Growth and quality identification
Two cherry tomato (Lycopersicon esculentum Mill.) lines, including a sucrose-accumulating (S) type and hexose-accumulating (H) type were selected for this study, and these fruits were divided into 5 stages (Ⅰ, Immature; Ⅱ, Mature; Ⅲ, Green Breaker; Ⅳ, Pink; Ⅴ, Red Ripe) according to the tomato growth period standards developed by the United States Department of Agriculture (USDA) and the growth curves of fruits of the two cherry tomato genotypes (Fig. S1).
Comparing the growth of H and S cherry tomatoes, we found that there were no visible differences in general plant morphology between two kinds of cherry tomatoes with respect to plant height and the number of leaves and fruits in the whole growth cycle, while differences were noted in the number of flowers (Fig. S2).
Assays involved in testing the quality and yield of the two kinds of cherry tomatoes indicated that the S cherry tomato had the higher total soluble sugar, Vitamin C and lycopene and lower organic acid at the fruit Red Ripe (V) stage than the H cherry tomato. There were no significant differences in fruit firmness or yield. It was easy to conclude that the S cherry tomato had better quality than the H type (Fig. 1).
Carbohydrates accumulation analysis
Sucrose, fructose, glucose and starch accumulation were analyzed in the developing fruits of sucrose-accumulating and hexose-accumulating cherry tomato lines. Regardless of any stage and tissue of the fruit, the S cherry tomato accumulated more sucrose than the H type, while the accumulation of glucose, fructose and starch in the H cherry tomato was significantly higher than that in the S type (Fig.2). Sucrose accumulation in whole fruit and different fruit tissues (exocarp, mesocarp, endocarp, placenta and septum) of S tomato showed a similar trend; the sucrose content reached a high point at Mature stage (Stage Ⅱ) first, then declined and reached its highest point at Red Ripe stage (Stage Ⅴ). The sucrose content in the S type fruit was dozens of times that in the H type fruit and there was little difference in sucrose content between different fruit tissues. The accumulation of glucose and fructose in the H type tomato fruit presented a similar trend as sucrose in the S cherry tomato and was high mainly in the later stages. It is worth mentioning that hexose also occupied a certain proportion in the S type cherry tomato, especially in the endocarp. In general, the starch content declined in early stages and slightly increased at the later stage and the H cherry tomato accumulated more starch than the S type in all tissues and stages.
Cytological path of phloem unloading of two cherry tomato fruits
The accumulation of sucrose or hexose in the fruit depends on the unloading of sugar from the phloem. To investigate the cytological bases of the accumulation of sugar during fruit development and ripening, we examined the phloem unloading pathway and the connection between the phloem and surrounding cells.
The CF tracer was used to detect the assimilate unloading pathway between the phloem and the surrounding parenchyma cells in the S cherry tomato. The results indicated that the pathway of fruit phloem unloading was altered during tomato development. CF was strictly restricted in the phloem at the early stages (Stage Ⅰ to Stage Ⅱ), which indicates an apoplastic pathway of fruit phloem unloading, while CF was distributed to surrounding parenchyma cells at the later stages (Stage Ⅲ to Stage Ⅴ), which means that the phloem unloading pathway was a symplastic pathway (Fig.3).
Observation of the plasmodesmata between the phloem and surrounding cells by transmission electron microscopy (TEM), revealed that in the early stages of the S cherry tomato fruit (Stage Ⅰ to Stage Ⅲ), there were only a few plasmodesmata between the sieve element/companion cell complex (SE/CCs) and parenchyma cells (PCs), and these plasmodesmata were blocked. However, there were many plasmodesmata at the later stages (Stage Ⅳ to Stage Ⅴ), implying that the phloem unloading pathway of the S cherry tomato changed from symplastic to apoplastic during fruit development, which was consistent with the results of the CFDA tracer (Fig.4 and Table S2).
Otherwise, the phloem unloading pathway of the H cherry tomato changed from apoplastic to symplastic during fruit development, because there were many plasmodesmata in the early stages and few plasmodesmata in the later stages between SE/CCs and PCs.
Expression analysis of sugar transporters and related metabolic enzymes
To further analyze the sugar accumulation mechanism in tomato fruits, the expression of three tomato sucrose transporters and the activities of several sugar metabolism enzymes were studied.
The mRNA expression levels of sucrose transporter (SUT) genes in the two cherry tomatoes had no obvious patterns (Fig.5). However, the expression of LeSUT1 was higher in the H tomato than in the S tomato, which implies that there may be more sucrose unloading from the phloem. The activities of sugar-related metabolic enzymes indicated that the H cherry tomato had higher activities of acid invertase (AI) than the S type at almost all fruit stages and tissues, and reached to the highest level at the Pink stage (Stage IV), which means that a large amount of sucrose hydrolysis may occur during this stage (Fig.6A).
The activity of sucrose phosphate synthase (SPS) was higher in the S cherry tomato than in the H type (Fig.6B), indicating that more sucrose was synthesized. However, sucrose synthase (SS), a reversible enzyme that broke down and synthesized sucrose, had higher levels in the early stages of fruits, implying that SS played an important role in the hydrolysis of sucrose in the early stages (Fig.6C). The S type tomato had higher SS activities than the H type tomato, but a similar trend in fruit tissues as the H type tomato. In contrast to AI, SS had the lowest activity in Stage IV. It is possible that the hydrolysis of sucrose during this stage mainly depends on AI.
These results indicated that LeSUT1 and AI played more important roles in the H cherry tomato, responsible for unloading sucrose from the phloem and breaking down sucrose into glucose and fructose. SPS and SS may play more roles in the S tomato, responsible for maintaining the homeostasis of sucrose by hydrolysis and synthesis. It is likely that sucrose accumulation in the S tomato mainly relied on symplastic transport at later stages.
To further analyze the mechanism of sugar accumulation in the S and H cherry tomatoes, the iTRAQ method for proteome sequencing of ripe fruits of two types of tomato was performed (Fig. S3). Mass spectrometry to identify differential proteins indicated that total 420 differentially expressed proteins were identified between S and H tomatoes, and 235 proteins were upregulated, while 185 proteins were downregulated (Fig.7A).
The COG analysis indicated that these differential proteins were mainly related to metabolic pathways, biosynthesis of secondary metabolism, biosynthesis of amino acids, carbon metabolism, starch and sucrose metabolism, sugar metabolism, photosynthesis, and so on (Fig.7B). Further KEGG analysis indicated that in sucrose-accumulating tomatoes, most differential proteins involved in starch and sucrose metabolism and photosynthesis were downregulated, while most differential proteins involved in fatty acid degradation were significantly upregulated (Fig. S4). These results showed that in addition to sugar metabolism, other metabolic pathways also changed accordingly with the different sugar accumulations between the two types of tomatoes. Differential proteins involved in carbon metabolism and photosynthesis were shown in Fig.7C and D in detail. It is worth noting that acid invertase was significantly downregulated in the sucrose-accumulating tomato, which was consistent with the results of the enzyme activity assay.