Age-dependent changes in leaf size in apple are governed by a cytokinin-integrated module.

Plants undergo various age-dependent changes in leaf morphology during the transition from the juvenile to the adult stage. However, the precise molecular mechanisms governing these changes in apple (Malus domestica) remain unknown. Here, we showed that CYTOKININ OXIDASE/DEHYDROGENASE5 (MdCKX5), an age-dependent gene, encodes a functional CKX enzyme and serves as the common downstream target of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MdSPL14 and WRKY transcription factor MdWRKY24 to control the degradation of cytokinin (CK). As the target of mdm-microRNA156a, MdSPL14 interacts with MdWRKY24 to coordinately repress the transcription of MdCKX5 by forming the age-mediated mdm-miR156a-MdSPL14-MdWRKY24 module, which regulates age-dependent changes in CK during the juvenile-to-adult phase transition. We further demonstrated that MdARR6, a type-A ARABIDOPSIS RESPONSE REGULATOR (ARR), is a negative feedback regulator in the CK signaling pathway. Silencing of MdARR6 in apple resulted in large leaves with smaller epidermal cells and a greater number of epidermal cells. Biochemical analysis showed that the mdm-miR156a-MdSPL14-MdWRKY24 module acts as a transcriptional repressor to directly regulate MdARR6 expression, thus controlling the age-dependent changes in leaf size by reducing CK responses. These findings established a link between the age pathway and CK signaling and revealed the molecular mechanism underlying age-dependent changes during the juvenile-to-adult phase transition; our results also provide targets for the genetic improvement of the vegetative phase transition in apple.

MdASMT9-mediated melatonin biosynthesis enhances basal thermotolerance in apple plants.

High temperatures negatively impact the yield and quality of fruit crops. Exogenous melatonin (MT) application has been shown to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains unclear. The present study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthesis gene N-acetylserotonin methyltransferase 9 (MdASMT9). Endogenous MT protected transgenic plants from heat stress by increasing antioxidant enzyme activity and scavenging reactive oxygen species (ROS), and protecting the chloroplasts from damage. Application of MT and overexpression of MdASMT9 also reduced abscisic acid accumulation through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus inducing stomatal opening for better heat dissipation. Furthermore, MT-enhanced autophagic activity through promoting MdWRKY33-mediated transcriptional enhancement of MdATG18a under heat stress. These findings provide new insights into the regulation of endogenous MT and its role in improving basal thermotolerance in perennial fruit trees.

Melatonin confers tolerance to nitrogen deficiency through regulating MdHY5 in apple plants.

Nitrogen (N) is an essential nutrient for crop growth and development, significantly influencing both yield and quality. Melatonin (MT), a known enhancer of abiotic stress tolerance, has been extensively studied. However, its relationship with nutrient stress, particularly N deficiency, and the underlying regulatory mechanisms of MT on N absorption remain unclear. In this study, exogenous MT treatment was found to improve the tolerance of apple plants to N deficiency. Apple plants overexpressing the MT biosynthetic gene N-acetylserotonin methyltransferase 9 (MdASMT9) were used to further investigate the effects of endogenous MT on low-N stress. Overexpression of MdASMT9 improved the light harvesting and heat transfer capability of apple plants, thereby mitigating the detrimental effects of N deficiency on the photosynthetic system. Proteomic and physiological data analyses indicated that MdASMT9 overexpression enhanced the trichloroacetic acid cycle and positively modulated amino acid metabolism to counteract N-deficiency stress. Additionally, both exogenous and endogenous MT promoted the transcription of MdHY5, which in turn bound to the MdNRT2.1 and MdNRT2.4 promoters and activated their expression. Notably, MT-mediated promotion of MdNRT2.1 and MdNRT2.4 expression through regulating MdHY5, ultimately enhancing N absorption. Taken together, these findings shed light on the association between MdASMT9-mediated MT biosynthesis and N absorption in apple plants under N-deficiency conditions.

Fruit crops combating drought: Physiological responses and regulatory pathways.

Drought is a common stress in agricultural production. Thus, it is imperative to understand how fruit crops respond to drought and to develop drought-tolerant varieties. This paper provides an overview of the effects of drought on the vegetative and reproductive growth of fruits. We summarize the empirical studies that have assessed the physiological and molecular mechanisms of the drought response in fruit crops. This review focuses on the roles of calcium (Ca2+) signaling, abscisic acid (ABA), reactive oxygen species signaling, and protein phosphorylation underlying the early drought response in plants. We review the resulting downstream ABA-dependent and ABA-independent transcriptional regulation in fruit crops under drought stress. Moreover, we highlight the positive and negative regulatory mechanisms of microRNAs in the drought response of fruit crops. Lastly, strategies (including breeding and agricultural practices) to improve the drought resistance of fruit crops are outlined.

Introducing melatonin to the horticultural industry: physiological roles, potential applications, and challenges.

Melatonin (N-acetyl-5-methoxytryptamine) is an emerging biomolecule that influences horticultural crop growth, flowering, fruit ripening, postharvest preservation, and stress protection. It functions as a plant growth regulator, preservative and antimicrobial agent to promote seed germination, regulate root system architecture, influence flowering and pollen germination, promote fruit production, ensure postharvest preservation, and increase resistance to abiotic and biotic stresses. Here, we highlight the potential applications of melatonin in multiple aspects of horticulture, including molecular breeding, vegetative reproduction, production of virus-free plants, food safety, and horticultural crop processing. We also discuss its effects on parthenocarpy, autophagy, and arbuscular mycorrhizal symbiosis. Together, these many features contribute to the promise of melatonin for improving horticultural crop production and food safety. Effective translation of melatonin to the horticultural industry requires an understanding of the challenges associated with its uses, including the development of economically viable sources.

Overexpression of the tyrosine decarboxylase gene MdTyDC in apple enhances long-term moderate drought tolerance and WUE.

Drought stress affects the apple yield and quality. Tyrosine decarboxylase (TyDC) plays a fundamental role in many secondary metabolite reactions in higher plants (including those involving dopamine). Our aims of this study are: 1) to identify the role of TyDC in dopamine derivative biosynthesis and its function in long-term moderate drought conditions; and 2) to explore the role of MdTyDC in plant growth and development as well as the drought stress response. Wild type and three independently apple plants overexpression of MdTyDC were treated for long-term moderate drought stress. The growth and physiological parameters of apple plant, photosynthetic capacity, antioxidant enzymes activity, water use efficiency (WUE), stomatal behavior, amino acid content and dopamine content were detected under long-term moderate drought stress. Overexpression of MdTyDC (OE) in apple showed better growth performance, higher photosynthetic capacity and higher capacity for photochemical reactions than wild type lines (WT). Under long-term moderate drought stress, OE lines showed higher WUE, increased ABA content, decreased stomatal aperture, higher antioxidant activity, lower accumulation of ROS and increases in amino acids, such as proline, phenylalanine and lysine. In addition, qRT-PCR revealed higher gene expression of MdTyDC and dopamine content in OE compared with WT lines under long-term moderate drought stress. These results indicate that MdTyDC confers long-term moderate drought tolerance by improving photosynthetic capacity, WUE, antioxidant activity, dopamine content and changing the contents of amino acids (such as proline accumulation).

Heterologous Expression of the Melatonin-Related Gene HIOMT Improves Salt Tolerance in Malus domestica.

Melatonin, a widely known indoleamine molecule that mediates various animal and plant physiological processes, is formed from N-acetyl serotonin via N-acetylserotonin methyltransferase (ASMT). ASMT is an enzyme that catalyzes melatonin synthesis in plants in the rate-determining step and is homologous to hydroxyindole-O-methyltransferase (HIOMT) melatonin synthase in animals. To date, little is known about the effect of HIOMT on salinity in apple plants. Here, we explored the melatonin physiological function in the salinity condition response by heterologous expressing the homologous human HIOMT gene in apple plants. We discovered that the expression of melatonin-related gene (MdASMT) in apple plants was induced by salinity. Most notably, compared with the wild type, three transgenic lines indicated higher melatonin levels, and the heterologous expression of HIOMT enhanced the expression of melatonin synthesis genes. The transgenic lines showed reduced salt damage symptoms, lower relative electrolyte leakage, and less total chlorophyll loss from leaves under salt stress. Meanwhile, through enhanced activity of antioxidant enzymes, transgenic lines decreased the reactive oxygen species accumulation, downregulated the expression of the abscisic acid synthesis gene (MdNCED3), accordingly reducing the accumulation of abscisic acid under salt stress. Both mechanisms regulated morphological changes in the stomata synergistically, thereby mitigating damage to the plants' photosynthetic ability. In addition, transgenic plants also effectively stabilized their ion balance, raised the expression of salt stress-related genes, as well as alleviated osmotic stress through changes in amino acid metabolism. In summary, heterologous expression of HIOMT improved the adaptation of apple leaves to salt stress, primarily by increasing melatonin concentration, maintaining a high photosynthetic capacity, reducing reactive oxygen species accumulation, and maintaining normal ion homeostasis.

MdTyDc Overexpression Improves Alkalinity Tolerance in Malus domestica.

Tyrosine is decarboxylated to tyramine by TYDC (Tyrosine decarboxylase) and then hydroxylated to dopamine, which is involved in plant response to abiotic stress. However, little is known about the function of MdTyDc in response to alkaline stress in plants. In our study, it was found that the expression of MdTyDc was induced by alkaline stress. Therefore, the apple plants overexpressing MdTyDc was treated with alkali stress, and we found that MdTyDc played an important role in apple plants' resistance to alkali stress. Our results showed that the restriction on the growth, the decrease of membrane permeability and the accumulation of Na+ were alleviated to various degrees in MdTyDc transgenic plants under alkali stress. In addition, overexpression of MdTyDc enhanced the root activity and photosynthetic capacity, and improved the enzyme activity related to N metabolism, thus promoting N absorption. It is noteworthy that the dopamine content of these three transgenic lines is significantly higher than that of WT. In summary, these findings indicated that MdTyDc may enhance alkaline tolerance of apples by mediating dopamine content, mainly by maintaining high photosynthetic capacity, normal ion homeostasis and strong nitrogen absorption capacity.

Exogenous dopamine and overexpression of the dopamine synthase gene MdTYDC alleviated apple replant disease.

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.

Functions of dopamine in plants: a review.

Dopamine (3-hydroxytyramine or 3,4-dihydroxyphenethylamine) has many functions in animals, but also shows several other functions in plants. Since the discovery of dopamine in plants in 1968, many studies have provided insight into physiological and biochemical functions, and stress responses of this molecule. In this review, we describe the biosynthesis of dopamine, as well as its role in plant growth and development. In addition, endogenous or exogenously applied dopamine improved the tolerance against several abiotic stresses, such as drought, salt, and nutrient stress. There are also several studies that dopamine contributes to the plant immune response against plant disease. Dopamine affects the expression of many abiotic stresses related genes, which highlights its role as a multi-regulatory molecule and can coordinate many aspects of plant development. Our review emphasized the effects of dopamine against environmental stresses along with future research directions, which will help improve the yield of eco-friendly crops and ensure food security.

Physiological and transcriptome analyses of the effects of exogenous dopamine on drought tolerance in apple.

Water shortage is one of the main limiting factors in apple (Malus domestica Borkh.) production. Although dopamine is produced in plants and has been linked with response to abiotic stress, the underlying mechanism remains unknown. In this study, physiological analyses revealed that pretreatment with 100 µM dopamine alleviated drought stress in apple seedlings. Dopamine inhibited the degradation of photosynthetic pigments and increased net photosynthetic rate under drought stress. Dopamine also reduced H2O2 content, possibly through direct scavenging and by mediating the antioxidant enzyme activity. Seedlings pretreated with dopamine had higher sucrose and malic acid contents but lower starch accumulation in their leaves. RNA-Seq analysis identified 1052 differentially expressed genes (DEGs) between non-treated and dopamine-pretreated plants under drought. An in-depth analysis of these DEGs revealed that dopamine regulated the expression of genes related to metabolism of nitrogen, secondary compounds, and amino acids under drought stress. In addition, dopamine may improve apple drought tolerance by activating Ca2+ signaling pathways through increased expression of CNGC and CAM/CML family genes. Moreover, analysis of transcription factor expression suggested that dopamine affected drought tolerance mainly through the regulation of WRKY, ERF, and NAC transcription factors.

Melatonin increases the performance of Malus hupehensis after UV-B exposure.

Melatonin, an evolutionarily conserved molecule, is implicated in numerous physiological processes in plants. To explore the potential roles of melatonin in response to UV-B radiation, we examined the influence of exogenous melatonin on Malus hupehensis Rehd. seedlings under two levels of UV-B radiation. Under UV-B stress, seedlings showed significant reduction in plant growth, biomass production, and root system development. However, 1 µM melatonin solution markedly alleviated these effects, especially at the higher dosage of UV-B radiation. The inhibitory effects of UV-B radiation on photosynthetic parameters, chlorophyll fluorescence parameters, stomatal apertures, chlorophyll levels and leaf membrane damages were also markedly alleviated with melatonin application. Melatonin treatment was also associated with higher activity and expression of genes encoding antioxidant enzymes (ascorbate peroxidase, catalase and peroxidase) and greater decline of H2O2 content in leaves exposed to UV-B. Moreover, exogenous melatonin treatment and UV-B stress increased the concentration of endogenous melatonin. The content of several phenolic compounds, including chlorogenic acid, phloridzin and quercetin-3-galactoside, also increased under UV-B stress, and these were further elevated significantly with melatonin addition. This study provides insight into the role(s) of endogenous melatonin in response to UV-B stress, and will facilitate application of exogenous melatonin in agriculture.

The mitigation effects of exogenous melatonin on replant disease in apple.

Melatonin mediates many physiological processes in plants. The problem of apple replant disease is unsolved. Our study objectives were to evaluate the regulatory effect of melatonin on plant resistance to this challenge and investigate the preliminary mechanism by which melatonin helps alleviate the effects of this disease. Two-year-old trees of "Fuji" apple (Malus domestica), grafted onto rootstock M.26, were grown in "replant" soil for 6 months in the absence or presence of a 200 µmol/L melatonin supplement. The addition of melatonin to the soil significantly increased the rates of plant growth and net photosynthesis and chlorophyll concentrations under replant conditions. This molecule elevated the levels of K in leaves and roots and enhanced the activity of soil enzymes. Such supplementation also changed the composition of the bacterial and fungal communities in the soil. We concluded that the application of melatonin to a replant soil can protect their chloroplasts from oxidative damage and release the apple root from membrane damage, and also lead to increased soil enzyme activity and soil quality while altering the composition of bacterial and fungal communities. These changes can then promote seedling growth, stimulate photosynthesis, and elevate K levels, thereby alleviating the effects of apple replant disease.

Effects of Exogenous Dopamine on the Uptake, Transport, and Resorption of Apple Ionome Under Moderate Drought.

The frequency and intensity of water deficits is expected to increase because of global warming. Drought stress is often one of the most limiting factors for plant growth. We conducted greenhouse pot experiments to address how dopamine affects the drought-resistance traits of apple trees at the physiological and molecular levels. Our factorial design consisted of dopamine and no-dopamine applications combined with well-watered and moderate-drought conditions. Seedling biomass, photosynthesis rates, chlorophyll concentrations, and stomatal apertures were markedly reduced under stress but dopamine treatment mitigated the inhibiting effects of drought on plant growth and helped maintain strong photosynthesis, chlorophyll levels, and stomatal functioning. Concentrations of most macro-, micro-, and trace elements decreased in response to drought. This stress also diminished the uptake and transport of elements in the leaves and stems, but increased the partitioning of elements in the roots. Nutrient resorption proficiency decreased while nutrient resorption efficiency increased for most analyzed elements. Exogenous dopamine significantly increased the concentrations, uptake, and transport of nutrients under drought stress, and also altered their distribution within the whole plant. However, this molecule had a negative effect on nutrient resorption. Although transcript levels of a key chlorophyll degradation gene, pheide a oxygenase, and senescence-associate gene 12 were elevated upon drought treatment, dopamine significantly suppressed the upregulation of those genes under such stress conditions. These observations indicate that dopamine has an important anti-senescence effect that might be helpful for regulating nutrient uptake, transport, and resorption, and ultimately influencing overall plant growth. Thus, understanding the role of dopamine in drought tolerance introduces new possibilities to use this compound for agricultural purposes.

Effects of Exogenous Melatonin on Methyl Viologen-Mediated Oxidative Stress in Apple Leaf.

Oxidative stress is a major source of damage of plants exposed to adverse environments. We examined the effect of exogenous melatonin (MT) in limiting of oxidative stress caused by methyl viologen (MV; paraquatin) in apple leaves (Malus domestica Borkh.). When detached leaves were pre-treated with melatonin, their level of stress tolerance increased. Under MV treatment, melatonin effectively alleviated the decrease in chlorophyll concentrations and maximum potential Photosystem II efficiency while also mitigating membrane damage and lipid peroxidation when compared with control leaves that were sprayed only with water prior to the stress experiment. The melatonin-treated leaves also showed higher activities and transcripts of antioxidant enzymes superoxide dismutase, peroxidase, and catalase. In addition, the expression of genes for those enzymes was upregulated. Melatonin-synthesis genes MdTDC1, MdT5H4, MdAANAT2, and MdASMT1 were also upregulated under oxidative stress in leaves but that expression was suppressed in response to 1 mM melatonin pretreatment during the MV treatments. Therefore, we conclude that exogenous melatonin mitigates the detrimental effects of oxidative stress, perhaps by slowing the decline in chlorophyll concentrations, moderating membrane damage and lipid peroxidation, increasing the activities of antioxidant enzymes, and changing the expression of genes for melatonin synthesis.