Insights into Loquat Flowering Regulation Through Analysis of Alternative Splicing of Flowering-Time Genes and Functions of EjCO1 Isoforms

Loquat (Eriobotrya japonica), an important subtropical fruit crop, blooms in autumn/winter, which is distinctive compared with other fruit trees such as apple, pear, and peach in Rosaceae. Currently, alternative splicing (AS) of flowering time genes remains understudied in loquat. In this study, full-length transcriptome sequencing of mixed tissues composed of leaves and shoot apical meristems/flower buds was performed and analyzed. A total of 94,194 high-quality isoforms and 44,186 complete open reading frames (ORFs) were obtained out of the 41.79 Gb of subread data. Further analysis revealed 25,988 AS events among 7461 genes, of which the most abundant type was intron retention (IR) occupying 55.32%. Importantly, 197 loquat genes homologous to Arabidopsis or Rosaceae flowering time genes were found to be alternatively spliced, including an important player CONSTANS (EjCO1) that contained three different isoforms (EjCO1-1, EjCO1-2, and EjCO1-3). To investigate the effect of AS on gene function, we overexpressed the three EjCO1 isoforms in Arabidopsis. The results showed that overexpression of EjCO1-1 and EjCO1-3 significantly promoted early flowering of transgenic Arabidopsis plants, whereas overexpressing EjCO1-2 did not significantly change the flowering time. Dual-luciferase reporter assays showed that EjCO1-1 and EjCO1-3 could significantly activate the expression of FLOWERING LOCUS T (EjFT2), while EjCO1-2 had no significant effect on the promoter activity of EjFT2. The results from this study systematically cataloged AS events of flowering time genes and illustrated the important effect of AS on gene functions, which provides insights into the molecular regulation of flowering time by AS in loquat.