Research articleResponse of sugar metabolism in apple leaves subjected to short-term drought stress
Introduction
Carbohydrates produced from photosynthesis in plant leaves provide energy and building blocks for plant growth and productivity. In addition, soluble carbohydrates (e.g., sucrose, fructose, glucose and sorbitol) are known to act as important osmoregulation substances to maintain cell turgor under osmotic stress (Subbarao et al., 2000), e.g., drought and salt. Drought stress significantly affects carbohydrate-modulated gene expression in plant cells (Gifford and Bremner, 1981; Boyer, 1982; Koch, 1996; Bartels and Sunkar, 2005), and changed soluble sugars under drought stress act as signal molecules to regulate the expression of many key genes involved in plant defense responses and metabolic processes, consequently controlling plant resistance and growth (Rosa et al., 2009). Regulation of soluble carbohydrate concentrations in plant cells is an important pathway of plant adaptation or resistance to water deficit, but the understanding of this pathway is still limited.
Soluble carbohydrate concentrations in leaves are highly regulated by the balance between synthesis, degradation and export. Sucrose is a main soluble product of photosynthesis in most plants and is synthesized in green leaves and transported into sink tissues or cell compartments by sucrose transporters (Ruan, 2014). One of the key enzymes involved in sucrose synthesis is sucrose-phosphate synthase (SPS) (Huber and Huber, 1992). Sucrose can be converted to fructose (Fru) and glucose (Glc) by invertase or to Fru and UDP-glucose (UDPG) by sucrose synthase (SUSY) (Ruan, 2014). Although SUSY can catalyze both sucrose synthesis and decomposition, the latter is its main role (Geigenberger and Stitt, 1993). The resulting Glc and Fru are then phosphorylated to glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P), respectively, by hexokinase (HK) or fructokinase (FK) (Granot, 2007).
The interconversions between F6P, G6P, G1P, and UDPG are enzymatically catalyzed in readily reversible reactions. Both F6P and UDPG produced in sugar metabolism can be combined to resynthesize Suc via sucrose phosphate synthase (SPS) and sucrose-phosphate phosphatase (SPP, EC 3.1.3.24) (Hoffmann-Thoma et al., 2010). Suc, Glc and Fru and other soluble sugars that have not been metabolized are transported into vacuoles by special transporter proteins located on the vacuole membrane. Once inside the vacuole, Suc can also be converted to Glc and Fru by vacuolar acid invertase (AINV) (Ruan, 2014). This sugar metabolism system in cells regulates sugar concentrations and maintains the balance in osmotic potential and turgor between the cytosol and other subcellular compartments.
Most stress conditions (especially drought) lead to the accumulation of carbohydrates in leaves, which can play an important role in osmoprotection, osmotic adjustment, carbon storage and radical scavenging in plants (Parvaiz and Satyawati, 2008). Under stress conditions, a change in the sugar concentrations would be a result of the regulation of sugar metabolism and export. Despite the reduced carbon fixation in drought-stressed leaves, plants accumulate a large amount of water-soluble carbohydrates such as glucose, fructose, sucrose, stachyose, mannitol and pinitol; the types of soluble carbohydrates vary among species (Bohnert et al., 1995; Pinheiro et al., 2001; Bartels and Salamini, 2001; Villadsen et al., 2005; Valliyodan and Nguyen, 2006). The accumulation of water-soluble carbohydrates is widely regarded as an adaptive response of plants to drought stress. These soluble carbohydrates are not only used as osmolytes for maintaining leaf cell turgor and protecting membrane integrity (Bartels and Sunkar, 2005; Verslues et al., 2006) but also act as nutrient and metabolite signaling molecules, modulating the expression of a large number of metabolic genes through sugar-sensing mechanisms (Ho et al., 2001; Price et al., 2004). The accumulation of these sugars represents extensive carbon redistribution in plants when carbon fixation is reduced. To elucidate the regulation of carbohydrate metabolism involved in carbon redistribution for the accumulation of soluble sugars in plant leaves under drought stress, a systematic metabolic pathway-based analysis would provide insight into the roles of carbohydrate metabolism in response to drought stress. Although sugar contents and enzyme activity in pathways of carbohydrate metabolism have been investigated under drought stress in many plants (Ranney et al., 1991; Wang and Stutte, 1992; Escobar-Gutiérrez et al., 1998; Xu et al., 2001), a systematic understanding of gene expression, enzyme activity and sugar concentration is limited, and it is not well known which genes are important in regulating sugar metabolism under drought stress as several isoenzymes are coded by different homologous genes (Li et al., 2018).
In apple and many other Rosaceae fruit trees, sorbitol is a primary end product of photosynthates produced in leaves (Beruter, 2004). It was suggested that water stress favors a functional role for sorbitol as an osmoticum in apple (Wang and Stutte, 1992; Bianco et al., 2012; Wu et al., 2014), whereas other soluble sugars exhibited different responses to drought stress in different apple cultivars (Wang et al., 1995; Li et al., 2005). However, our knowledge is very limited to why these carbohydrate contents in apple leaves would change to adapt to drought stress.
The objectives of this study were to understand how apple plants adjust carbohydrate metabolic flow to increase sugar osmolytes under the conditions of reduced photosynthesis and to identify which genes are involved in the regulation of sorbitol and sugar metabolism in apple leaves during drought stress and after re-watering. We focused mainly on the regulation of enzyme activities and gene expression levels involved in the synthesis of sorbitol and the subsequent conversion to sucrose and hexoses in drought-stressed apple leaves using a systematic metabolic pathway-based expression analysis. These analyses provide a complete picture of the regulation and relationships of various genes and enzymes involved in these metabolic pathways and the role of important isoenzymes in the alteration of carbohydrate metabolism in drought-stressed apple leaves.
Section snippets
Plant materials
One-year-old ‘Greensleeves’ apple (M. domestica Borkh.) trees grafted on seedling rootstocks (M. hupehensis (Pamp) Rehd.var.pinyiensis Jiang, which is an apomictic species) were used in this study. Grafted plants were planted in plastic pots (28 × 21 cm) that were filled with a mixture of sand, organic-eral fertilizer, and soil (1:1:3, v:v:v). All pots initially weighed the same (9.5 kg) to facilitate calculations and to maintain a specified field capacity via the weighing method. All trees
Water stress and Pn
Midday leaf water potentials decreased in drought-stressed trees as the water withholding duration increased, whereas controls exhibited constant leaf water potentials (Fig. 1). According to Hsiao et al. (1976), water-stressed plants show a mild water deficit after 2 days without watering, moderate stress after 4 days without watering, and severe stress after 6 days without watering, as indicated by their leaf water potential, compared to well-watered controls. Two days after the
Discussion
Leaf water potential has been used to determine the degree of plant water deficit or water status, and osmotic adjustment is an adaptive process that greatly assists in the maintenance of turgor under water deficit (Turner et al., 1978). The significant decrease of leaf water potential and continued increase of relative electrical conductivity during the experimental treatments (Fig. 1) suggest that the trees were subjected to an increasing degree of drought stress. As the leaf water potential
Author contributions
JJY and JZ performed the majority of experiments. CL performed essential experiments. ZZ analysed and discussed data. FWM and MJL supervised work. All authors contributed to final manuscript.
Conflicts of interest
The authors declare that they have no conflict of interest.
Acknowledgements
This work was supported by the Program for the National Natural Science Foundation of China (No. 31672128; 31872043) and by the earmarked fund for the China Agricultural Research System (CARS-28). The authors thank Mr. Xuanchang Fu and Xiaowei Ma for maintaining the plants.
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