Drought stress affects plant growth and development. Zeaxanthin, as the key substance of the xanthophyll cycle and the precursor to abscisic acid (ABA), whose synthesis depends on light, can quickly respond to drought stress and enhance plant drought tolerance. However, the molecular mechanism by which zeaxanthin adapts to drought stress is still unclear. In this study, the application of exogenous zeaxanthin via spraying was confirmed to alleviate the damage caused by drought stress in apple seedlings, and MdBCH1 and MdLCYB2, which encode proteins that catalyze zeaxanthin synthesis, enhanced drought resistance by accelerating the lutein cycle and ABA synthesis. Furthermore, a transcription factor, MdPIF4, was identified as effectively reducing the contents of zeaxanthin and ABA and negatively regulating drought resistance. Further studies confirmed that MdPIF4 inhibits zeaxanthin biosynthesis by binding to the promoters of MdBCH1 and MdLCYB2. Therefore, this study revealed the regulation of drought resistance and zeaxanthin homeostasis in apple by the MdPIF4-MdBCH1/MdLCYB2 module. These findings provide insights into the molecular mechanisms of drought resistance in plants.

Drought stress is one of the important factors limiting the growth of the apple industry. Although 5-hydroxytryptamine (5-HT), an ancient indole amine, is widely present in plants, the role of 5-HT in drought stress in apples is unknown. We tested the impact of 5-HT on long-term drought stress in apple seedlings. Our findings revealed that 1 μM of exogenous 5-HT could improve the resistance of apple seedlings to short- and long-term drought stress. These plants experienced better root growth, more efficient water use, and less reactive oxygen species (ROS). The higher ABA levels mediated by 5-HT in leaves increased stomatal density and improved the photosynthetic capacity of leaves. Furthermore, exogenous 5-HT boosted the expression of drought stress-related genes, enabling apple plants to adapt to drought stress.

Abstract Soil salinization represents a significant constraint on global agricultural development. Although several NAC gene families have been implicated in the response to salt stress, the mechanisms underlying salt stress tolerance remain unclear, particularly in perennial fruit trees. In this study, we identify MdJUB1, a NAC transcription factor, as a key regulator of salt stress response in apple. Under salt stress, MdJUB1 increases γ-aminobutyric acid (GABA) content, thereby positively regulating salt stress tolerance in apple. Further analysis using yeast one-hybrid, luciferase assays, and electrophoretic mobility shift assays demonstrated that MdJUB1 directly binds to the promoter of MdGAD1, promoting GABA synthesis. Additionally, under salt stress, overexpression of MdJUB1 increased the Na+/K+ ratio in apple. MdJUB1 also binds to the promoters of MdNHX1, MdHKT1, and MdSOS3, activating their expression. Using yeast two-hybrid screening, we identified MdDOF2.4 as an interactor of MdJUB1 and found that MdDOF2.4 positively regulates salt stress tolerance. Importantly, the interaction between MdDOF2.4 and MdJUB1 enhances the expression of downstream genes, including MdGAD1, MdNHX1, MdHKT1, and MdSOS3. Our results demonstrate that MdJUB1 is involved in the regulation of GABA levels and the maintenance of Na+/K+ homeostasis, which may contribute to enhanced salt tolerance in apple.

Serotonin (5-hydroxytryptamine, 5-HT), an indole derivative, has a variety of physiological and developmental functions in plants. Anthocyanins play a crucial role in fruit coloring. Although light controls melatonin biosynthesis in plants, how serotonin, a melatonin precursor, contributes to anthocyanin accumulation at the molecular level remains unknown. In this study, we investigated the potential function of the 5-HT synthetase MdT5H5 in inhibiting anthocyanin accumulation in response to light. In apple fruits, light increased MdT5H5 expression and 5-HT accumulation. Anthocyanin accumulation was reduced in light-induced calli overexpressing MdT5H5, which was also supported by the observation that anthocyanins accumulate in apple fruits when MdT5H5 is transiently silenced. In addition, we identified MdbZIP23 as a repressor of MdT5H5, which promotes anthocyanin accumulation in apple fruits. These results suggest that MdbZIP23 negatively regulates MdT5H5-mediated anthocyanin accumulation, providing insight into the molecular mechanisms of photo-induced anthocyanin accumulation in apple fruits.

Alkali stress is an important factor that restricts the growth and yield of crops. Apple (Malus baccata Borkh.) rootstocks have attracted widespread attention because of their wide distribution and ability to exist in various forms. This study reported the tolerance of several M. baccata accessions to alkaline stress using the sand culture method. Nitrogen (N), phosphorus (P), and potassium (K) contents in the roots of 22 M. baccata accessions decreased after 40 days of alkali stress exposure. N and P contents in the leaves of most M. baccata seedlings decreased, whereas K content increased. In addition, the new leaf number and fresh and dry weights of the M. baccata accessions decreased significantly, indicating that alkali stress inhibits the growth of M. baccata seedlings. The 22 M. baccata accessions were divided into three categories: high, moderate, and low tolerance based on the cluster analysis of the resistance coefficients of six growth indices combined with the average resistance coefficient evaluation of the growth indices. These delineations are important for screening M. baccata resources. This study provides a basis for the growth of the apple industry in areas such as northwest China, which suffer from severe salinization.

Soil alkalization is an adverse factor limiting plant growth and yield. As a signaling molecule and secondary metabolite, γ-aminobutyric acid (GABA) responds rapidly to alkaline stress and enhances the alkaline resistance of plants. However, the molecular mechanisms by which the GABA pathway adapts to alkaline stress remain unclear. In this study, a transcription factor, MdNAC104 is identified, from the transcriptome of the alkaline-stressed roots of apple, which effectively reduces GABA levels and negatively regulates alkaline resistance. Nevertheless, applying exogenous GABA compensates the negative regulatory mechanism of overexpressed MdNAC104 on alkaline resistance. Further research confirms that MdNAC104 repressed the GABA biosynthetic gene MdGAD1/3 and the GABA transporter gene MdALMT13 by binding to their promoters. Here, MdGAD1/3 actively regulates alkaline resistance by increasing GABA synthesis, while MdALMT13 promotes GABA accumulation and efflux in roots, resulting in an improved resistance to alkaline stress. This subsequent assays reveal that MdSINA2 interacts with MdNAC104 and positively regulates root GABA content and alkaline resistance by ubiquitinating and degrading MdNAC104 via the 26S proteasome pathway. Thus, the study reveals the regulation of alkaline resistance and GABA homeostasis via the MdSINA2-MdNAC104-MdGAD1/3/MdALMT13 module in apple. These findings provide novel insight into the molecular mechanisms of alkaline resistance in plants.

Soil salinization had become a global ecological problem, which restricts the plant growth, and the quantity and quality of fruits. As a signaling molecule, γ-Aminobutyric acid (GABA) mediates a series of physiological processes and stress responses. Our previous research showed that GABA could alleviate drought, low phosphorus, cadmium stresses in apples, but the further research about its physiological mechanisms under salt stress was even more needed. The present study showed that the inhibition of salt stress on plant growth might be effectively alleviated by the treatment of 0.5 mM GABA, and the osmotic balance and photosynthetic capacity of plants could be maintained. Exogenous GABA could effectively inhibit the enrichment of reactive oxygen species and the uptake of Na+, while maintaining ion homeostasis. The experiment results indicated GABA could markedly promote the expression amount of Na+ and K+ transport-related genes (e.g., HKT1, AKT1, NHX1, SOS1, SOS2, and SOS3) in apples under salt stress. Overexpression and interference (RNAi) of MdGAD1 in apple roots, which is a crucial enzyme in the GABA biosynthesis, affected the salt tolerance of plants. Transgenic apple plants with roots of overexpression MdGAD1 showed less relative electrolyte leakage and more expression level of related ion transport genes than CK group, but RNAi MdGAD1 led to the opposite results. These results indicated that GABA accumulation could effectively strengthen the resistance of apple plants to salt stress and alleviate the injury of apple seedlings resulted from salinity.

Serotonin (5-hydroxytryptamine, 5-HT), a tryptophan derivative, is an important functional component in fruits. Although 5-HT was found in most fruits, the accumulation characteristics of its biosynthesis in apple fruits remain unclear. Here, we conducted a large-scale survey of 148 apple fruits in 5-HT content, and investigated the biosynthesis differences of 5-HT. Our results displayed a broad skewed normal distribution of 5-HT in apple fruits. 5-HT accumulated in all tissues of apple plants, with the highest content in the seeds and the higher content in the pulp than that of the peel. A significant increase of 5-HT at ripening stage was observed in the fruits with high 5-HT content, however, the 5-HT content did not change significantly during fruit development in the fruits with low 5-HT content. The bagging reduced the 5-HT biosynthesis compared to the fruits exposed to light conditions. In addition, the expression level of MdT5H5 was positively correlated with the 5-HT content, suggesting that MdT5H5 may play a key regulatory role in the 5-HT biosynthesis in apple fruits.

Low temperatures can seriously affect apple yield and can also cause chilling injury to apple fruit. γ-aminobutyric acid (GABA) plays an important role in improving plant stress resistance. Some studies have reported that GABA can improve cold resistance in plants, only through exogenous treatment; however, the molecular mechanism of its resistance to low temperature is still unknown. This result suggested that exogenous GABA treatment of both apple seedlings and fruit could improve the resistance of apple to low temperatures. MdGAD1, a key gene involved in GABA synthesis, was overexpressed in tomato plants and apple callus to improve their cold tolerance. Both yeast one-hybrid and luciferase assay showed that MdCBF3 could bind to the MdGAD1 promoter to activate its expression and promote GABA synthesis. These results revealed a molecular mechanism utilizing the MdCBF3-MdGAD1 regulatory module that can enhance cold resistance by increasing endogenous GABA synthesis in apple.

Cord blood (CB) CD34+ cells have the potential to be used to achieve artificial hematopoiesis because of their ability to expand and differentiate in multiple directions. However, the mechanism and molecular changes underlying such differentiation are still unclear. The differentiation of CB CD34+ cells is generally driven by subtle changes in gene expression. A crucial method for examining gene expression is quantitative real-time polymerase chain reaction, but the accuracy of the results is dependent on the use of reliable reference genes. Here, the transcription levels of 10 novel candidate reference genes (EIF4G2, DYNC1H1, LUC7L3, CD46, POLR1D, WSB1, GAPVD1, HGS, LGALS8, and RBM5) and 8 traditional reference genes (GAPDH, YWHAZ, ACTB, B2MG, TBP, HMBS, PPIA, HPRT1) in CB CD34+ cells under different oxygen concentrations were screened and evaluated by using the geNorm and NormFinder algorithms. Comprehensive analysis conducted by RefFinder online tool showed that TBP (a traditional reference gene) and EIF4G2 (a novel reference gene) had the most stable expression, whereas GAPDH and HMBS were the least suitable reference genes under these conditions. These results may serve as a basis for selecting reference genes with stable expression for more accurate normalization under different oxygen concentration stimulation during CB CD34+ cells differentiation.

Carotenoids play an important role in the coloring and nutritional value of apple (Malus spp.) fruits. Here, six carotenoids, including lutein, zeaxanthin, β-carotene, β-cryptoxanthin, violaxanthin, and neoxanthin, were detected in 105 fruits of apple germplasm resources, which showed a skewed distribution in both the peel and pulp. There were more carotenoids in the peel than in the pulp, and lutein and β-carotene were the primary carotenoids that were present. The expression levels of most carotenoid pathway genes in germplasm fruits during fruit development were higher in the fruits that had an abundance of carotenoids. A linear relationship analysis showed that the expression levels of MdCRTISO and MdLCYE were highly correlated with the content of carotenoids. The leaves accumulated the greatest number of carotenoids, while the roots had the lowest amount. MdCRTISO and MdLCYE were highly expressed in the fruits compared to other tissues. Transgenic calli and transiently transformed fruits confirmed that MdCRTISO and MdLCYE affected the biosynthesis of carotenoids owing to their effects on the expression of other genes for enzymes in the carotenoid pathway. Our findings will extend the understanding of carotenoid biosynthesis in apple and excavate apple germplasm resources with rich carotenoids to breed high-quality apples.

As the immobility of inorganic phosphorus (P) in soil, the acquisition of P by sessile plants is limited. γ-Aminobutyric acid (GABA) as a signal molecule and a metabolite can regulate plants to cope with various stresses. However, whether GABA could contribute to the adaption to low P stress in apple plants remains unclear. This study combined different methods to detect the induction of auxin (IAA) synthesis by GABA to improve the tolerance of apple seedlings to low P stress (including growth and development analysis, reactive oxygen species (ROS) clearance effect, root structure analysis, multiple factorial analysis (MFA), gene expression analysis). Exogenous GABA improved the growth of apple seedlings under low P conditions, reduced the ROS accumulation, and promoted the photosynthetic capacity. GABA contributed to the root system architecture and the development of mature area of root tips. In addition, the intervention of exogenous GABA interfered with the homeostasis of endogenous IAA and activated the expression of P starvation induction (PSI) gene, leading to the significantly increase of the P uptake in apple plants. Meanwhile, transgenic roots with overexpressing MdGAD1 enhanced the tolerance of apple seedlings to low P stress, improved the root development through regulating IAA signaling pathway, and significantly improved the P uptake of apple seedlings under low P conditions. All results suggested that GABA could contribute to the adaption of apple seedlings to low P conditions, by decreasing ROS accumulation, maintaining photosynthetic capacity, and increasing IAA level to improve the root development and the P absorption.

The fungal disease Glomerella leaf spot (GLS) seriously impacts apple production. As a nonprotein amino acid, γ-aminobutyric acid (GABA) is widely involved in biotic and abiotic stresses. However, it is not clear whether GABA is involved in a plant's response to GLS, nor is its molecular mechanism understood. Here, we found that exogenous GABA could significantly alleviate GLS, reduce lesion lengths, and increase antioxidant capacity. MdGAD1 was identified as a possible key gene for GABA synthesis in apple. Further analysis indicated that MdGAD1 promoted antioxidant capacity to improve apple GLS resistance in transgenic apple calli and leaves. Yeast one-hybrid analysis identified the transcription factor MdWRKY33 upstream of MdGAD1. Electrophoretic mobility shift assay, β-glucuronidase activity, and luciferase activity further supported that MdWRKY33 bound directly to the promoter of MdGAD1. The content of GABA and the transcription level of MdGAD1 in the MdWRKY33 transgenic calli were higher than that of the wild type. When MdWRKY33 transgenic calli and leaves were inoculated with GLS, MdWKRY33 positively regulated resistance to GLS. These results explained the positive regulatory effects of GABA on apple GLS and provided insight into the metabolic regulatory network of GABA.

Glomerella leaf spot (GLS), caused by the fungal phytopathogen Glomerella cingulata, is one of the most devas-tating premature defoliation diseases affecting apple production. Chitosan (CHT) is a degraded product of chitin and is used for bacteriostasis and postharvest preservation. Herein, we investigated the effects of CHT on improving the apple resistance to GLS. We identified that spraying with 0.5 g center dot L-1 exogenous CHT was the optimal concentration to reduce the G. cingulata disease severity on detached leaves of apple seedlings. Further study demonstrated that the exogenous CHT application significantly enhanced the superoxide dismutase (SOD) activity, elevated alicylic acid (SA) and jasmonic acid (JA) contents, increased certain kinds of phenolic com-pound contents and up-regulated the expression of different disease resistance-related genes in inoculated leaves than plants without CHT pretreatment. Furthermore, the exogenous CHT application significantly reduced the contents of abscisic acid (ABA), 1-aminocyclopropanecarboxylic acid (ACC), and auxin (IAA) of G. cingulata infection. Additionally, the CHT spraying treatment reduced the amino acid contents compared with the control plants. The results indicated that exogenous spraying of CHT improved the resistance of apples to GLS through enhancing plant immunity.

Apple is a major fruit crop grown worldwide and provides humans with an essential diet and health benefits. One of the health benefits is related to the accumulation of fruit anthocyanin, which also provides fruit with an attractive red colour. It is known that an MdMYB10 allele containing a transposable element (TE) insertion in its promoter (termed Red-TE allele) underlies anthocyanin accumulation and red colouration in the fruit skin of cultivated apples. However, the distribution of this Red-TE allele in wider Malus germplasm accessions is not clear. In this study, we showed that MdMYB10 RNA in fruit skin was specifically expressed from the Red-TE allele by using allele-specific expression analysis of transcriptome data. Apple cultivars and hybrids with homozygous Red-TE alleles showed stronger red colour than those with heterozygous alleles after analysing 65 cultivars and 337 hybrids. Furthermore, both hetero- and homozygous plants growing in the same high-temperature conditions had different colourations. However, the Red-TE allele was not detected in 16 wild apple accessions showing red skin, indicating that the red skin colour of these wild apples was not conferred by the Red-TE allele. These findings provide guidance for selecting cultivars able to develop consistent red colouration under high growth temperature conditions and open the opportunity for identifying novel genetic variants underpinning fruit red colouration in wild apple species.

Marssonina apple blotch, caused by the pathogen Marssonina coronaria, is one of the most important pre-mature defoliation diseases that affected apple production. gamma-aminobutyric acid (GABA) is a non-protein amino acid and as an endogenous metabolite and signal molecule plays a key role in the stress response in plants. Herein, we investigated the effects of GABA in improving apple resistance to Marssonina apple blotch. Soil drenching of 0.5 mM exogenous GABA was identified as the optimal concentration to reduce M. coronaria disease severity on twomonths-old apple seedlings (Malus x domestica cv. 'Royal Gala') in a growth chamber. Further study demonstrated that the application of exogenous GABA significantly reduced the chlorophyll content degradation and photosynthetic rate than inoculated and non-GABA treated plants. After inoculation with M. coronaria, the GABA content in apple leaves increased significantly in infected plants. The soil drenching of GABA further elevated the salicylic acid (SA) content and activities of chitinase and beta-1,3-glucanase compared with the inoculated nonGABA treated plants. Furthermore, phenylalanine ammonia lyase (PAL) activity and the total phenol content of GABA-treated plants were further increased compared with the inoculated non-GABA treated plants. As a component of natural plant metabolism, exogenous soil drenching of GABA maybe a promising strategy through enhancing plants own immunity to control Marssonina apple blotch prevalence.

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.

Drought stress is an important factor limiting apple production. gamma-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.

GABA, a four-carbon non-protein amino acid, plays an important role in animals and plants. We previously found GABA could alleviate alkali stress in apple seedlings. However, its physiological mechanism under heavy metal cadmium (Cd) stress need to be further studied. Thus, we explored its biological role in response to Cd stress. It was verified that 0.5 mM GABA could effectively alleviate Cd toxicity. Using NMT technique, we found that exogenous GABA could significantly reduce the net Cd2+ fluxes in apple roots, and Cd content was significantly lower than that in roots under Cd stress. Further analysis indicated exogenous GABA could significantly reduce the expression of genes related to the uptake and transport of Cd in apples under Cd stress. In addition, exogenous GABA could significantly increase the content of amino acids in apple roots under Cd stress. GAD is a key enzyme in GABA synthesis, we obtained transgenic apple roots of overexpression MdGAD1. Compared with the control, transgenic roots accumulated less Cd, maintained lower Cd uptake by roots, and lower expression of related transport genes. These results showed that GABA could effectively alleviate Cd toxicity in apple seedlings and provide a new perspective of GABA to alleviate Cd stress.