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

Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3' splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3' splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in Arabidopsis thaliana.

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.

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.

FLOWERING LOCUS T paralogs control the annual growth cycle in Populus trees.

In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.1 This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.1-4 The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.3,5 Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy.3,5 In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle.

The splicing factor 1-FLOWERING LOCUS M module spatially regulates temperature-dependent flowering by modulating FLOWERING LOCUS T and LEAFY expression.

KEY MESSAGE: The AtSF1-FLM module spatially controls temperature-dependent flowering by negatively regulating the expression of FT and LFY in the leaf and shoot apex, respectively. Alternative splicing mediated by various splicing factors is important for the regulation of plant growth and development. Our recent reports have shown that a temperature-dependent interaction between Arabidopsis thaliana splicing factor 1 (AtSF1) and FLOWERING LOCUS M (FLM) pre-mRNA introns controls the differential production of FLM-ß transcripts at different temperatures, eventually resulting in temperature-responsive flowering. However, the molecular and genetic interactions between the AtSF1-FLM module and floral activator genes remain unknown. Here, we aimed to identify the interactions among AtSF1, FLM, FLOWERING LOCUS T (FT), and LEAFY (LFY) by performing molecular and genetic analyses. FT and TWIN SISTER OF FT (TSF) expression in atsf1-2 mutants significantly increased in the morning and middle of the night at 16 and 23 °C, respectively, under long-day conditions. In addition, ft mutation suppressed the early flowering of atsf1-2 and atsf1-2 flm-3 mutants and masked the temperature response of atsf1-2 flm-3 mutants, suggesting that FT is a downstream target gene of the AtSF1-FLM module. LFY expression significantly increased in the diurnal samples of atsf1-2 mutants and in the shoot apex regions of atsf1-2 ft-10 mutants at different temperatures. The chromatin immunoprecipitation (ChIP) assay revealed that FLM directly binds to the genomic regions of LFY but not of APETALA1 (AP1). Moreover, lfy mutation suppressed the early flowering of flm-3 mutants, suggesting that LFY is another target of the AtSF1-FLM module. Our results reveal that the AtSF1-FLM module spatially modulates temperature-dependent flowering by regulating FT and LFY expressions.

Populus SVL Acts in Leaves to Modulate the Timing of Growth Cessation and Bud Set.

SHORT VEGETATIVE PHASE (SVP) is an important regulator of FLOWERING LOCUS T (FT) in the thermosensory pathway of Arabidopsis. It is a negative regulator of flowering and represses FT transcription. In poplar trees, FT2 is central for the photoperiodic control of growth cessation, which also requires the decrease of bioactive gibberellins (GAs). In angiosperm trees, genes similar to SVP, sometimes named DORMANCY-ASSOCIATED MADS-BOX genes, control temperature-mediated bud dormancy. Here we show that SVL, an SVP ortholog in aspen trees, besides its role in controlling dormancy through its expression in buds, is also contributing to the regulation of short day induced growth cessation and bud set through its expression in leaves. SVL is upregulated during short days in leaves and binds to the FT2 promoter to repress its transcription. It furthermore decreases the amount of active GAs, whose downregulation is essential for growth cessation, by repressing the transcription of GA20 oxidase. Finally, the SVL protein is more stable in colder temperatures, thus integrating the temperature signal into the response. We conclude that the molecular function of SVL in the photoperiodic pathway has been conserved between Arabidopsis and poplar trees, albeit the physiological process it controls has changed. SVL is thus both involved in regulating the photoperiod response in leaves, modulating the timing of growth cessation and bud set, and in the subsequent temperature regulation of dormancy in the buds.

Role of Arabidopsis Splicing factor SF1 in Temperature-Responsive Alternative Splicing of FLM pre-mRNA.

Small changes in temperature affect plant ecological and physiological factors that impact agricultural production. Hence, understanding how temperature affects flowering is crucial for decreasing the effects of climate change on crop yields. Recent reports have shown that FLM-ß, the major spliced isoform of FLOWERING LOCUS M (FLM)-a flowering time gene, contributes to temperature-responsive flowering in Arabidopsis thaliana. However, the molecular mechanism linking pre-mRNA processing and temperature-responsive flowering is not well understood. Genetic and molecular analyses identified the role of an Arabidopsis splicing factor SF1 homolog, AtSF1, in regulating temperature-responsive flowering. The loss-of-function AtSF1 mutant shows temperature insensitivity at different temperatures and very low levels of FLM-ß transcript, but a significantly increased transcript level of the alternative splicing (AS) isoform, FLM-δ. An RNA immunoprecipitation (RIP) assay revealed that AtSF1 is responsible for ambient temperature-dependent AS of FLM pre-mRNA, resulting in the temperature-dependent production of functional FLM-ß transcripts. Moreover, alterations in other splicing factors such as ABA HYPERSENSITIVE1/CBP80 (ABH1/CBP80) and STABILIZED1 (STA1) did not impact the FLM-ß/FLM-δ ratio at different temperatures. Taken together, our data suggest that a temperature-dependent interaction between AtSF1 and FLM pre-mRNA controls flowering time in response to temperature fluctuations.

The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms.

KEY MESSAGE: The Arabidopsis splicing factors, AtU2AF65, AtU2AF35, and AtSF1 shuttle between nuclei and cytoplasms. These proteins also move rapidly and continuously in the nuclei, and their movements are affected by ATP depletion. The U2AF65 proteins are splicing factors that interact with SF1 and U2AF35 proteins to promote U2snRNP for the recognition of the pre-mRNA 3' splice site during early spliceosome assembly. We have determined the subcellular localization and movement of these proteins' Arabidopsis homologs. It was found that Arabidopsis U2AF65 homologs, AtU2AF65a, and AtU2AF65b proteins interact with AtU2AF35a and AtU2AF35b, which are Arabidopsis U2AF35 homologs. We have examined the mobility of these proteins including AtSF1 using fluorescence recovery after photobleaching and fluorescence loss in photobleaching analyses. These proteins displayed dynamic movements in nuclei and their movements were affected by ATP depletion. We have also demonstrated that these proteins shuttle between nuclei and cytoplasms, suggesting that they may also function in cytoplasm. These results indicate that such splicing factors show very similar characteristics to their human counterparts, suggesting evolutionary conservation.

RRM domain of Arabidopsis splicing factor SF1 is important for pre-mRNA splicing of a specific set of genes.

KEY MESSAGE: The RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing of FLOWERING LOCUS M pre-mRNA and a heat shock transcription factor HsfA2 pre-mRNA. Splicing factor 1 (SF1) plays a crucial role in 3' splice site recognition by binding directly to the intron branch point. Although plant SF1 proteins possess an RNA recognition motif (RRM) domain that is absent in its fungal and metazoan counterparts, the role of the RRM domain in SF1 function has not been characterized. Here, we show that the RRM domain differentially affects the full function of the Arabidopsis thaliana AtSF1 protein under different experimental conditions. For example, the deletion of RRM domain influences AtSF1-mediated control of flowering time, but not the abscisic acid sensitivity response during seed germination. The alternative splicing of FLOWERING LOCUS M (FLM) pre-mRNA is involved in flowering time control. We found that the RRM domain of AtSF1 protein alters the production of alternatively spliced FLM-ß transcripts. We also found that the RRM domain affects the alternative splicing of a heat shock transcription factor HsfA2 pre-mRNA, thereby mediating the heat stress response. Taken together, our results suggest the importance of RRM domain for AtSF1-mediated alternative splicing of a subset of genes involved in the regulation of flowering and adaptation to heat stress.

Functional conservation of rice OsNF-YB/YC and Arabidopsis AtNF-YB/YC proteins in the regulation of flowering time.

KEY MESSAGE: Rice Os NF - YB and Os NF - YC complement the late flowering phenotype of Arabidopsis nf - yb double and nf - yc triple mutants, respectively. In addition, OsNF-YB and OsNF-YC interact with AtNF-YC and AtNF-YB, respectively. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors play important roles in plant development and abiotic stress. In Arabidopsis thaliana, two NF-YB (AtNF-YB2 and AtNF-YB3) and five NF-YC (AtNF-YC1, AtNF-YC2, AtNF-YC3, AtNF-YC4, and AtNF-YC9) genes regulate photoperiodic flowering by interacting with other AtNF-Y subunit proteins. Three rice NF-YB (OsNF-YB8, OsNF-YB10, and OsNF-YB11) and five rice OsNF-YC (OsNF-YC1, OsNF-YC2, OsNF-YC4, OsNF-YC6, and OsNF-YC7) genes are clustered with two AtNF-YB and five AtNF-YC genes, respectively. To investigate the functional conservation of these NF-YB and NF-YC genes in rice and Arabidopsis, we analyzed the flowering phenotypes of transgenic plants overexpressing the respective OsNF-YB and OsNF-YC genes in Arabidopsis mutants. Overexpression of OsNF-YB8/10/11 and OsNF-YC2 complemented the late flowering phenotype of Arabidopsis nf-yb2 nf-yb3 and nf-yc3 nf-yc4 nf-yc9 mutants, respectively. The rescued phenotype of 35S::OsNF-YC2 nf-yc3 nf-yc4 nf-yc9 plants was attributed to the upregulation of FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). In vitro and in planta protein-protein analyses revealed that OsNF-YB8/10/11 and OsNF-YC1/2/4/6/7 interact with AtNF-YC3/4/9 and AtNF-YB2/3, respectively. Our data indicate that some OsNF-YB and OsNF-YC genes are functional equivalents of AtNF-YB2/3 and AtNF-YC3/4/9 genes, respectively, and suggest functional conservation of Arabidopsis and rice NF-Y genes in the control of flowering time.

OsNF-YC2 and OsNF-YC4 proteins inhibit flowering under long-day conditions in rice.

MAIN CONCLUSION: OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes ( Ehd1, Hd3a , and RFT1 ) in rice. Plant NUCLEAR FACTOR Y (NF-Y) transcription factors control numerous developmental processes by forming heterotrimeric complexes, but little is known about their roles in flowering in rice. In this study, it is shown that some subunits of OsNF-YB and OsNF-YC interact with each other, and among them, OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response of rice. Protein interaction studies showed that the physical interactions occurred between the three OsNF-YC proteins (OsNF-YC2, OsNF-YC4 and OsNF-YC6) and three OsNF-YB proteins (OsNF-YB8, OsNF-YB10 and OsNF-YB11). Repression and overexpression of the OsNF-YC2 and OsNF-YC4 genes revealed that they act as inhibitors of flowering only under long-day (LD) conditions. Overexpression of OsNF-YC6, however, promoted flowering only under LD conditions, suggesting it could function as a flowering promoter. These phenotypes correlated with the changes in the expression of three rice flowering-time genes [Early heading date 1 (Ehd1), Heading date 3a (Hd3a) and RICE FLOWERING LOCUS T1 (RFT1)]. The diurnal and tissue-specific expression patterns of the subsets of OsNF-YB and OsNF-YC genes were similar to those of CCT domain encoding genes such as OsCO3, Heading date 1 (Hd1) and Ghd7. We propose that OsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response by interacting directly with OsNF-YB8, OsNF-YB10 or OsNF-YB11 proteins in rice.

A homolog of splicing factor SF1 is essential for development and is involved in the alternative splicing of pre-mRNA in Arabidopsis thaliana.

During initial spliceosome assembly, SF1 binds to intron branch points and interacts with U2 snRNP auxiliary factor 65 (U2AF65). Here, we present evidence indicating that AtSF1, the Arabidopsis SF1 homolog, interacts with AtU2AF65a and AtU2AF65b, the Arabidopsis U2AF65 homologs. A mutant allele of AtSF1 (At5g51300) that contains a T-DNA insertion conferred pleiotropic developmental defects, including early flowering and abnormal sensitivity to abscisic acid. An AtSF1 promoter-driven GUS reporter assay showed that AtSF1 promoter activity was temporally and spatially altered, and that full AtSF1 promoter activity required a significant proportion of the coding region. DNA chip analyses showed that only a small proportion of the transcriptome was altered by more than twofold in either direction in the AtSF1 mutant. Expression of the mRNAs of many heat shock proteins was more than fourfold higher in the mutant strain; these mRNAs were among those whose expression was increased most in the mutant strain. An RT-PCR assay revealed an altered alternative splicing pattern for heat shock transcription factor HsfA2 (At2g26150) in the mutant; this altered splicing is probably responsible for the increased expression of the target genes induced by HsfA2. Altered alternative splicing patterns were also detected for the transcripts of other genes in the mutant strain. These results suggest that AtSF1 has functional similarities to its yeast and metazoan counterparts.