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Soil salinization, which leads to the excessive accumulation of Na+ in plants, affects plant growth and development and reduces the yield of fruit crops. To minimize Na+ toxicity, the Na+/H+ antiporters (NHX) mediate the transport of Na+ into vacuoles to promote Na+ compartmentalization or reduce the Na+ concentration in the cytoplasm by promoting Na+ efflux. NHX family members have been identified in many plant species. However, few studies have reported on NHX family members in kiwifruit. In this study, eight NHX family members were identified in the Actinidia chinensis genome. These AcNHXs were clustered into three groups based on phylogenetic analysis. Further analysis of gene structures and conserved motifs of these AcNHX proteins supported the clustering patterns. Collinearity analysis showed that segmental duplication has played an important role in the expansion of the AcNHX family. Multiple cis -acting elements involved in abiotic stress or hormonal responses were detected in the promoters of AcNHX genes. Gene expression analysis revealed that the responses of AcNHXs to salt stress varied. AcNHX3 and AcNHX7 were shown to play a role in regulating salt tolerance in yeast and Arabidopsis , respectively. These findings provide valuable information for future studies aimed at examining the roles of AcNHX proteins in regulating salt tolerance in kiwifruit, as well as the underlying molecular mechanisms. • The NHX family members in kiwifruit was characterized. • Gene expression analysis showed that AcNHXs significantly responded to salt stress. • The AcNHX3 and AcNHX7 genes were cloned and their roles in regulating salt tolerance in yeast and Arabidopsis were clarified. [ABSTRACT FROM AUTHOR]

Drought seriously threatens the growth, development, and yield of crops. WRKY transcription factors (TFs) play important roles in abiotic stress responses. However, the study of WRKY TFs in woody plant apple (Malus domestica) in terms of drought tolerance regulation is limited. This study aimed to functionally characterize an apple group IIc WRKY gene MdWRKY56. The transcription abundance of MdWRKY56 was significantly upregulated under drought stress. The subcellular localization analysis revealed that the MdWRKY56 protein localized to the nucleus. Transcriptional activation experiments proved that MdWRKY56 had transcriptional activity in yeast, and its N-terminal played a decisive role in this activity. The functional analysis using MdWRKY56 transgenic Arabidopsis , apple calli, and apple plants indicated that the MdWRKY56 TF functioned positively in the modulation of osmotic and drought stress tolerance, with the MdWRKY56-overexpressed transgenic materials showing lower electrolyte leakage, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation, and higher fresh weight, proline content, and antioxidant enzyme activities. In conclusion, our findings indicated that MdWRKY56 served as an important positive regulator of drought stress tolerance in apple. • The transcription abundance of MdWRKY56 was significantly upregulated under drought stress. • Transcriptional activation activity and nuclear localization of MdWRKY56 were confirmed. • Overexpression of MdWRKY56 enhanced tolerance to osmotic and drought stresses in transgenic materials. [ABSTRACT FROM AUTHOR]

• Cloning and characterization of an epidermal patterning factor (EPF) gene MdEPF2 from apple. • Functional analysis of MdEPF2 in regulating stomatal development in transgenic tomato. • MdEPF2 -overexpressing tomato showed enhanced drought resistance and water use efficiency. Increasing drought frequency and diminishing groundwater resources are critical environmental constraints that seriously reduce global plant production. However, these limitations might be partially addressed by improving crop drought tolerance and water-use efficiency (WUE). We isolated an epidermal patterning factor (EPF), MdEPF2 , from apple (Malus domestica). Transcript levels of that gene were higher after plants were treated with abscisic acid and drought. To investigate its function in drought tolerance, we ectopically expressed MdEPF2 in Solanum lycopersicum cultivar 'Micro-Tom'. When compared with the wild type (WT), stomatal density was significantly lower in the leaves from the transgenics, which enabled those plants to avoid dehydration. Under drought stress, transgenic plants had higher values for relative leaf water content, chlorophyll, photosynthetic rates, and WUE than did WT. The former also had lower accumulations of reactive oxygen species and malonyldialdehyde but greater activities of antioxidant enzymes, thereby leading to less oxidative damage. Our results suggested that MdEPF2 is a functional ortholog of EPF2 in Arabidopsis and can be potentially used in apple breeding to improve WUE and drought tolerance in this economically important fruit crop. [ABSTRACT FROM AUTHOR]

Highlights • Overexpression of MhYTP2 improves apple water-use efficiency. • MhYTP2 decreases the transpiration rate through activation of ABA and ethylene signaling. • MhYTP2 plays more pivotal roles in rootstock than in scion. Abstract A critical component of crop breeding is the identification of genes involved in improving water-use efficiency (WUE). We characterized MhYTP2 , a gene cloned from Malus hupehensis that encodes an RNA-binding protein, to examine its role in WUE. Under both well-watered and drought conditions, overexpression of this gene enhanced WUE in transgenic plants and they accumulated more biomass than untransformed wild-type plants. This increase in WUE was probably due to enhancement of photosynthetic rate and root water uptake, and a reduction in stomatal aperture that resulted from elevated level of ABA and activated ethylene signaling. MhYTP2 was predominantly expressed in apple roots. We noted with interest that the rate of photosynthesis, instantaneous WUE, and root water potential were higher when 35S:MhYTP2 plants were used as rootstocks rather than as scions in our grafting experiments. Our results indicated that MhYTP2 has a more crucial role in the roots than in the aerial parts of the plant, and it implies that this gene can be used for improving WUE by apple rootstocks and, potentially, rootstocks of other graft-suitable crops. [ABSTRACT FROM AUTHOR]

Highlights • The effect of drought-induced stress can be alleviated by melatonin. • Melatonin positively influenced the growth and physiological parameter of Malus. • Melatonin increased 15N uptake, utilization and accumulation. • Activity of enzymes involved in N-metabolism can be increased by melatonin. • Melatonin increased nutrient uptake by increasing related genes transcription. Abstract To examine the potential roles of melatonin in nutrient uptake, we investigated the influence of its long-term exogenous application on ‘Naganofuji No.2′ apple ( Malus domestica Borkh.) under moderate drought conditions. Both growth and the uptake of macro- and micronutrients were generally decreased in stressed plants. However, the application of exogenous melatonin significantly mitigated this growth reduction and enabled plants to maintain uptake fluxes. This addition of melatonin also markedly alleviated the inhibitory effects of drought on photosynthesis, stomatal apertures, chlorophyll levels, and relative water content, and it controlled the burst of relative electrolyte leakage and hydrogen peroxide. Our investigation with stable isotopes further verified that exogenous melatonin was associated with significant increases in the uptake, utilization, and accumulation of δ15N under drought conditions. Stress sharply reduced the activity of enzymes involved in nitrogen metabolism (NR, NiR, GS, and GOGAT), but the application of melatonin substantially reversed that response. We also examined whether melatonin might control the expression of genes for N-metabolism and transport. Here, the transcript levels of metabolic genes ( NR , NiR , GS , Fd-GOGAT , and NADH-GOGAT ) and uptake genes ( AMT1;2 , AMT1;5 , AMT1;6 , AMT2;1 , NRT1;1 , NRT2;4 , NRT2;5 , and NRT2;7 ) were greatly up-regulated in the leaves. Exogenous melatonin treatment also significantly increased the concentration of endogenous melatonin. Thus, understanding the role of melatonin in nutrient uptake introduces new possibilities to use this compound for agriculture purposes and provides a valuable foundation for enhancing plant tolerance and adaptability to future drought stress. [ABSTRACT FROM AUTHOR]

It is unclear how plants optimize their preference for different forms of inorganic nitrogen to enhance their survival and fitness in challenging environments. Using a hydroponics culture system, we monitored morphological, physiological, and molecular changes in Malus hupehensis seedlings when drought conditions (induced by 5% polyethylene glycol, or PEG) were combined with a low or normal supply of N (0.05 mM or 1 mM NH 4 NO 3 , respectively). We found here that PEG-induced drought stress negatively inhibited the growth of Malus hupehensis seedlings and resulted in higher NH 4 + /NO 3 − ratios in their roots and leaves. The net ammonium influx at the surface of fine roots rose dramatically under drought treatment, while that of nitrate was less changed. This was confirmed by higher absorption of stable 15 NH 4 + isotope than of 15 NO 3 − under drought conditions. Consistently, two ammonium transporter genes closely related to ammonium uptake ( AMT4;2 and AMT4;3 ) were notably up-regulated in response to drought stress, while transcript levels of most genes related to nitrate uptake/reduction and nitrogen assimilation decreased. Therefore, in acclimating to drought, Malus hupehensis plants exhibited relatively increased uptake of NH 4 + compared to NO 3 − . In addition, reducing N supply may alleviate the adverse affect of drought stress on the growth of Malus hupehensis . [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an element that is not only unnecessary for plant growth, but can also induce irreversible damage to plants. Dopamine (DA) and tyrosine decarboxylase (TyDC), a key protein in its synthesis, play important roles in responses to biotic and abiotic stress in plants. To explore the role of dopamine in apple under Cd stress, Malus hupehensis seedlings and TyDC transgenic plants were treated with 0 and 300 μmol L−1 CdCl 2 solutions. We demonstrated that the application of dopamine alleviated Cd stress-induced growth retardation, and the chlorophyll content increased by 22.5%. The root vigor and photosynthesis increased by 1.10 and 1.01 times, respectively. Dopamine significantly increased the activity of antioxidant enzymes to reduce the reactive oxygen species (ROS) level. The accumulation of Cd2+ in the roots and shoots of apple plants was decreased by 33.7% and 28.6% under dopamine treatment, and the phytohormone levels were noticeably increased. Dopamine and MdTyDC alleviated Cd stress by markedly increasing the content of free amino acids and phenolic compounds following Cd exposure. In addition, exogenous dopamine and MdTyDC overexpression alleviated Cd stress by regulating the expression of genes involved in Cd uptake, transport, and detoxification, including HA7 , NRAMP1 , NRAMP3, HMA4 , PCR2 , ABCC1 , MHX , NAS1 , and MT2. Overall, our findings indicate that dopamine can eliminate ROS by increasing antioxidant enzyme activity and reducing Cd accumulation. In addition, dopamine also increased the levels of phytohormones, amino acids, and phenols under Cd stress and regulated the transcription of Cd transporters to enhance Cd tolerance in apple. • DA and MdTyDC alleviate Cd stress and reduce Cd concentration in apple. • DA and MdTyDC remove ROS induced by Cd stress. • DA and MdTyDC increase free amino acids and phenols level. • DA and MdTyDC regulate expression level of Cd transportation genes. [ABSTRACT FROM AUTHOR]

Salinity is a significant environmental challenge that detrimentally affects the yield and quality of plants. The Argonaute (AGO) family proteins are core effectors of RNAi pathways in eukaryotes. Different AGOs bind different types of sRNAs and participate in different silencing pathways and physiological processes. The Argonaute1 (AGO1) protein binds microRNAs and post-transcriptionally inhibits gene expression in the cytoplasm. In addition, AGO1 directly binds to the promoter region of genes to promote transcription in the stress responses. In this study, we isolated the AGO family gene MdAGO1 from apple (Malus domestica). MdAGO1 encodes a protein located in the nucleus and cytoplasm. MdAGO1 expression was significantly induced by salt stress. We employed MdAGO1 -RNAi transgenic apple plants, MdAGO1 overexpressing transgenic apple calli, and Arabidopsis thaliana to investigate the role of MdAGO1 in salt stress. We exposed all of the genotypes to salt stress and showed that MdAGO1 played a role in scavenging reactive oxygen species, maintaining ion balance, and increasing the accumulation of polyamines in the plants under salt stress. In addition, the MdAGO1 -RNAi apple lines exhibited low photosynthetic capacity under salt stress. Taken together, these results reveal that MdAGO1 positively regulates apple salt tolerance. • MdAGO1 enhances apple salt tolerance by promoting ROS scavenging. • MdAGO1 positively regulates ion balance in apple under salt stress. • MdAGO1 promotes the accumulation of polyamines in apple under salt stress. [ABSTRACT FROM AUTHOR]

Nitrogen (N) is the main nutrient element limiting plant growth and crop yield. Plant proline-rich proteins (PRPs) are a group of structural proteins that are rich in proline and hydroxyproline. Previously, we identified 9 PRPs from the apple genome and found that they were induced by different stress treatments. In this study, we showed that the expression of MdPRP6 was inhibited by low N stress. Transgenic apple plants with decreased MdPRP6 expression were generated by RNA interference (Ri) to explore the biological role of MdPRP6 under low N stress. Ri transgenic apple plants grew better than wild-type (WT) plants under low N stress, as indicated by their well-developed roots; greater height, stem diameter, number of leaves, and biomass; and higher photosynthetic capacity and N content. N and starch metabolism were more active in Ri plants than in WT plants. Ri plants accumulated greater amounts of starch and soluble sugar under low N stress, which was consistent with the higher expression levels of related genes in Ri plants relative to WT plants. The accumulation of anthocyanins was higher in Ri plants than in WT plants under low N stress, which helped plants resist the effects of low N stress. The indole acetic acid (IAA) content was higher in the roots of Ri plants, which strengthened the root system. Overall, our data suggested that MdPRP6 negatively regulates low N stress tolerance by modulating anthocyanin accumulation and IAA metabolism in the roots of apple plants. • RNAi of MdPRP6 resulted in more LR in transgenic apples under LN stress. • RNAi of MdPRP6 conferred more accumulation of anthocyanins in transgenic apples under LN stress. • The tolerance of LN stress conferred by MdPRP6 was related to the soluble sugars content. [ABSTRACT FROM AUTHOR]

Nitrogen (N) deficiency limits the production of apple (Malus domestica) on the Loess Plateau, which is the largest apple-producing region in China. Autophagy is a conserved degradation and recycling pathway in eukaryotes that has been reported to play a key role in the responses of plants to N deficiency. We previously identified the core autophagy-related gene MdATG5a from apple and showed that overexpression of this gene improves the drought tolerance of apple plants. Here, we showed that MdATG5a overexpression enhances root growth and the root N uptake of apple plants under low-N conditions and contributes to improving N use efficiency. MdATG5a positively regulated root N uptake by activating the shikimate pathway to increase levels of phenylalanine and tryptophan, which promoted the accumulation of flavonoids and IAA in apple roots under low-N conditions. In addition, MdATG5a was demonstrated to affect the expression of genes involved in root development in response to N deficiency. Moreover, we also found that autophagy activity was higher in the roots of MdATG5a -overexpressing apple plants compared with wild-type plants under low-N conditions. Overall, the results of our study provide new insights into the association between MdATG5a -mediated autophagy and the root N uptake of apple plants under low-N conditions. • MdATG5a overexpression improved nitrogen usage in apple under low-N conditions. • MdATG5a positively regulated root N uptake in apple under low-N stress. • MdATG5a promoted the flavonoids accumulation in apple roots under low-N stress. • MdATG5a increased the IAA level in apple roots under low-N stress. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is an important phytohormone for plant growth, development, and the response to various types of abiotic stress. Pyrabactin Resistance 1 (PYR1)/PYR1-Like (PYL)/Regulatory Component of ABA Receptor (RCAR) family proteins (PYLs) are ABA receptors that have been studied in many plant species. However, no detailed functional studies of these genes in regulating apple drought tolerance have been reported. Here, we isolated a homolog of the ABA receptor PYL9 in Arabidopsis, MdPYL9, from apple (Malus domestica) and analyzed its role in regulating drought tolerance in transgenic apple plants. Gene structure and protein sequence analysis showed that MdPYL9 shares a high similarity with AtPYL9. Protein sequence comparison and 3D structure prediction identified several conserved motifs and residues that are important for ABA binding in MdPYL9, including the helix-grip structure and the gate and latch loops. Expression analysis showed that MdPYL9 was significantly induced by drought treatment. Overexpression of MdPYL9 conferred enhanced tolerance to drought stress in transgenic apple plants, including a higher photosynthetic rate and relative water content and lower MDA content and electrolyte leakage compared with wild-type plants. Lower accumulation of reactive oxygen species (ROS) and higher activity of antioxidant enzymes suggest that MdPYL9 overexpression could promote ROS scavenging and thus reduce oxidative damage in transgenic apple plants under drought stress. Yeast two-hybrid assays indicated that there were both ABA-dependent and ABA-independent interactions between MdPYL9 and the clade A subgroup members of the PP2C family (PP2CAs) in apple. Expression analysis identifed two PP2CA genes, MdHAI1 and MdHAI2 , that were highly expressed under drought treatment by not expressed under normal conditions. These results provide new insight that could be used to aid future studies examining the function of MdPYLs and the mechanism by which they regulate apple drought tolerance. • Overexpression of MdPYL9 enhances the drought tolerance of transgenic apple plants. • MdPYL9 interacts with MdPP2CAs in both ABA-dependent and ABA-independent ways. • MdHAI1 and MdHAI2 may play a key negative role in drought induced ABA signaling. [ABSTRACT FROM AUTHOR]

• MdCCX2 functions positively in modulation of salt tolerance. • MdCCX2 inhibits the Na+ accumulation in transgenic plants under salt stress. • MdCCX2 promotes the ROS scavenging of transgenic plants under salt stress. High salinity is a major environmental constraint that limits global agricultural productivity. The CCX (cation/calcium exchanger) proteins are involved in ion transport and homeostasis and salt stress response. However, the mechanism by which CCX proteins mediate plant salt tolerance is largely unknown, and no detailed functional studies of apple CCX proteins have been reported. Here, we cloned a CCX family gene MdCCX2 (MD12G1011800) from apple (Malus domestica). Its expression was strongly induced by NaCl treatment. As a plasma membrane-localized protein, MdCCX2 -overexpression suppressed the sensitivity of Na+-sensitive yeast mutant to NaCl and reduce the Na+ content in yeast cells under NaCl treatment. To further explore its role in salt stress, we transformed MdCCX2 into Arabidopsis , apple calli, and apple plants. The results of growth phenotype comparison and stress-related physiological index determination under NaCl treatment showed that MdCCX2 -overexpression improved the salt tolerance of these transgenic materials. The significantly reduced Na+ content and increased activity of antioxidant enzymes suggested that MdCCX2 could enhance plant salt tolerance by inhibiting sodium accumulation and promoting ROS scavenging. These results provide a solid basis for further study of the mechanisms by which CCX proteins regulate apple salt tolerance. [ABSTRACT FROM AUTHOR]

• This study revealed the HD-Zip I transcription factor MdHB-7 contributes to the water deficit response in transgenic apple. • Through drought stress treatments and phenotypic assessments, we found that MdHB-7 expression enhanced the drought tolerance of apple plants. • Our results lay a foundation for further research on the mechanisms by which HD-Zip I proteins promote drought tolerance. Drought severely restricts global crop yield and quality. These limitations may be partially addressed through genetic engineering to improve crop drought tolerance. The HD-Zip I transcription factors play important roles in the regulation of plant drought tolerance, but their specific functions in the regulation of apple drought response remain unclear. In this study, we found that MdHB-7 was expressed at different levels in a variety of apple tissues and was significantly induced by abscisic acid (ABA) and drought stress treatments. To investigate the functions of MdHB-7 , we created transgenic apple plants that exhibited either MdHB-7 overexpression or RNAi-induced MdHB-7 suppression. Through drought stress treatments and phenotypic assessments, we found that MdHB-7 expression enhanced the drought tolerance of apple plants. Overexpression of MdHB-7 promoted endogenous ABA accumulation, stomatal closure, and reactive oxygen species (ROS) detoxification in response to drought, whereas suppression of MdHB-7 expression had the opposite effect. Our results lay a foundation for further research on the mechanisms by which HD-Zip I proteins promote drought tolerance. [ABSTRACT FROM AUTHOR]

• MdTyDC overexpression resulted in higher dopamine contents in apple plants and apple calli. • Increased antioxidant activity and ROS scavenging alleviated salt tolerance. • Overexpression of MdTyDC mediated ion homeostasis. Dopamine mediates numerous physiological processes in plants. Tyrosine decarboxylase (TyDC, EC 4.1.1.28) is a common enzyme prevalent in plants and mediates numerous secondary reactions, but little is known about the role of TyDC in the dopamine derivative biosynthesis and function in salt tolerance. Here, apple and apple calli were utilized to study the function of MdTyDC under salt conditions. Overexpression of MdTyDC (OE) significantly increased dopamine content in apple and apple calli. The OE apple lines exhibited increased growth performance and higher photosynthetic capacity and contained more photosynthetic pigments than those of the wild-type lines (WT) under salt conditions. The OE lines had lower H 2 O 2 and O 2 .− according to DAB and NBT-staining compared to the WT lines. Overexpressing MdTyDC enhanced antioxidant enzyme activities and maintained ion homeostasis, as well as increased the expression levels of associated genes. Furthermore, overexpressing MdTyDC enhanced proline content, increased fresh weight, and reduced malondialdehyde (MDA) content in apple calli; however, RNA interference lines showed higher MDA content and lower fresh weight and proline content. Our findings demonstrate that overexpressing MdTyDC induced higher dopamine levels and increased scavenging of reactive oxygen species and the Na+/K+ balance; thus, enhancing salt tolerance. [ABSTRACT FROM AUTHOR]

• Dopamine improved AMF colonization in apple trees under salt stress. • Dopamine and AMF act synergistically to promote apple growth under salt stress. • Overexpression of MdTYDC improved AMF symbiosis in apple trees. • Overexpression of MdTYDC promoting synthesis of flavonoids. Dopamine is common in animals and plants where it plays a variety of important roles, including roles in stress responses. Salt stress commonly occurs in China and seriously affects plant growth and arbuscular mycorrhizal (AM) symbiosis. So, we studied the influence of dopamine on AM fungi (AMF) colonization in apple trees (Malus domestica Borkh.) under salt stress. Apple trees were inoculated with the AM fungi, Funneliformis mosseae , and grown for three weeks. Roots were irrigated with 100 μM dopamine or water and after a 10 day pre-culture period, 200 mM NaCl was introduced (or not). Dopamine improved apple trees salt tolerance by promoting AM symbiosis through increased carbohydrate content. AMF and dopamine worked synergistically to maintain plant cell membrane stability and improve photosynthesis under high salinity, which improved the salt tolerance of the plants. AMF and dopamine also significantly increased root length, surface area, average diameter, and number of root forks, which increased the surface area in contact with soil nutrients and water under salt stress. Dopamine synthetase MdTYDC (tyrosine decarboxylase) transgenic apples were used to study the role of dopamine on AM symbiosis. Transcriptome analyses showed that overexpression of MdTYDC improved AMF symbiosis by promoting synthesis of flavonoids such as luteolin, kaempferol and quercetin. This is the first report that exogenous dopamine promoted AM symbiosis under salt stress, the synergistic effect of dopamine and AMF improved apple salt resistance, and overexpression of MdTYDC promoted AMF symbiosis. [ABSTRACT FROM AUTHOR]

• MdATG8i -overexpressing (OE) apple plants exhibited higher autophagic activity and enhanced tolerance under salt stress. • MdATG8i -OE apple plants showed reduced shrinkage of stoma and less damaged photosynthetic ability under salt stress. • MdATG8i -OE apple plants exhibited an increased accumulation of proline, arginine and polyamines under salt stress. Autophagy has been widely reported to play critical roles in plant adaption to various abiotic and biotic stressors. Although many studies have shown that autophagy-deficient mutants exhibit sensitivity to salt stress, our understanding of the detailed function of autophagy in response to salt stress remains limited. In this study, we found that the expression of MdATG8i was induced by salt stress. We treated transgenic apple plants overexpressing MdATG8i with salt stress, and explored its biological role in response to salt treatment. The results showed that the growth limitation, reactive oxygen species generation and Na+ accumulation caused by salt stress were alleviated in transgenic plants. The shrinkage of stomata and the damage to photosynthetic ability caused by salt stress were diff ;erentially mitigated. These changes were accompanied by the elevated autophagic activity and the accumulation of proline, arginine and its downstream polyamine products in two transgenic lines, which play important roles in plant tolerance to salt stress. In summary, these results revealed that MdATG8i -mediated autophagy enhanced salt tolerance in apple, primarily by alleviating the decrease in carbon assimilation and the accumulation of compatible osmolytes. [ABSTRACT FROM AUTHOR]