Soluble Sugars are the Main Differential Metabolites between SS and IS
The rice cultivar 'Jinhui No. 809' exhibited significant differences in grain filling between SS and IS. In the present study, the untargeted metabolomics analysis revealed a marked metabolites difference between the SS and IS. Except for a few metabolites including sucrose, trehalose, and hydroxyproline, the majority of the differential metabolites in IS had a higher level at 10 DAF than at 25 DAF, that is, as grain filling progressed, the levels of most soluble sugars and acid substances decreased, indicating that grain filling is a process converting soluble sugars, such as fructose and glucose, to polysaccharides, as well as converting amino acids to proteins. Between SS (active grain filling stage) and IS (developmental stagnancy phase) at 10 DAF, the concentrations of most soluble sugars and amino acids were higher in IS than in SS, indicating that the conversion of sugars and amino acids between SS and IS was unsynchronized. Interesting, a total of 35 differential metabolites have been identified between 10 DAF SS and 25 DAF IS, which may be related to the lower maximum grain-filling rate of IS compared with the SS.
A previous study showed that the rice grain-filling process is mainly a process of starch accumulation [15] and that the starch in the grains is mostly converted from sucrose [16]. The present results showed that the concentrations of soluble sugars were significantly higher than those of other metabolites. This indicated that soluble sugars were the major metabolites in the grains, and most sugars were related to the conversion of sucrose. As the foundation of substance and energy metabolism, sugars themselves and their intermediate metabolites are structural components for energy and storage substances, as well as the intermediates for synthesizing other organic molecules [17]. Sugars can also act as signaling molecules and can interact with inorganic regulatory networks to promote or inhibit plant growth [18–20]. The total concentration of the four types of soluble sugars showed that at the early grain-filling stage, the concentration of soluble sugar in SS was higher than in IS. However, this pattern was reversed at the late stage of grain filling. During the early stage of fertilization, the concentrations of endogenous hormones such as ABA and IAA are at high levels in SS [21, 22]. During this time, the ability to allocate photosynthate was also high in SS; photoassimilates generated in the leaves were continuously transported into the grains. As a result, the concentrations of soluble sugars in SS were higher than in IS. As the grain-filling process progressed, the activity of enzymes involved in the conversion of sugar to starch gradually increased in SS, and the concentration of sugar in the grains continued to decrease. However, at the early grain-filling stage, most genes involved in the conversion of sucrose to starch exhibited lower expression levels in IS than in SS [7, 8, 23]. The activity of enzymes involved in the conversion of sugar to starch was also lower in IS [24, 25], with a low efficiency in converting soluble sugars to starch. Thus, the level of soluble sugars in IS was lower than that in SS. At the mid- and late stages of grain filling, the expression level of genes related to the conversion of sucrose to starch, as well as the activity of enzymes involved in this process, gradually increased in IS [7, 9]. As a result, the level of soluble sugars continuously decreased. Since grain filling in SS was almost completed at this time, the level of soluble sugars in IS at the mid- and late stages of grain filling was always higher than that in SS at the same stage. However, the higher sugar content in IS was not favorable for the transport of photosynthate to IS, leading to a low seed setting rate in IS. These results indicated that the difference in the soluble sugar concentrations was an important cause of the difference in grain filling between SS and IS.
The Difference in Trehalose Content was an Important Cause of the Difference in the Conversion Rate of Sucrose to Starch between SS and IS
A comparison of the changing patterns of the 4 types of soluble sugars between SS and IS during grain filling revealed that the levels of trehalose and sucrose were consistent with the pattern of the grain-filling rate, which peaked early in SS and late in IS during the grain filling stage, suggesting that changes in the trehalose and sucrose contents may play important roles in regulating the grain-filling rate of SS and IS. Previous studies have shown that applying an appropriate concentration of exogenous sucrose induced the expression of sucrose synthase genes and increased the activity of sucrose synthase, thereby increasing the 1000-grain weight [26, 27]. In this study, 100 mM of exogenous sucrose solution was applied to IS at 10 DAF. Results showed that the seed setting rate and 1000-grain weight increased, but not significantly. This is possibly because the exogenous sucrose concentration did not reach a concentration high enough to promote grain filling. Applying the same concentration of trehalose solution significantly increased the 1000-grain weight and seed setting rate of IS.
Trehalose and sucrose are both disaccharides; their metabolism is also similar. Trehalose, a non-reducing sugar composed of two glucose molecules, is present in many plants and participates in signal regulatory processes [28] [29]. In this study, we found that the timing when the trehalose level reached its peak in SS and IS was later than that of sucrose. The sucrose concentration in IS rapidly increased after 10 DAF, but the trehalose concentration remained at a low level. At this time, grain filling in IS had not been activated. At 15 DAF, the level of trehalose in IS began to slowly increase, followed by the activation of grain filling in IS. Starch contents gradually increased in the grains, and the trehalose level in IS reached its maximum at 25 DAF, consistent with the timing of the grain-filling rate reaching its maximum (25 DAF) in IS. In addition, the results from this study showed that the exogenous application of sucrose did not affect the trehalose content in IS. However, after the application of exogenous trehalose, the level of sucrose significantly decreased and the starch content significantly increased in the grains. This indicated that, compared to sucrose, trehalose may play a more important role in regulating grain filling and can promote the conversion of sucrose to starch.
Results from qRT-PCR showed that trehalose induced the expression of key genes involved in sucrose conversion and starch synthesis in IS. Among them, the expression of GBSSI, SUS4, and SSI increased significantly. The enzyme activities of SuSase, StSase, and AGPase also significantly increased. In Arabidopsis, exogenous trehalose has been found to induce the expression of APL3, a gene involved in starch synthesis, resulting in starch accumulation in the cotyledon [30]. These results showed that certain concentrations of trehalose in the grain-filling stage can activate the expression of genes related to starch synthesis and promote the conversion of sucrose to starch. The difference in trehalose content between SS and IS is likely an important reason leading to the differential expression of genes related to starch synthesis.
TPS-2 and TPP-1 are Key Genes that Cause the Difference in the Trehalose Content between SS and IS
Trehalose synthesis in plants is a two-step process: the production of trehalose-6-phosphate (T6P) from UDP-Glucose and Glucose-6-phosphate, catalyzed by TPS, and consecutive dephosphorylation of T6P to trehalose, catalyzed by TPP [28]. In this study, we found that the changes in TPS-2 and TPP-1 gene expression during grain filling in IS were similar to the pattern of changes in the trehalose content, that is, the expression levels of TPS-2 and TPP-1 were low at the at the early grain-filling stage and increased from the mid-grain-filling stage. Furthermore, the expression patterns of TPS-2 and TPP-1 also showed a similar pattern as the changes in the trehalose content between SS and IS, which were higher in SS at early grain filling stage and lower in late grain-filling stage. Therefore, the differential expression of TPS-2 and TPP-1 between SS and IS is predicted to be an important reason leading to the difference in the trehalose content between SS and IS. In addition, a TPS isoform was identified as a target protein of the 14-3-3 protein [31]. 14-3-3 proteins bind to phosphorylated motifs and function in multiple developmental processes by regulating the activity of a wide variety of target proteins [32]. In vivo phosphorylation of Ser22 and Thr49 of the TPS protein by AMPK and SnRK1s resulted in the binding of TPS to the 14-3-3 protein [33]. Interesting, our previous study determined that the 14-3-3 protein showed temporal and spatial differences in expression patterns between SS and IS; specifically, reducing the expression of 14-3-3 protein during grain filling effectively promoted starch synthesis in IS [34]. Based on these results, it was speculated that through the binding of phosphorylated TPS-2 or TPP1 protein, the 14-3-3 protein could regulate trehalose synthesis in SS and IS. However, the mechanism needs to be further investigated.