Two Arabidopsis Splicing Factors, U2AF65a and U2AF65b, Differentially Control Flowering Time by Modulating the Expression or Alternative Splicing of a Subset of FLC Upstream Regulators

Abstract

We investigated the transcriptomic changes in the shoot apices during floral transition in Arabidopsis mutants of two closely related splicing factors: AtU2AF65a (atu2af65a) and AtU2AF65b (atu2af65b). The atu2af65a mutants exhibited delayed flowering, while the atu2af65b mutants showed accelerated flowering. The underlying gene regulatory mechanism of these phenotypes was unclear. We performed RNA-seq analysis using shoot apices instead of whole seedlings and found that the atu2af65a mutants had more differentially expressed genes than the atu2af65b mutants when they were compared to wild type. The only flowering time gene that was significantly up- or down-regulated by more than two-fold in the mutants were FLOWERING LOCUS C (FLC), a major floral repressor. We also examined the expression and alternative splicing (AS) patterns of several FLC upstream regulators, such as COOLAIR, EDM2, FRIGIDA, and PP2A-b’ɤ, and found that those of COOLAIR, EDM2, and PP2A-b’ɤ were altered in the mutants. Furthermore, we demonstrated that AtU2AF65a and AtU2AF65b genes partially influenced FLC expression by analyzing these mutants in the flc-3 mutant background. Our findings indicate that AtU2AF65a and AtU2AF65b splicing factors modulate FLC expression by affecting the expression or AS patterns of a subset of FLC upstream regulators in the shoot apex, leading to different flowering phenotypes.

Regulation of Flowering Time and Other Developmental Plasticities by 3’ Splicing Factor-Mediated Alternative Splicing in Arabidopsis thaliana

“…U2AF65 and U2AF35 also exist as two homologs (AtU2AF65a and AtU2AF65b, and AtU2AF35a and AtU2AF35b, respectively) in Arabidopsis [27,32]. The recent evidence suggests that AtU2AF35a/b and AtU2AF65a/b play important roles in regulating flowering time by regulating the AS of key flowering genes (Figure 1, Table 1) [24,25,27]. The atu2af35a and atu2af35b mutants showed late-flowering phenotypes under both long-day (LD) and short-day (SD) conditions [27].…”

“…Several 3' splicing factors have been implicated in the regulation of flowering time and development in Arabidopsis thaliana (Table 1). It is well-known that the AS of key player genes including transcriptional control of MADS-box transcription factor, FLOWERING LOCUS C (FLC) [24,25], temperature-dependent AS of FLOWERING LOCUS M (FLM) [26], FLOWERING CONTROL LOCUS A (FCA) [27]; and the circadian clock genes, including PSEUDO-RESPONSE REGULATOR 7 (PRR7) and PRR9 [28] are involved in regulating flowering time control. The AS of these genes, modulated by splicing factors, comprising the large spliceosome assembly machinery, is crucial for proper pre-mRNA splicing.…”

“…Furthermore, mutations in AtU2AF35 altered the expression level of the flowering time gene FCA due to altered AS in FCA isoforms, suggesting that the increased abnormal FCA transcripts could not repress the FLC expression (Figure 1a). Loss-of-function mutants of the two isoforms of AtU2AF65 also reveal their functional roles in floral transition [24,25,33]. atu2af65a and atu2af65b mutants showed late-and early-flowering phenotypes, respectively, which correlated with altered expression levels of the flowering time genes, including FLC and FLOWERING LOCUS T (FT) in the leaves [24].…”

“…Loss-of-function mutants of the two isoforms of AtU2AF65 also reveal their functional roles in floral transition [24,25,33]. atu2af65a and atu2af65b mutants showed late-and early-flowering phenotypes, respectively, which correlated with altered expression levels of the flowering time genes, including FLC and FLOWERING LOCUS T (FT) in the leaves [24]. In addition, RNA-sequencing (RNA-seq) analysis in the shoot apex regions of wild-type (Col-0), atu2af65a, and atu2af65b plants revealed that the expression levels or AS patterns of COOLAIR long non-coding RNAs (lncRNAs), EDM2, or , the FLC upstream regulators, were changed in the shoot apices of atu2af65a mutants [24], suggesting that AtU2AF65a regulated the FLC expression through COOLAIR RNA-mediated FLC repression, and the reduced expression of EDM2 and in vernalization-mediated flowering (Figure 1b).…”

Advances in alternative splicing identification: deep learning and pantranscriptome

“…For example, AS of the NAC transcription factor 109 ( NACTF109 ) during maize embryo development regulates seed dormancy by controlling ABA content in seeds ( Thatcher et al., 2016 ). FLOWERING LOCUS C (FLC) is an important repressor of flowering in Arabidopsis ( Andersson et al., 2008 ; Sharma et al., 2020 ), and AtU2AF65b is a splicing factor involved in ABA-mediated regulation of flowering time in Arabidopsis by splicing FLC pre-mRNA ( Xiong et al., 2019 ; Lee et al., 2023 ). JASMONATE ZIM-DOMAIN (JAZ) is a key regulators of jasmonate (JA) signaling in plants ( Yan et al., 2009 ).…”