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Background  The asynchronous filling between superior spikelets (SS) and inferior spikelets (IS) in rice has become a research hotspot. The stagnant development and poor grain filling of IS limit yields and the formation of good quality rice. A large number of studies on this phenomenon have been carried out from the genome, transcriptome and proteome level, indicating that asynchronous filling of SS and IS filling is a complex, but orderly physiological and biochemical process involving changes of a large number of genes, protein expression and modification. However, the analysis of metabolomics differences between SS and IS is rarely reported currently.    Results  This study utilized untargeted metabolomics and identified 162 metabolites in rice spikelets. Among them, 17 differential metabolites associated with unsynchronized grain filling between SS and IS, 27 metabolites were related to the stagnant development of IS and 35 metabolites related to the lower maximum grain-filling rate of IS compared with the SS. We found that soluble sugars were an important metabolite during grain filling for SS and IS. Absolute quantification was used to further analyze the dynamic changes of 4 types of soluble sugars (sucrose, fructose, glucose, and trehalose) between SS and IS. The results showed that sucrose and trehalose were closely associated with the dynamic characteristics of grain filling between SS and IS. The application of exogenous sugar showed that trehalose functioned as a key sugar signal during grain filling of IS. Trehalose regulated the expression of genes related to sucrose conversion and starch synthesis, thereby promoting the conversion of sucrose to starch. The difference in the spatiotemporal expression of  TPS-2  and  TPP-1  between SS and IS was an important reason that led to the asynchronous change in the trehalose content between SS and IS.    Conclusions  The results from this study are helpful for understanding the difference in grain filling between SS and IS at the metabolite level. In addition, the present results can also provide a theoretical basis for the next step of using metabolites to regulate the filling of IS.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12870-021-03219-8.

GC-MS-based metabolite profiling of key differential metabolites between superior and inferior spikelets of rice during the grain filling stage