Sucrose phosphate synthase (SPS) is a rate-limiting enzyme that works in conjunction with sucrose-6-phosphate phosphatase (SPP) for sucrose synthesis, and it plays an essential role in energy provisioning during growth and development in plants as well as improving fruit quality.  However, studies on the systematic analysis and evolutionary pattern of the SPS gene family in apple are still lacking.  In the present study, a total of seven MdSPS and four MdSPP genes were identified from the Malus domestica genome GDDH13 v1.1.  The gene structures and their promoter cis-elements, protein conserved motifs, subcellular localizations, physiological functions and biochemical properties were analyzed.  A chromosomal location and gene-duplication analysis demonstrated that whole-genome duplication (WGD) and segmental duplication played vital roles in MdSPS gene family expansion.  The Ka/Ks ratio of pairwise MdSPS genes indicated that the members of this family have undergone strong purifying selection during domestication.  Furthermore, three SPS gene subfamilies were classified based on phylogenetic relationships, and old gene duplications and significantly divergent evolutionary rates were observed among the SPS gene subfamilies.  In addition, a major gene related to sucrose accumulation (MdSPSA2.3) was identified according to the highly consistent trends in the changes of its expression in four apple varieties (‘Golden Delicious’, ‘Fuji’, ‘Qinguan’ and ‘Honeycrisp’) and the correlation between gene expression and soluble sugar content during fruit development.  Furthermore, the virus-induced silencing of MdSPSA2.3 confirmed its function in sucrose accumulation in apple fruit.  The present study lays a theoretical foundation for better clarifying the biological functions of the MdSPS genes during apple fruit development.

Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research.  However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits.  In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function.  However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed.  Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’.  The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L⁻¹ sucrose, 8 g L⁻¹ agar, 5 mg L⁻¹  6-benzylaminopurine (6-BA), 2 mg L⁻¹ thidiazuron (TDZ), and 1 mg L⁻¹ 1-naphthlcetic acid (NAA), pH 5.8).  A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L⁻¹) and kanamycin (15 mg L⁻¹).  We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material.  These findings make a significant contribution to apple biology research

Myo-inositol and its derivatives play important roles in the tolerance of higher plants to abiotic stresses, and myo-inositol-1-phosphate synthase (MIPS) is the rate-limiting enzyme in myo-inositol biosynthesis.  In this study, we found that increased myo-inositol biosynthesis enhanced drought tolerance in MdMIPS1-overexpressing apple lines under short-term progressive drought stress.  The effect of myo-inositol appeared to be mediated by the increased accumulation of osmoprotectants such as glucose, sucrose, and proline, and by the increased activities of antioxidant enzymes that eliminate reactive oxygen species.  Moreover, enhanced water-use efficiency (WUE) was observed in MdMIPS1-overexpressing apple lines under long-term moderate water deficit conditions that mimicked the water availability in the soil of the Loess Plateau.  Enhanced WUE may have been associated with the synergistic regulation of osmotic balance and stomatal aperture mediated by increased myo-inositol biosynthesis.  Taken together, our findings shed light on the positive effects of MdMIPS1-mediated myo-inositol biosynthesis on drought tolerance and WUE in apple.

Dopamine is a catecholamine and an anti-oxidant which functions in responses to stress and it interacts with plant hormones to mediate plant development.  At present, there are few studies on the functions of dopamine in apple.  This study developed a method for dopamine determination which was used to analyze dopamine in Malus germplasm, in order to clarify the tissue distribution, developmental changes, diurnal variations, and stress responses in apple trees.  First, the proposed method was verified.  The linear range of quantification was robust from 0.1 to 20 ng mL^–1.  The instrumental, inter-day precision, and sample repeatability relative standard deviations were 1.024, 5.607, and 7.237%, respectively.  The spiked recovery was greater than 100%, indicating the feasibility of the method and its suitability for the rapid analysis of dopamine in Malus.  Next, the dopamine content was measured in 322 Malus tissues.  The results showed that the dopamine level in Malus was low and the average dopamine content in leaf was higher than in peel and flesh.  The dopamine had a skewed distribution that deviated to the right in cultivars and wild accessions.  Finally, the tissue specificity, developmental changes, diurnal changes, and responses to stress were analyzed.  In cultivar ‘Pinova’ (Malus domestica), the dopamine concentration was the highest in leaf buds and lowest in flesh.  The dopamine contents in leaf and flesh decreased with the growth and development of cultivar ‘Liangxiang’ (Malus domestica).  The dopamine content of apple leaves was higher after either drought or salinity stress as compared to the control.  In this study, a dopamine detection method for apple was established based on HPLC-MS and shown to be a robust approach.  This study provides a framework for future research on elucidating the tissue distribution, developmental changes, diurnal variation, and stress responses of dopamine in apple trees.

Auxin (indole-3-acetic acid, IAA) has a considerable impact on the regulation of plant carbohydrate levels and growth, but the mechanism by which it regulates sugar levels in plants has received little attention.  In this study, we found that exogenous IAA altered fructose (Fru), glucose (Glc), and sucrose (Suc) concentrations in shoot tips mainly by regulating MdSUSY1, MdFRK2, MdHxK1 and MdSDH2 transcript levels.  Additionally, we used 5-year-old ‘Royal Gala’ apple trees to further verify that these genes play primary roles in regulating sink strength.  The results showed that MdSUSY1, MdFRK2, MdHxK1/3 and MdSDH2 might be major contributors to sink strength regulation.  Taken together, these results provide new insight into the regulation of the carbohydrate metabolism mechanism, which will be helpful for regulating sink strength and yield.

The plant hydraulic network faces several challenges under drought stress.  Hydraulic conductivity is one of the major indicators of the hydraulic network’s response to drought stress.  Here, we review our current understanding of the factors directly affecting hydraulic conductivity and the methods used to measure it.   

Soil alkalinity is a major factor that restricts the growth of apple roots. To analyze the response of apple roots to alkali stress, the root structure and endogenous hormones of two apple rootstocks, Malus prunifolia (alkali-tolerant) and Malus hupehensis (alkali-sensitive), were compared. To understand alkali tolerance of M. prunifolia at the molecular level, transcriptome analysis was performed. When plants were cultured in alkaline conditions for 15 d, the root growth of M. hupehensis with weak alkali tolerance decreased significantly. Analysis of endogenous hormone levels showed that the concentrations of indole-3-acetic acid (IAA) and zeatin riboside (ZR) in M. hupehensis under alkali stress were lower than those in the control. However, the trend for IAA and ZR in M. prunifolia was the opposite. The concentration of abscisic acid (ABA) in the roots of the two apple rootstocks under alkali stress increased, but the concentration of ABA in the roots of M. prunifolia was higher than that in M. hupehensis. The expression of IAA-related genes ARF5, GH3.6, SAUR36, and SAUR32 and the Cytokinin (CTK)-related gene IPT5 in M. prunifolia was higher than those in the control, but the expression of these genes in M. hupehensis was lower than those in the control. The expression of ABA-related genes CIPK1 and AHK1 increased in the two apple rootstocks under alkali stress, but the expression of CIPK1 and AHK1 in M. prunifolia was higher than in M. hupehensis. These results demonstrated that under alkali stress, the increase of IAA, ZR, and ABA in roots and the increase of the expression of related genes promoted the growth of roots and improved the alkali tolerance of apple rootstocks.

Sucrose synthases (SUS) are a family of enzymes that play pivotal roles in carbon partitioning, sink strength and plant development.  A total of 11 SUS genes have been identified in the genome of Malus domestica (MdSUSs), and phylogenetic analysis revealed that the MdSUS genes were divided into three groups, named as SUS I, SUS II and SUS III, respectively.  The SUS I and SUS III groups included four homologs each, whereas the SUS II group contained three homologs.  SUS genes in the same group showed similar structural characteristics, such as exon number, size and length distribution.  After assessing four different tissues, MdSUS1s and MdSUS2.1 showed the highest expression in fruit, whereas MdSUS2.2/2.3 and MdSUS3s exhibit the highest expression in shoot tips.  Most MdSUSs showed decreased expression during fruit development, similar to SUS enzyme activity, but both MdSUS2.1 and MdSUS1.4 displayed opposite expression profiles.  These results suggest that different MdSUS genes might play distinct roles in the sink-source sugar cycle and sugar utilization in apple sink tissues.

Radiation sensitivity proteins-23 (RAD23) are DNA repair factors participate in the ubiquitin/proteasome system (UPS).  Although the genome-wide analysis of RAD23 family members has been conducted in some species, little is known about RAD23 genes in apple (Malus×domestica Borkh.).  We analyzed this gene family in M. domestica in terms of genomic locations, protein and promoter structures, and expressions in response to stresses.  Various members showed a ubiquitous pattern of expression in all selected apple parts.  Their expressions were altered under chilling, heat, and hydrogen peroxide treatments, as well as abscisic acid (ABA) treatment and water deficiency, suggesting their possible roles in plant stress responses.  These results provide essential information about RAD23 genes in apple and will contribute to further functional studies

    Alkaline soils have a great influence on apple production in Northern China. Therefore, comprehensive evaluations of tolerance to such stress are important when selecting the most suitable apple rootstocks. We used hydroponics culturing to test 17 genotypes of apple rootstocks after treatment with 1:1 Na₂CO₃ and NaHCO₃. When compared with the normally grown controls, stressed plants produced fewer new leaves, and had shorter roots and shoots and lower fresh and dry weights after 15 d of exposure to alkaline conditions. Their root/shoot ratios were also reduced, indicating that the roots had been severely damaged. For all stressed rootstocks, electrolyte leakage (EL) and the concentration of malondialdehyde (MDA) increased while levels of chlorophyll decreased. Changes in root activity (up or down), as well as the activities of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) were rootstock-dependent, possibly reflecting their differences in alkali tolerance. Using alkali injury index (AI), adversity resistance coefficients (ARC), cluster analysis, and evaluation of their physiological responses, we classified these 17 genotypes into three groups: (1) high tolerance: Hubeihaitang, Wushanbianyehaitang, Laoshanhaitang Ls2, Xiaojinbianyehaitang, and Fupingqiuzi; (2) moderate tolerance: Pingyitiancha, Laoshanhaitang Ls3, Hubeihaitang A1, Deqinhaitang, Balenghaitang, Maoshandingzi, Shandingzi, and Xinjiangyepingguo; or (3) low tolerance: Pingdinghaitang, Hongsanyehaitang, Xiaojinhaitang, and Sanyehaitang. These results will significantly contribute to the selection of the most suitable materials for rootstocks with desired levels of tolerance to alkali stress.

The effects of light intensity on photosynthesis and photoprotective mechanisms under progressive drought were studied on apple trees (Malus domestica Borkh.) Fuji. The potted trees were exposed to drought stress for 12 days and different light conditions (100, 60 and 25% sunlight). During the progressive drought, the relative water content (RWC) in leaf declined and was faster in full light than in 60 and 25% sunlight. However, the decrease in the net photosynthetic rate (Pn), stomatal conductance (Gs) and Rubisco activity were slower under 100% sunlight condition than other light conditions. After the 6 days of drought, the maximum PSII quantum yield (Fv/Fm), the capacity of electrons move beyond QA − (1–VJ) and electron move from intersystem to PSI acceptor side (1–VI)/(1–VJ) decreased, with greater decline extent in brighter light. While RWCs were >75%, the variations in different light intensities of Gs and Rubisco activity at identical RWC, suggested the direct effects of light. While the little difference in the state of photosynthetic electron transport chain among tested light intensities indicates the results of faster water loss rate of light. Our results also demonstrated that the enhancement the de-epoxidations of xanthophyll cycle, activities of ascorbate peroxidase (APX) and catalase (CAT) were directly regulated by light intensity. While the higher photorespiration rate (Pr) under stronger light condition was mainly caused by faster water loss rate of light.

To understand how drought stress affects CO2 assimilation and energy partitioning in apple (Malus domestica Borkh.), we investigated photosynthesis and photo-protective mechanisms when irrigation was withheld from potted Fuji trees. As the drought progressing, soil relative water content (SRWC) decreased from 87 to 24% in 15 d; this combined the decreasing in leaf relative water content (LRWC), net photosynthesis rate (Pn) and stomatal conductance (Gs). However, the concentrations of chlorophylls (Chl) remained unchanged while Pn values were declining. Photochemistry reactions were slightly down-regulated only under severe drought. Rubisco activity was significantly decreased as drought conditions became more severe. The actual efficiency of photosystem II (ΦPSII) was diminished as drought became more intense. Consequently, xanthophyll-regulated dissipation of thermal energy was greatly enhanced. Simultaneously, the ratio of ΦPSII to the quantum yield of carbon metabolism, which is measured under non-photorespiratory conditions, increased in parallel with drought severity. Our results indicate that, under progressive drought stress, the reduction in photosynthesis in apple leaves can be attributed primarily to stomatal limitations and the inhibited capacity for CO2 fixation. Xanthophyll cycle-dependent thermal dissipation and the Mehler reaction are the most important pathways for dispersing excess energy from apple leaves during periods of drought stress.

Plants that grow well while accumulating and transporting less potassium (K) perform better than more-sensitive plants when under deficiency conditions, which makes low-K-input and environmentally friendly agriculture possible. We conducted hydroponics and sand culture experiments to evaluate the efficiency of various apple (Malus domestica Borkh) rootstocks in their K uptake and utilization. Five genotypes were selected which are widely used in China -M. hupehensis Rehd, M. prunifolia Borkh, M. robusta Rehd, M. sieversii Roem, and M. rockii Rehd. Plant heights, root and shoot dry weights, and K concentrations were recorded. These genotypes differed markedly in dry weights, absolute and relative K concentrations, absolute and relative K accumulations, and their K efficiency ratio under deficient K conditions. The last parameter, expressed as relative shoot dry weight, was strongly and positively correlated with the other four parameters in each genotype. Therefore, we suggest that this parameter could serve as an index when selecting K-efficient genotypes. In this study, we have determined that M. sieversii and M. rockii are K-inefficient genotypes; M. prunifolia is K-efficient genotype; M. hupehensis and M. robusta have moderate levels of potassium efficiency.