N increased markedly in the transgenic lines under long-term moderate drought stress. Drought stress sharply reduced the activity of enzymes involved in nitrogen metabolism, but ectopic expression of HIOMT largely reversed that response. The expression levels of genes of nitrogen metabolism and uptake were more upregulated in transgenic apple plants than the wild-type. Overall, our study demonstrates that ectopic expression of HIOMT enhanced the tolerance of apple plants to drought stress, and transgenic apple plants showed improved growth due to higher nutrient utilization efficiency under drought conditions. [ABSTRACT FROM AUTHOR]

Drought is a major abiotic stress limiting the growth and production of apple trees worldwide. The receptor-like kinase FERONIA is involved in plant growth, development and stress responses; however, the function of FERONIA in apple under drought stress remains unclear. Here, the FERONIA receptor kinase gene MdMRLK2 from apple (Malus domestica) was shown to encode a plasma membrane-localized transmembrane protein and was significantly induced by abscisic acid and drought treatments. 35S::MdMRLK2 apple plants showed less photosystem damage and higher photosynthetic rates compared with wild-type (WT) plants, after withholding water for 7 days. 35S::MdMRLK2 apple plants also had enhanced energy levels, activated caspase activity and more free amino acids, than the WT, under drought conditions. By performing yeast two-hybrid screening, glyceraldehyde-3-phosphate dehydrogenase and MdCYS4, a member of cystatin, were identified as MdMRLK2 interaction partners. Moreover, under drought conditions, the 35S::MdMRLK2 apple plants were characterized by higher abscisic acid (ABA) content. Overall, these findings demonstrated that MdMRLK2 regulates apple drought tolerance, probably via regulating levels of energetic matters, free amino acids and ABA. [ABSTRACT FROM AUTHOR]

As a precursor of aromatic compounds, fatty acids play important roles in apple fruit quality; however, the genetic and molecular basis underlying fatty acid synthesis and metabolism is largely unknown. In this study, we conducted a genome-wide association study (GWAS) of seven fatty acids using genomic data of 149 Malus accessions and identified 232 significant signals (–log10 P >5) associated with 99 genes from GWAS of four fatty acids across 2 years. Among these, a significant GWAS signal associated with linoleic acid was identified in the transcriptional regulator SUPERMAN-like (SUP) MD13G1209600 at chromosome 13 of M. × domestica. Transient overexpression of MdSUP increased the contents of linoleic and linolenic acids and of three aromatic components in the fruit. Our study provides genetic and molecular information for improving the flavor and nutritional value of apple. [ABSTRACT FROM AUTHOR]

Drought stress is an important factor limiting apple production. γ-Aminobutyric acid (GABA) exists widely in plants and participates in the response to abiotic stress as a metabolite or signaling molecule. The role of exogenous GABA in apple plants, response to long-term drought stress remains unclear. Our study confirmed that exogenous GABA affects the drought resistance of apple plants under long-term drought stress. We found that 1 mM exogenous GABA improved the resistance of apple seedlings to long-term drought stress. The plants showed better growth, less reactive oxygen radical accumulation, less damage to cell membranes and greater active photosynthetic capacity. Under long-term drought stress, exogenous GABA facilitated GABA shunt, resulting in more accumulation of organic acids, namely citric acid, succinic acid and malic acid, in roots and stems of apple seedlings. In addition, exogenous GABA upregulated the expression of cellulose-related genes and lignin-related genes, and activated secondary cell wall-related transcription factors to synthesize more cellulose and lignin. A multiple factorial analysis confirmed that the GABA shunt and the biosynthesis of cellulose and lignin substantially contributed to the growth of apple seedlings with the application of exogenous GABA under long-term drought stress. Our results suggested that exogenous GABA improved the resistance of apple seedlings to long-term drought stress by enhancing GABA shunt and secondary cell wall biosynthesis. [ABSTRACT FROM AUTHOR]

The apple rootstock Malus prunifolia (Willd.) Borkh. is widely used for apple production. Because polyploid plants are often more tolerant to abiotic stress than diploids, we wondered whether polyploidy induction in M. prunifolia might improve its stress tolerance, particularly to high salinity. We used a combination of colchicine and dimethyl sulfoxide (DMSO) to induce chromosome doubling in M. prunifolia and identified the resulting polyploids by stomatal observations and flow cytometry. We found the best way to induce polyploidy in M. prunifolia was to use 2% DMSO and 0.05% colchicine for 2 days for leaves or 0.02% colchicine for stem segments. The results of hydroponic salt treatment showed that polyploid plants were more salt tolerant and had greater photosynthetic efficiency, thicker leaf epidermis and palisade tissues, and shorter but denser root systems than diploids. During salt stress, the polyploid leaves and roots accumulated less Na+, showed upregulated expression of three salt overly sensitive (SOS) pathway genes, and produced fewer reactive oxygen species. The polyploid plants also had considerably higher ABA and jasmonic acid levels than diploid plants under salt stress. Under normal growth conditions, gibberellins (GAs) levels were much lower in polyploid leaves than in diploid leaves; however, after salt treatment, polyploid leaves showed upregulation of essential GAs synthesis genes. In summary, we developed a system for the induction of polyploidy in M. prunifolia and response to salt stress of the resulting polyploids, as reflected in leaf and root morphology, changes in Na+ accumulation, antioxidant capacity and plant hormone levels. [ABSTRACT FROM AUTHOR]

Improving apple water-use efficiency (WUE) is increasingly desirable in the face of global climate change. Melatonin is a pleiotropic molecule that functions in plant development and stress tolerance. In apple, exogenous application of melatonin has been largely investigated, but melatonin biosynthesis and its physiological roles remain elusive. In the plant biosynthetic pathway of melatonin, the last and key step is that N -acetylserotonin methyltransferase (ASMT) converts N -acetylserotonin into melatonin. Here, we identified an apple ASMT gene, MdASMT9 , using homology-based cloning and in vitro enzyme assays. Overexpression of MdASMT9 significantly increased melatonin accumulation in transgenic apple lines. Moreover, an enhanced WUE was observed in the MdASMT9 -overexpressing apple lines. Under well-watered conditions, this increase in WUE was attributed to an enhancement of photosynthetic rate and stomatal aperture via a reduction in abscisic acid biosynthesis. By contrast, under long-term moderate water deficit conditions, regulations in photoprotective mechanisms, stomatal behavior, osmotic adjustment and antioxidant activity enhanced the WUE in transgenic apple lines. Taken together, our findings shed light on the positive effect of MdASMT9 on improving WUE of apple by modulating melatonin biosynthesis. [ABSTRACT FROM AUTHOR]

The high accumulation of phloridzin makes apple (Malus domestica) unique in the plant kingdom, which suggests a vital role of its biosynthesis in physiological processes. In our previous study, silencing MdUGT88F1 (a key UDP-GLUCOSE: PHLORETIN 2'-O-GLUCOSYLTRANSFERASE gene) revealed the importance of phloridzin biosynthesis in apple development and Valsa canker resistance. Here, results from MdUGT88F1 -silenced lines showed that phloridzin biosynthesis was indispensable for normal chloroplast development and photosynthetic carbon fixation by maintaining MdGLK1/2 (GOLDEN2-like1/2) expression. Interestingly, increased phloridzin biosynthesis did not affect plant (or chloroplast) development, but reduced nitrogen accumulation, leading to chlorophyll deficiency, light sensitivity, and sugar accumulation in MdUGT88F1- overexpressing apple lines. Further analysis revealed that MdUGT88F1-mediated phloridzin biosynthesis negatively regulated the cytosolic glutamine synthetase1-asparagine synthetase-asparaginase (GS1-AS-ASPG) pathway of ammonium assimilation and limited chlorophyll synthesis in apple shoots. The interference of phloridzin biosynthesis in the GS1-AS-ASPG pathway was also assumed to be associated with its limitation of the carbon skeleton of ammonium assimilation through metabolic competition with the tricarboxylic acid cycle. Taken together, our findings shed light on the role of MdUGT88F1-mediated phloridzin biosynthesis in the coordination between carbon and nitrogen accumulation in apple trees. [ABSTRACT FROM AUTHOR]

Drought stress is an environmental factor that seriously threatens plant growth, development and yield. VQ proteins are transcriptional regulators that have been reported to be involved in plant growth, development and the responses to biotic and abiotic stressors. However, the relationship between VQ proteins and drought stress has not been well documented in plants. In this study, overexpressing the apple VQ motif-containing protein (MdVQ37) gene in apple plants markedly reduced the tolerance to drought. Physiological and biochemical studies further demonstrated lower enzymatic activities and decreased photosynthetic capacity in transgenic lines compared with wild-type (WT) plants under drought stress. Ultrastructural analysis of leaves showed that the leaves and palisade tissues from the transgenic lines were significantly thinner than those from WT plants. Salicylic acid (SA) analysis indicated that overexpression of MdVQ37 increased the accumulation of 2,5-DHBA by up-regulating the expression of the SA catabolic gene, which ultimately resulted to a significant reduction in endogenous SA content and the disruption of the SA-dependent signaling pathway under drought stress. Applying SA partially increased the survival rate of the transgenic lines under drought stress. These results demonstrate that the regulatory function of apple MdVQ37 is implicated in drought stress, through a change in leaf development and SA homeostasis. This study provides novel insight into understanding the multiple functions of VQ proteins. [ABSTRACT FROM AUTHOR]

High temperature (HT) is one of the most important environmental stress factors and seriously threatens plant growth, development, and production. VQ motif-containing proteins are transcriptional regulators that have been reported to regulate plant growth and developmental processes, including responses to biotic and abiotic stresses. However, the relationships between VQ motif-containing proteins and HT stress have not been studied in depth in plants. In this study, transgenic apple (Malus domestica) plants overexpressing the apple VQ motif-containing protein-coding gene (MdVQ37) were exposed to HT stress, and the transgenic lines exhibited a heat-sensitive phenotype. In addition, physiological and biochemical studies revealed that, compared with WT plants, transgenic lines had lower enzymatic activity and photosynthetic capacity and lower amounts of nonenzymatic antioxidant system metabolites under HT stress. Transcriptome analysis revealed 1379 genes whose expression differed between the transgenic lines and WT plants. GO and KEGG pathway analyses showed that transcription factor activity and plant hormone signaling pathways were differentially influenced and enriched in the transgenic lines. Salicylic acid (SA) content analysis indicated that overexpression of MdVQ37 reduced the content of endogenous SA by regulating the expression of SA catabolism-related genes, which ultimately resulted in disruption of the SA-dependent signaling pathway under HT stress. The application of SA slightly increased the survival rate of the transgenic lines under HT stress. Taken together, our results indicate that apple MdVQ37 has a regulatory function in basal thermotolerance by modulating the activity of transcription factors and SA homeostasis. Overall, this study provides novel insights that improve our understanding of the various functions of VQ motif-containing proteins. [ABSTRACT FROM AUTHOR]

Apple replant disease (ARD) is a soil-borne disease that leads to economic losses due to reduced plant growth and diminished fruit yields. Dopamine is involved in interactions between plants and pathogens. However, it remains unclear whether dopamine can directly stimulate defense responses to ARD. In this study, an exogenous dopamine treatment and dopamine synthetase MdTYDC (tyrosine decarboxylase) transgenic plants were used to verify the role of dopamine in treating ARD. First, 2-year-old apple trees (Malus domestica cv. Fuji), grafted onto rootstock M26, were grown in replant soils. The addition of dopamine (100 μM) to the soil promoted seedling growth and changed the accumulation of mineral elements in plants in replant soils. Such supplementation improved the activity of invertase, urease, proteinase and phosphatase under replant conditions. Sequencing analysis of 16S rDNA and internal transcribed spacer (ITS) rDNA revealed that dopamine had a slight influence on bacterial diversity but had an obvious effect on the fungal diversity in replant soils. The application of dopamine to replant soil changed the composition of bacterial and fungal communities. Second, overexpression of MdTYDC in apple plants alleviated the effects of ARD. MdTYDC transgenic lines exhibited mitigated ARD through inhibited degradation of photosynthetic pigment, maintaining the stability of photosystems I and II and improving the antioxidant system. Furthermore, overexpression of MdTYDC improved arbuscular mycorrhizal fungi colonization by improving the accumulation of soluble sugars under replant conditions. Together, these results demonstrated that dopamine enhances the tolerance of apples to ARD. [ABSTRACT FROM AUTHOR]

Flavonoids play important roles in regulating plant growth and development. In this study, three kaempferol 3-O-glycosides were identified and mainly accumulated in flowers but not in leaves or fruits of Malus. In Malus, flower petal color is normally white, but some genotypes have red flowers containing anthocyanin. Anthocyanin biosynthesis appears to be in competition with kaempferol 3-O-glycosides production and controlled by the biosynthetic genes. The white flower Malus genotypes had better-developed seeds than the red flower genotypes. In flowers, the overexpression of MYB10 in Malus domestica enhanced the accumulation of anthocyanin, but decreased that of kaempferol 3-O-glycosides. After pollination the transgenic plants showed slower pollen tube growth and fewer developed seeds. Exogenous application of different flavonoid compounds suggested that kaempferol 3-O-glycosides, especially kaempferol 3-O-rhamnoside, regulated pollen tube growth and seed set rather than cyanidin or quercetin 3-O-glycosides. It was found that kaempferol 3-O-rhamnoside might regulate pollen tube growth through effects on auxin, the Rho of plants (ROP) GTPases, calcium and the phosphoinositides signaling pathway. With the inhibition of auxin transport, the transcription levels of Heat Shock Proteins (HSPs) and ROP GTPases were downregulated while the levels were not changed or even enhanced when blocking calcium signaling, suggesting that HSPs and ROP GTPases were downstream of auxin signaling, but upstream of calcium signaling. In summary, kaempferol glycoside concentrations in pistils correlated with auxin transport, the transcription of HSPs and ROP GTPases, and calcium signaling in pollen tubes, culminating in changes to pollen tube growth and seed set. [ABSTRACT FROM AUTHOR]

Drought leads to reductions in plant growth and crop yields. Arbuscular mycorrhizal fungi (AMF), which form symbioses with the roots of the most important crop species, alleviate drought stress in plants. In the present work, we identified 14 GH3 genes in apple (Malus domestica) and provided evidence that MdGH3-2 and MdGH3-12 play important roles during AM symbiosis. The expression of both MdGH3-2 and MdGH3-12 was upregulated during mycorrhization, and the silencing of MdGH3-2/12 had a negative impact on AM colonization. MdGH3-2/12 silencing resulted in the downregulation of five genes involved in strigolactone synthesis, and there was a corresponding change in root strigolactone content. Furthermore, we observed lower root dry weights in RNAi lines under AM inoculation conditions. Mycorrhizal transgenic plants showed greater sensitivity to drought stress than WT, as indicated by their higher relative electrolytic leakage and lower relative water contents, osmotic adjustment ability, ROS scavenging ability, photosynthetic capacity, chlorophyll fluorescence values, and abscisic acid contents. Taken together, these data demonstrate that MdGH3-2/12 plays an important role in AM symbiosis and drought stress tolerance in apple. [ABSTRACT FROM AUTHOR]

Water deficit is one of the major limiting factors for apple (Malus domestica) production on the Loess Plateau, a major apple cultivation area in China. The identification of genes related to the regulation of water use efficiency (WUE) is a crucial aspect of crop breeding programs. As a conserved degradation and recycling mechanism in eukaryotes, autophagy has been reported to participate in various stress responses. However, the relationship between autophagy and WUE regulation has not been explored. We have shown that a crucial autophagy protein in apple, MdATG8i, plays a role in improving salt tolerance. Here, we explored its biological function in response to long-term moderate drought stress. The results showed that MdATG8i-overexpressing (MdATG8i-OE) apple plants exhibited higher WUE than wild-type (WT) plants under long-term moderate drought conditions. Plant WUE can be increased by improving photosynthetic efficiency. Osmoregulation plays a critical role in plant stress resistance and adaptation. Under long-term drought conditions, the photosynthetic capacity and accumulation of sugar and amino acids were higher in MdATG8i-OE plants than in WT plants. The increased photosynthetic capacity in the OE plants could be attributed to their ability to maintain optimal stomatal aperture, organized chloroplasts, and strong antioxidant activity. MdATG8i overexpression also promoted autophagic activity, which was likely related to the changes described above. In summary, our results demonstrate that MdATG8i-OE apple lines exhibited higher WUE than WT under long-term moderate drought conditions because they maintained robust photosynthesis, effective osmotic adjustment processes, and strong autophagic activity. [ABSTRACT FROM AUTHOR]

Numerous environmental and endogenous signals control the highly orchestrated and intricate process of plant senescence. Ethylene, a well-known inducer of senescence, has long been considered a key endogenous regulator of leaf and flower senescence, but the molecular mechanism of ethylene-induced ovule senescence has not yet been elucidated. In this study, we found that blockage of fertilization caused ovule abortion in the pear cultivar '1913'. According to transcriptome and phytohormone content data, ethylene biosynthesis was activated by pollination. At the same time, ethylene overaccumulated in ovules, where cells were sensitive to ethylene signals in the absence of fertilization. We identified a transcription factor in the ethylene signal response, ethylene-insensitive 3-like (EIL1), as a likely participant in ovule senescence. Overexpression of PbEIL1 in tomato caused precocious onset of ovule senescence. We further found that EIL1 could directly bind to the promoter of the SENESCENCE-ASSOCIATED CYSTEINE PROTEINASE 1 (PbCysp1) gene and act upstream of senescence. Yeast one-hybrid and dual-luciferase assays revealed the interaction of the transcription factor and the promoter DNA sequence and demonstrated that PbEIL1 enhanced the action of PbCysp1. Collectively, our results provide new insights into how ethylene promotes the progression of unfertilized ovule senescence. [ABSTRACT FROM AUTHOR]

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9 -cis -epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees. [ABSTRACT FROM AUTHOR]

Most land plant species have the ability to establish a symbiosis with arbuscular mycorrhizal (AM) fungi. These fungi penetrate into root cortical cells and form branched structures (known as arbuscules) for nutrient exchange. We cloned the MdIAA24 from apple (Malus domestica) following its up-regulation during AM symbiosis. Results demonstrate the positive impact of the overexpression (OE) of MdIAA24 in apple on AM colonization. We observed the strigolactone (SL) synthesis genes, including MdD27 , MdCCD7 , MdCCD8a , MdCCD8b and MdMAXa , to be up-regulated in the OE lines. Thus, the OE lines exhibited both a higher SL content and colonization rate. Furthermore, we observed that the OE lines were able to maintain better growth parameters under AM inoculation conditions. Under drought stress with the AM inoculation, the OE lines were less damaged, which was demonstrated by a higher relative water content, a lower relative electrolytic leakage, a greater osmotic adjustment, a higher reactive oxygen species scavenging ability, an improved gas exchange capacity and an increased chlorophyll fluorescence performance. Our findings demonstrate that the OE of MdIAA24 in apple positively regulates the synthesis of SL and the formation of arbuscules as a drought stress coping mechanism. [ABSTRACT FROM AUTHOR]

Soil alkalization affects apple production in northwest China. Autophagy is a highly conserved degradative protein pathway in eukaryotes. Autophagy in plants can be activated by various abiotic factors. We previously identified the positive role of the autophagy-related gene MdATG18a in drought, nitrogen deficiency and resistance to Diplocarpon mali infection in apple. However, it is still unclear whether ATG18a is related to alkaline stress. In this study, we used hydroponic culture to simulate alkaline stress and found that the overexpression of MdATG18a significantly improved the tolerance of apple to alkaline stress. The overexpression of MdATG18a increased biomass, photosynthetic rate and antioxidant capacity of transgenic plants compared with wild-type plants under alkaline stress. The overexpression of MdATG18a promoted γ-aminobutyric acid (GABA) shunt via an increase in glutamate (GABA precursor) and GABA contents and upregulation of GABA shunt-related genes. In addition, the overexpression of MdATG18a significantly upregulated the expression of other core ATG genes and increased the formation of autophagosomes under alkaline stress. In conclusion, these results suggest that the overexpression of MdATG18a in apple enhances alkaline tolerance and the GABA shunt, which may be owing to the increase in autophagic activity. [ABSTRACT FROM AUTHOR]

The function of serrate (SE) in miRNA biogenesis in Arabidopsis is well elucidated, whereas its role in plant drought resistance is largely unknown. In this study, we report that MdSE acts as a negative regulator of apple (Malus × domestica) drought resistance by regulating the expression levels of MdMYB88 and MdMYB124 and miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, mdm-miR319, and mdm-miR399. MdSE interacts with MdMYB88 and MdMYB124, two positive regulators of apple drought resistance. MdSE decreases the transcript and protein levels of MdMYB88 and MdMYB124, which directly regulate the expression of MdNCED3, a key enzyme in abscisic acid (ABA) biosynthesis. Furthermore, MdSE is enriched in the same region of the MdNECD3 promoter where MdMYB88/MdMYB124 binds. Consistently, MdSE RNAi transgenic plants are more sensitive to ABA-induced stomatal closure, whereas MdSE OE plants are less sensitive. In addition, under drought stress, MdSE is responsible for the biogenesis of mdm-miR399, a negative regulator of drought resistance, and negatively regulates miRNAs, including mdm-miR156, mdm-miR166, mdm-miR172, and mdm-miR319, which are positive regulators of drought resistance. Taken together, by revealing the negative role of MdSE, our results broaden our understanding of the apple drought response and provide a candidate gene for apple drought improvement through molecular breeding. [ABSTRACT FROM AUTHOR]

MdMYB88 and MdMYB124 have been demonstrated to be responsible for lignin accumulation in apple under drought stress. In this study, using a metabolomic approach, we identified differentially accumulated phenylpropanoid and flavonoid metabolites in MdMYB88/124 transgenic RNAi plants under control and long-term drought stress conditions in apple roots. We confirmed the regulation of phenylalanine by MdMYB88 and MdMYB124 via UPLC-MS in apple roots under both control and drought conditions. Using Electrophoretic Mobility Shift Assay (EMSA) and ChIP-quantitative PCR (qPCR) analyses, we found that MdMYB88 positively regulates the MdCM2 gene, which is responsible for phenylalanine biosynthesis, through binding to its promoter region. Under long-term drought conditions, MdMYB88/124 RNAi plants consistently accumulated increased amounts of H2O2 and MDA, while MdMYB88 and MdMYB124 overexpression plants accumulated decreased amounts of H2O2 and MDA. We also examined the accumulation of metabolites in the phenylpropanoid biosynthesis pathway in the leaves of MdMYB88 and MdMYB124 transgenic apple plants after long-term drought stress. We found that metabolites responsible for plant defense, including phenylpropanoids and flavonoids, accumulated less in the RNAi plants but more in the overexpression plants under both control and drought conditions. We further demonstrated that MdMYB88/124 RNAi plants were more sensitive to Alternaria alternata f. sp. mali and Valsa mali, two pathogens that currently severely threaten apple production. In contrast, MdMYB88 and MdMYB124 overexpression plants were more tolerant to these pathogens. The cumulative results of this study provided evidence for secondary metabolite regulation by MdMYB88 and MdMYB124, further explained the molecular roles of MdMYB88 and MdMYB124 in drought resistance, and provided information concerning molecular aspects of their roles in disease resistance. [ABSTRACT FROM AUTHOR]

High temperature is an abiotic stress factor that threatens plant growth and development. Autophagy in response to heat stress involves the selective removal of heat-induced protein complexes. Previously, we showed that a crucial autophagy protein from apple, MdATG18a, has a positive effect on drought tolerance. In the present study, we treated transgenic apple (Malus domestica) plants overexpressing MdATG18a with high temperature and found that autophagy protected them from heat stress. Overexpression of MdATG18a in apple enhanced antioxidase activity and contributed to the production of increased beneficial antioxidants under heat stress. Transgenic apple plants exhibited higher photosynthetic capacity, as shown by the rate of CO2 assimilation, the maximum photochemical efficiency of photosystem II (PSII), the effective quantum yield, and the electron transport rates in photosystems I and II (PSI and PSII, respectively). We also detected elevated autophagic activity and reduced damage to chloroplasts in transgenic plants compared to WT plants. In addition, the transcriptional activities of several HSP genes were increased in transgenic apple plants. In summary, we propose that autophagy plays a critical role in basal thermotolerance in apple, primarily through a combination of enhanced antioxidant activity and reduced chloroplast damage. [ABSTRACT FROM AUTHOR]

Drought and nitrogen (N) deficiency are common factors that limit apple production in the Loess Plateau region of China. Different apple cultivars respond to drought and low N differently; however, the mechanism that underlies the difference in nitrogen-use efficiency (NUE) under drought conditions is not well understood. In this study, by comparing the physiological responses of two apple (Malus domestica Borkh.) cultivars with contrasting NUE, 'Qinguan' (higher NUE) and 'Honeycrisp' (lower NUE), under low N and drought conditions, we discovered that, 'Qinguan' had larger stomatal apertures, higher chlorophyll fluorescence levels, more active N metabolism and antioxidant enzymes, higher abscisic acid and auxin concentrations, larger root size and more efficient N uptake mediated by higher expression of MdNRT2.4 in rootstock than that of 'Honeycrisp'. Additionally, we experimentally confirmed that MdNRT2.4 enhanced low N and osmotic stress tolerance in Arabidopsis when being overexpressed. Taken together, our findings shed light on the mechanism that underlies the difference in NUE of apple under drought and N-deficient conditionss and provide MdNRT2.4 as a candidate gene for future genetic engineering. [ABSTRACT FROM AUTHOR]

Nutrient element deprivation, such as iron (Fe) deficiency stress, is a major factor limiting plant survival and proliferation in marginal soils. To cope with a low Fe environment, plants have evolved elaborate mechanisms underlying Fe homeostasis via intricate transcriptional and post-transcriptional regulation. Here, we characterized the Fe deficiency-inducible MYB transcription factor MdMYB58 in apple plants. Overexpressing MdMYB58 resulted in the accumulation of Fe in the root of transgenic Arabidopsis and apple calli when they were exposed to low Fe available conditions. Further investigation revealed that MdMYB58 bound to the promoter of MdMATE43, and its homolog FRD3 in Arabidopsis. Transient expression and stable transgenic assays in apple calli indicated that MdMYB58 transcriptionally repressed MdMATE43 mRNA, as well as FRD3 in Arabidopsis. Interestingly, AtMYB58, the homolog of MdMYB58, possessed higher binding activities to MdMATE43 and FRD3, which suggests a potentially conserved feature of MYB58 binding to MATE transporters in plants. Additionally, MYB-MATE-mediated regulation of Fe homeostasis may be related to the PYE-related Fe deficiency regulatory network via MdSAT1, a member of the IVa subfamily of bHLH transcription factors. Co-overexpression of MdSAT1 competitively weakened MdMYB58-overexpression induced repression of MdMATE43 transcript abundancy by protein–protein interaction. Taken together, the newly identified MYB-bHLH transcription factor expands our understanding of multilevel molecular mechanisms that plants use to coordinate Fe demand with Fe uptake, transport, and tissue partitioning under low Fe conditions. [ABSTRACT FROM AUTHOR]

Tolerance to drought stress is significantly enhanced following pre-treatment with melatonin in plants of two Malus species. Mechanisms involve regulation of ABA metabolism, free-radical detoxification, and improved functioning of stomata.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. [ABSTRACT FROM AUTHOR]

DNA-binding one zinc-finger (Dof) proteins constitute a family of transcription factors with a highly conserved Dof domain that contains a C2C2 zinc-finger motif. Although several studies have demonstrated that Dof proteins are involved in multiple plant processes, including development and stress resistance, the functions of these proteins in drought stress resistance are largely unknown. Here, we report the identification of the MdDof54 gene from apple and document its positive roles in apple drought resistance. After long-term drought stress, compared with nontransgenic plants, MdDof54 RNAi plants had significantly shorter heights and weaker root systems; the transgenic plants also had lower shoot and root hydraulic conductivity, as well as lower photosynthesis rates. By contrast, compared with nontransgenic plants, MdDof54-overexpressing plants had higher photosynthesis rates and shoot hydraulic conductivity under long-term drought stress. Moreover, compared with nontransgenic plants, MdDof54-overexpressing plants had higher survival percentages under short-term drought stress, whereas MdDof54 RNAi plants had lower survival percentages. MdDof54 RNAi plants showed significant downregulation of 99 genes and significant upregulation of 992 genes in response to drought, and 366 of these genes were responsive to drought. We used DAP-seq and ChIP-seq analyses to demonstrate that MdDof54 recognizes cis-elements that contain an AAAG motif. Taken together, our results provide new information on the functions of MdDof54 in plant drought stress resistance as well as resources for apple breeding aimed at the improvement of drought resistance. [ABSTRACT FROM AUTHOR]

As a versatile compound, myo-inositol plays vital roles in plant biochemistry and physiology. We previously showed that exogenous application of myo-inositol had a positive role in salinity tolerance in Malus hupehensis Rehd. In this study, we used MdMIPS (the rate-limiting gene of myo-inositol biosynthesis) transgenic apple lines to gain new insights into the physiological role of myo-inositol in apple. Decreasing myo-inositol biosynthesis in apple lines by RNA silencing of MdMIPS1/2 led to extensive programmed cell death, which manifested as necrosis of both the leaves and roots and, ultimately, plant death. Necrosis was directly caused by the excessive accumulation of reactive oxygen species, which may be closely associated with the cell wall polysaccharide-mediated increase in salicylic acid and a compromised antioxidant system, and this process was enhanced by an increase in ethylene production. In addition, a high accumulation of sorbitol promoted necrosis. This synergetic interplay between salicylic acid and ethylene was further supported by the fact that increased myo-inositol accumulation significantly delayed leaf senescence in MdMIPS1-overexpressing apple lines. Taken together, our results indicated that apple myo-inositol regulates reactive oxygen species-induced programmed cell death through salicylic acid-dependent and ethylene-dependent pathways. [ABSTRACT FROM AUTHOR]

Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport "Red Zaosu" (Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type "Zaosu" revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from "Red Zaosu" compared with "Zaosu". Moreover, the GA levels in "Red Zaosu" seedlings were lower than those in "Zaosu" seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in "Red Zaosu". Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in "Red Zaosu". Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in "Red Zaosu" induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit. [ABSTRACT FROM AUTHOR]

Drought stress can negatively impact apple fruit quality and yield. Apple microRNAs (miRNAs) participate in apple tree and fruit development, as well as in biotic stress tolerance; however, it is largely unknown whether these molecules are involved in the drought response. To identify drought-responsive miRNAs in Malus, we first examined the drought stress tolerance of ten F1 progenies of R3 (M. × domestica) × M. sieversii. We performed Illumina sequencing on pooled total RNA from both drought-tolerant and drought-sensitive plants. The sequencing results identified a total of 206 known miRNAs and 253 candidate novel miRNAs from drought-tolerant plants and drought-sensitive plants under control or drought conditions. We identified 67 miRNAs that were differentially expressed in drought-tolerant plants compared with drought-sensitive plants under drought conditions. Under drought stress, 61 and 35 miRNAs were differentially expressed in drought-tolerant and drought-sensitive plants, respectively. We determined the expression levels of seven out of eight miRNAs by stem-loop qPCR analysis. We also predicted the target genes of all differentially expressed miRNAs and identified the expression of some genes. Gene Ontology analyses indicated that the target genes were mainly involved in stimulus response and cellular and metabolic processes. Finally, we confirmed roles of two miRNAs in apple response to mannitol. Our results reveal candidate miRNAs and their associated mRNAs that could be targeted for improving drought tolerance in Malus species, thus providing a foundation for understanding the molecular networks involved in the response of apple trees to drought stress. [ABSTRACT FROM AUTHOR]