Sorbitol in loquat promotes early flower bud differentiation via the MADS-box transcription factor EjCAL

The sugar alcohol sorbitol plays an important signaling role in fruit trees. Here, we found that sorbitol significantly increased during flower bud differentiation (FBD) in loquat (Eriobotrya japonica Lindl.) from the physiological FBD stage (EjS1) to the morphological FBD stage (EjS2), and it then decreased in the panicle development stage (EjS3) compared to in EjS2, and in subsequent stages. Spraying sorbitol increased the sorbitol content and thereby promoted early FBD and increased the proportion of flower buds that completed FBD. A transcriptomics analysis showed that the expression of a MADS-box transcription factor (TF) family gene, EjCAL, was highly correlated with the FBD phenotypic data. EjCAL-overexpressing transgenic tobacco exhibited the early FBD phenotype. Using the EjCAL promoter as bait in a yeast-one hybrid (Y1H) assay, the TF ERF12 was identified. Chromatin immunoprecipitation (ChIP)-PCR confirmed that EjERF12 can bind to the EjCAL promoter, and {beta}-glucuronidase (GUS) activity assays demonstrated that EjERF12 can regulate EjCAL expression. Spraying loquat with sorbitol confirmed that EjERF12 and EjCAL expression were regulated by sorbitol. We also identified downstream functional genes (EjUF3GaT1, EjGEF2, and EjADF1) that might be involved in FBD. Finally, we found that the change in the level of hyperoside (a reproduction-related flavonoid) was consistent with that of sorbitol during FBD in loquat, and EjCAL can bind to the EjUF3GaT1 promoter and might thereby regulate hyperoside biosynthesis. Two early- and late-flowering varieties of loquat and EjCAL-overexpressing transgenic tobacco plants were used to confirm this hypothesis. One-sentence summarySorbitol promotes bud differentiation via EjCAL.