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.

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.

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.