Effects of salt stress on sucrose metabolism and growth in Chinese rose (Rosa chinensis)

Abstract Salt stress is a major abiotic stress with detrimental effects on plants. Sucrose, the main product of photosynthesis in plants, is used as a source of energy for the development of Chinese rose (Rosa chinensis). Hua Xianzi, a variety of Chinese rose, was investigated in this study. Phenotypic traits such as digital biomass, greenness average, leaf angle and leaf area, as well as physiological characters such as proline (Pro), malondialdehyde (MDA), H2O2, O2 - and antioxidant enzymes such as SOD (Superoxide Dismutase), POD (Peroxidase), CAT (Catalase) and APX (Ascorbate Peroxidase), changed significantly in response to salt stress (150 mmol/L NaCl for 72 h). The physiological changes were examined based on photosynthetic and fluorescence indicators. Salt stress increased the concentration of glucose, fructose and sucrose, upregulated the expression of Rc-SS1, Rc-SS2, Rc-SPS1, Rc-SPS2, Rc-αA1, Rc-αA2, Rc-αA3, Rc-βA1 and Rc-βA3 genes. Finally, it decreased the starch content in leaves. This study revealed that salt stress disrupted the equilibrium of sucrose metabolism in leaves. This finding provides the basis for further research into the mechanism of salt tolerance adaption.


Introduction
Abiotic stress refers to the adverse effects exerted by non-living factors on living organisms [1,2]. As an important environmental stress, salt stress is one of the most persistent problems in agricultural practices [3]. According to scientific estimates, over 800 million hectares of land, which is about 6% of the global landmass, are affected by salt [4]. The continuous global warming has increased the detrimental effects of salinity on plants [5]. Plants are sessile and must adapt to and cope with salt stress during growth and development [6]. Salt stress, unlike other abiotic stresses such as drought, heat and cold, affects the entire plant life cycle, affecting all stages from seed germination to plant growth and development [7]. In plants, salt stress induces osmotic stress, Na + and Cltoxicity, and increases the reactive oxygen species (ROS) levels [8]. Osmotic stress is caused by high salt concentration, particularly in roots [9]. Accumulation of Na + and Clcauses ion toxicity, which impairs plant growth and development [10]. Lastly, salt stress induces overproduction of ROS, which damages the cell membranes [11].
Carbohydrates are the most important energy sources for the normal growth and metabolism of plants. Carbohydrates also provide the carbon skeleton needed by other processes associated with plant growth and development [12,13]. Plants have two types of carbohydrates: structural and non-structural carbohydrates. Non-structural carbohydrates, which include sucrose, glucose, fructose and starch, are the main armamentarium of sucrose metabolism in plants.
For most plants, carbohydrates are mainly synthesized, stored and transported in the form of metabolized sucrose, such as glucose, fructose and starch [14]. Carbohydrates are mainly transported in the form of sucrose which provides energy for plant growth and development and balances the osmotic potential of plant cells, as well as enhancing crop resistance [15]. In plants, carbohydrates are mainly stored in the form of starch. Accordingly, starch and sucrose can be converted into each other through a series of enzymatic reactions. Furthermore, the metabolism of sucrose and starch is regulated by a series of enzymes. Photosynthesis generates triose phosphate, which is transported to the cytoplasm and converted to sucrose by a series of enzymes, such as sucrose phosphate synthase (SPS) and sucrose phosphate phosphorylase (SPP) [16]. Salt stress increases SPS activity resulting in an increase in sucrose content [17]. Sucrose and sucrose synthase (SuSy), the enzyme associated with drought stress, are correlated [18,19]. In plants, α-amylase and β-amylase are important enzymes in starch and sucrose metabolism and are involved in abiotic stress response [20]. Sucrose metabolism is one of the important pathways under salt stress in soybean (Glycine max) [21], sorghum (Sorghum bicolor) [22], Nitraria sibirica Pall [23] and quinoa (Chenopodium quinoa) [24].
The Chinese rose (Rosa chinensis) is a member of the Rosaceae family and one of the most popular species in the world due to its ornamental and economic values [25,26]. Roses are grown extensively because of their various uses, including medicinal, ornamental and fragrance, and they have been carefully selected and bred. In 2008, an estimated eight billion cut stems, 60-80 million potted, and 220 million roses, worth approximately 24 billion euros were reported [27]. The demand for high quality roses has been increasing [28]. However, as one of the most important ornamental plants in the world, roses are subjected to various abiotic stressors, such as salt.
In this study, we investigated the phenotypic and physiologic characteristics of roses in the vegetative stage under salt stress, particularly in the context of photosynthesis (Fv/Fm, q p , ΦPSII and NPQ) and sucrose metabolism (sucrose, glucose, fructose and starch). Moreover, genes and enzymes involved in sucrose metabolisms were analysed using quantitative real-time PCR (qRT-PCR). The results revealed the effects of salt stress on roses, especially in sucrose metabolism.

Plant materials and growth conditions
The plant materials (Rose variety: Hua Xianzi) was provided by the College of Horticulture, Northeast Agricultural university. Hua Xianzi is a local variety in Northeast China [29]. The two-year-old cuttings were planted in a greenhouse at the National engineering Technology Research Center for Coarse Cereals of Heilongjiang, China (46.0°N, 125.0°e) under the following conditions: One plant was set in one 15 cm diameter pot, which the mixture of plant nutrient soil and vermiculite was 3:1 by volume in each pot, 16-hour light/8-hour dark cycle, 25 ± 1 °C, 50 ± 5% relative humidity. Sampling was performed when plants had seven pairs of leaves and salt stress was simulated using 150 mmol/L NaCl [30]. Three plants were treated in each treatment as three biological replicates. The NaCl concentration was selected in preliminary experimental concentration screening. The control plants (CK) received 100 mL of water, while the same amount of salt solution (150 mmol/L NaCl) was added as the salt stress. The salt solution was used for watering one time; 0 h and 72 h after stress were set as sampling times, while part of the penultimate leaf was used for samples, whose fresh weight was 0.5 g.

Determination of phenotypic and physiological factors
Plant phenotypes were measured and analysed before and after different treatments using Planteye (F500, Phenospex, Netherlands) [31,32]. The reflectance under four wavelength channels, including red light (620-645 nm), green light (530-540 nm), blue light (460-485 nm) and near infra-red wave (820-850 nm), was measured and used to calculate the corresponding parameters to directly obtain relevant plant phenotype information.
The penultimate set of leaves, at a temperature of 25 °C and relative humidity of approximately 25%, were subjected to measurement for the rate of net photosynthesis, stomatal conductance, intercellular CO 2 concentration and transpiration rate using the Li-6400 portable photosynthesis assay system (Li-6400, LICOR, uSA), while the photosystem II Light energy Conversion efficiency (Fv/Fm), photochemical quenching coefficient (q p ), photosystem II actual photochemical efficiency (ΦPSII) and non-photochemical quenching coefficient (NPQ) were measured by a portable chlorophyll fluorometer (FMS-2, Hansatech, england). The samples were quickly frozen in liquid nitrogen, and then transferred to a −80 °C ultra-low temperature freezer for the determination of indicators. The physiological indicators of plants under different treatments, such as the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) were assessed using the methods described by Zhang et al. [33], in which the change in optical density (OD) per min was set as a unit (u) recorded as u g −1 . The proline (Pro) content was measured using an enzyme-linked immunosorbent assay (eLISA) kit (M0108A, Mengxi, Jiangsu, China). The malondialdehyde (MDA) content was measured using an eLISA kit (M0106A, Mengxi, Jiangsu, China) following the manufacturer's instructions. The hydrogen peroxide (H 2 O 2 ) content was measured using an eLISA kit (M0107A, Mengxi, Jiangsu, China), whereas the superoxide anion (O 2 -) was measured using an eLISA kit (M0114A, Mengxi, Jiangsu, China) following the manufacturer's instructions. The fructose content was measured using an eLISA kit (gT-1-Y, Keming, Jiangsu, China), whereas the glucose content was measured using eLISA kit (PT-1-Y, Keming, Jiangsu, China) followed the manufacturer's instructions. The sucrose content was measured using an enzyme-linked immunosorbent assay (eLISA) kit (ZHT-1-Y, Keming, Jiangsu, China). The starch content was measured using an eLISA kit (DF-1-Y, Keming, Jiangsu, China) according to the manufacturer's instructions. The activities of enzymes in sucrose metabolism were also determined using eLISA kits. The activity of SuSy was measured using eLISA kit (SSII-1-Y, Keming, Jiangsu, China); the activity of SPS, using eLISA kit (SPS-1-Y, Keming, Jiangsu, China) following the manufacturer's instructions; the activity of α-amylase, using eLISA kit (DFMA-1-Y, Keming, Jiangsu, China) and the activity of β-Amylase, using eLISA kit (DFMB-1-Y, Keming, Jiangsu, China) following the manufacturer's instructions.

RNA extraction and qRT-PCR analysis
SteadyPure Plant RNA extraction Kit (Ag21019, Accurate Biology, Hunan, China) was used to extract RNA from the samples, and the RNA was reverse-transcribed into cDNA using the Evo M-MLV RT Premix for qPCR (Ag11706, Accurate Biology, Hunan, China). The genes involved in sucrose metabolism were selected from the Kyoto encyclopedia of genes and genomes (Kegg) database (https://www.kegg.jp/) using Rcactin as the reference gene [29], which was the basis for Pre-Lab research. The primers of these genes were designed using Primer Premier 5 software [34], (Supplemental Table S1), and qRT-PCR was carried out using Roche Light Cycler (480II, Roche, Switzerland) and SYBR® green Premix Pro Taq HS qPCR Kit (Ag11701, Accurate Biology, Hunan, China). The reaction solution was held at 95 °C for 30 s, then amplification was performed for 40 cycles with the following cycle profile: 5 s denaturation step at 95 °C, 5 s annealing step at 60 °C. The experiments were performed in three biological replicates. The expression of these genes was visualized by Tbtools [35].

Data analysis
The data analysis was performed using Office 2010 and SPSS 19.0 [36]. Significant differences were determined using analysis of variance (ANOVA). The level of statistical significance was set at p < 0.05. The data are shown as the mean values with standard deviation (±SD); each point in each picture represented the data of each measurement in different treatments; Lower case letter(s) above the bars indicate significant differences (α = 0.05, LSD) among the different treatments.

Phenotypic changes under salt stress
Analysis of the phenotype indicated that exposure to salt stress for 72 h delayed digital biomass, greenness average and leaf area when compared to plants in CK treatments ( Figure 1). Furthermore, during salt stress, the leaf angle increased significantly (p < 0.05) while the light penetration depth decreased considerably. These results indicated that the plant responded to salt stress by changing phenotypes (Table 1).

Changes in adversity physiology under salt stress
When compared to plants in CK treatments, the osmotic regulation of plant tissues changed after 72 h of salt stress, the Pro level increased, and the concentration of MDA, H 2 O 2 and O 2 increased significantly (p < 0.05) (Figure 2A-D). Furthermore, exposure to salt stress increased the activities of antioxidant enzymes, including SOD, POD, CAT and APX significantly (p < 0.05) (Figure 2e-H). These findings indicated that the plants responded to salt stress.

Changes in photosynthetic physiology under salt stress
In the two treatments of photosynthesis indicators, the rate of net photosynthesis ( Figure 3A) and the stomatal conductance ( Figure 3B) decreased after exposure to salt stress for 72 h compared with control plants (CK). The intercellular CO 2 concentration and transpiration rate also decreased significantly (p < 0.05) under salt stress ( Figure 3C and D). Furthermore, the treatments of fluorescence indicators (the Fv/Fm, q p and ΦPSII) decreased significantly (p < 0.05), whereas the nonphotochemical chlorophyll fluorescence quenching (NPQ) increased remarkably (Figure 3e-H). These findings demonstrated that salt stress induced changes in photosynthesis and fluorescence indicators in plants.

Changes in sucrose metabolism under salt stress
When compared to the control plants (CK), those exposed to salt stress for 72 h had higher concentration of fructose; glucose and sucrose increased significantly after 72 h of salt stress (p < 0.05) in the sucrose metabolism pathway (Figure 4A-C). However, the starch content in plants exposed to salt stress for 72 h decreased considerably when compared to the other treatments (p < 0.05) ( Figure 4D). According to these results, the polysaccharide content in plants decreased, whereas monosaccharides (fructose and glucose) and disaccharides (sucrose) increased.
The data on enzyme activities in the sucrose metabolism pathway also revealed that salt stress significantly boosted the activities of sucrose synthase, sucrose phosphate synthase, α-amylase and β-amylase enzymes after 72 h (p < 0.05) ( Figure 5A-D). These results were in line with changes in the indicators of the sucrose metabolism pathway.

The expression level in sucrose metabolism genes under salt stress
The expression level of genes involved in sucrose metabolism was tested by qRT-PCR analysis. The result showed the expression of genes associated with the sucrose synthase enzyme, including Rc-SS1 and Rc-SS2; sucrose phosphate synthase enzyme, including Rc-SPS1 and Rc-SPS2; and α-amylase enzyme, including Rc-αA1 and Rc-αA2, increased significantly under stress. except for Rc-βA2, which had a significant reverse change, the expression of most genes in the β-amylase enzyme increased significantly ( Figure 6).

Phenotypic changes under drought stress
Several studies have shown that salt stress negatively affects plant growth and development [37][38][39]. In rose  [30], multi-leaf lettuce [40] and Nicotiana benthamiana [41], exposure to 150 mmol/L NaCl for 72 h has been used to simulate salt stress [42][43][44]. The phenotype, an important plant characteristic, is affected by salt stress, so HTP systems, which rely on frequent, non-invasive, automated imaging, can be used to investigate the photosynthetic capacity and performance of plants under salt stress [45,46]. Specific indices are used as indicators of crop stress [47][48][49]. In the present study, salt stress delayed the growth of digital biomass, greenness average and leaf area while increasing the leaf angle compared to the CK treatment. These phenotypic changes indicated that the Chinese rose responded to salt stress and activated defense mechanisms.

Physiology under drought stress
When exposed to salt stress, plants have a systemic response that includes stress sensing and signalling, osmotic effects, ion homeostasis and metabolism [48]. The concentration of Pro increases under salt stress, which affects ROS scavengers and minimizes ROS damage to promote plant adaptation [50,51]. Similarly, the concentration of MDA, which is an important indicator of the damage caused by salt stress [52], increases significantly under salt stress [53]. The level of reactive oxygen species (ROS), such as H 2 O 2 and O 2 − , also increases under salt stress [52], and can be used as evaluation indices of plants under stress [54]. Similarly, the activities of antioxidant enzymes, such as POD, SOD, APX and CAT, are influenced by salt stress [29] or are changed by salt stress [55]. In this study, the concentration of Pro, MDA, H 2 O 2 and O 2 increased significantly under salt stress, which was congruent with previous findings in rice (Oryza sativa) [56,57] and barley (Hordeum vulgare) [58]. Furthermore, the activities of antioxidant enzymes, including SOD, POD, CAT and APX increased significantly under salt stress (Figure 2e and F), which was similar to the reported values in cotton, tomato and pepper [53,59,60]. These physiological indicators (including osmotic stress indicators and ROS enzymes activities) revealed that the plants responded to salt stress by changing physiological processes.

Photosynthesis and sucrose metabolism under salt stress
Studies have shown that salt stress damages the plant photosystem. Ionic effects increase gradually as Na + accumulates, resulting in a decline in photosynthesis [48]. under salt stress, the rate of net photosynthesis, stomatal conductance, intercellular CO 2 concentration and transpiration decreased, which was similar to the findings in black locust (Robinia pseudoacacia) and  wheat (Triticum aestivum) [61]. Salt stress affects fluorescence indicators such as Fv/Fm, q p , ΦPSII and NPQ [62]. In this study, exposure to salt stress decreased Fv/Fm, q p and ΦPSII levels but increased the NPQ levels. These photosynthesis and fluorescence indicators indicated that the plants sensed and responded to salt stress.
According to previous studies, plant response to salt stress involves sucrose metabolism [63]. For example, sucrose metabolism is critical in resisting salt stress in rice (Oryza sativa) [64,65], sugar beet (Beta vulgaris) [63], cotton (Gossypium hirsutum) [66] and Chenopodium quinoa [67]. Sugars, such as sucrose, glucose and fructose, are involved in osmotic protection, and ROS-scavenging abilities accumulate under salt stress [68,69], and sugar concentrations increase in response to neutral salt stress [70,71]. Previously, it was reported that the concentration of glucose, fructose and sucrose increases under salt stress, whereas the content of starch decreases, which is similar to the findings in the present study. The activity of sucrose synthase (SS) and sucrose phosphate synthase (SPS) could be  affected by salt stress, and then, in turn, influence the developmental regulators of sucrose metabolic processes [72]. For example, the expression of both SPS and SS genes, which related to the SPS and SS enzymes, was significantly induced in perennial ryegrass [73], which was corroborated by the findings obtained in this study. Moreover, stimulated amylase activity, including α-amylase and β-amylase activity, plays an osmoprotectant role in overcoming salt stress. Here, the activities of amylase, both α-amylase and β-amylase, changed in response to salt stress, which was also observed in barley (Hordeum vulgare) and Vigna sinensis [74,75]. In this study, the sugar biochemical reactions were stimulated by salt stress, which improved the salt tolerance. Drought affects the expression levels of key metabolic genes and the activity of sugar-metabolizing enzymes. In this study, drought induced changes in sugar levels in plants (Figure 7). These indicators (including the physiological indicators of sugar metabolism and the expression of sugar metabolism genes) indicate that sucrose metabolism is likely a regulation mode in Chinese rose under salt stress.

Conclusions
Salt stress induced changes in the photosynthesis efficiency, ion homeostasis and physiological indicators in Chinese rose. Several phenotypic indicators, including digital biomass, greenness average, leaf angle and leaf area, were altered in response to salt stress. This finding was corroborated by changes in physiological indicators such as Pro, MDA, H 2 O 2 , O 2 -, SOD, POD, CAT and APX activities, as well as photosynthetic indicators including rate of net photosynthetic rate, stomatal conductance, intercellular CO 2 concentration, transpiration rate and fluorescence indices (Fv/Fm, q p , ΦPSII and NPQ). Salt stress was associated with upregulated expression of Rc-SS1, Rc-SS2, Rc-SPS1, Rc-SPS2, Rc-αA1, Rc-αA2, Rc-αA3, Rc-βA1 and Rc-βA3, promoted fructose, glucose and sucrose accumulation in leaves, and decreased starch content in leaves. In summary, these results indicate that salt stress disrupts sucrose metabolism in leaves.

Data availability statement
The data that support the findings reported in this study were available from the corresponding author upon reasonable request.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The study was supported by Science and Technology Development Plan Project of Jilin Province (20210506005ZP).