Yun Hee Jang

OsCO3, a CONSTANS-LIKE gene, controls flowering by negatively regulating the expression of FT-like genes under SD conditions in rice.

The photoperiod is an important environmental stress that determines flowering time. The CONSTANS (CO) and Heading date 1 (Hd1) genes are known to be central integrators of the photoperiod pathway in Arabidopsis and rice, respectively. Although they are both members of the CONSTANS-LIKE (COL) family and have two B-boxes and a CCT domain, rice also possesses novel COL genes that are not found in Arabidopsis. Here, we demonstrate that a novel COL gene, OsCO3, containing a single B-box and a CCT domain, modulates photoperiodic flowering in rice. The circadian expression pattern of OsCO3 mRNA oscillated in a different phase from Hd1 and was similar to that of OsCO3 pre-mRNA, suggesting that the diurnal expression pattern of OsCO3 transcripts may be regulated at the transcriptional level. Overexpression of OsCO3 specifically caused late flowering under short day (SD) conditions relative to wild-type rice plants. The expression of Hd3a and FTL decreased in these transgenic plants, whereas the expression of Hd1, Early heading date 1 (Ehd1), OsMADS51, and OsMADS50 did not significantly change. Our results suggest that OsCO3 primarily controls flowering time under SD conditions by negatively regulating Hd3a and FTL expression, independent of the SD-promotion pathway.

Abscisic Acid Does Not Disrupt Either the Arabidopsis FCA-FY Interaction or its Rice Counterpart in vitro

We examined the effect of (+)-ABA on the in vitro interaction of rice FCA and FY homologs, OsFCA and OsFY. From this analysis, we found no disruption of the OsFCA-OsFY complexes by ABA treatment. This result prompted us to examine the effect of ABA on the FCA-FY interaction. In these experiments, we could not reproduce the inhibitory effect of (+)-ABA on the interaction between FCA and FY. Based on these combined results, we believe that the inhibitory effect of (+)-ABA on the FCA-FY interaction should be cautiously reconsidered.

The sequence variation responsible for the functional difference between the CONSTANS protein, and the CONSTANS-like (COL) 1 and COL2 proteins, resides mostly in the region encoded by their first exons

Although the protein CONSTANS (CO) and its close relatives CONSTANS-like (COL) 1 and COL2 exhibit high amino acid sequence similarities, only the CO protein regulates floral induction in Arabidopsis. To investigate the structural basis for the functional differences between CO, COL1, and COL2 in flowering, we performed domain-swapping between CO, COL1, and COL2, and site-directed mutagenesis on the first exon of CO. The results suggest that the lack of flowering promotion activity by COL1 and COL2 is mainly attributed to the differences between CO and the COL1 and COL2 proteins in the amino acid sequence encoded by their first exons.

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.

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

AbstractMain conclusionOsNF-YC2 and OsNF-YC4 proteins regulate the photoperiodic flowering response through the modulation of three flowering-time genes (Ehd1, Hd3a, andRFT1) 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.

OsFCA Transcripts Show More Complex Alternative Processing Patterns than its Arabidopsis Counterparts

The FCA gene, which is a component of the autonomous pathway that regulates flowering time, is an important example of how alternative processing can control plant development. We have previously characterized the FCA homolog, OsFCA, from a japonica-type rice cultivar and demonstrated that the polyadenylation site within intron 3, which can generate non-functional FCA-β, was conserved in rice. In this study, we detected five alternatively processed variants of OsFCA pre-mRNA, four of which were equivalents of FCA-α, -β, -γ, and -δ, in japonica-type Korean rice cultivars. The fifth transcript, referred to as OsFCA-ɛ, was similar to OsFCA-γ, except a part of the OsFCA intron 16 was retained. Unlike the FCA-γ protein, the OsFCA-γ protein contains a glycine-rich region at its N-terminus. We detected the OsFCA transcripts missing the region encoding the glycine-rich domain in the indica-type rice, but not in the japonica-type rice. We also found that the OsFCA-δ and OsFCA-ɛ transcripts were expressed in almost all of the different tissue types examined. Taken together, these results indicate that the alternative processing of the OsFCA transcript is more complex than its Arabidopsis counterpart.

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

Key messageTheArabidopsissplicing 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.AbstractThe 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 messageThe RNA recognition motif of Arabidopsis splicing factor SF1 affects the alternative splicing ofFLOWERING LOCUS Mpre-mRNA and a heat shock transcription factorHsfA2pre-mRNA.AbstractSplicing 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-β 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.

Variation of Bolting at Cultivation of Different Regions and Molecular Characterization of FLC homologs in Angelica gigas Nakai

This study were carried out to find bolting response of cultivation in different regions and to isolate FLC (FLOWERING LOCUS C) homologs in Angelica gigas Nakai. The mean temperature of different regions, ordering in altitude, were as follows: 100 m > 350 m > 530 m > 700 m. The largest amount of rainfall was occurred in the region of 350 m while the longest time of sunshine was occurred in the region of 100 m. The content of soil chemical properties in regions showed pH 6.2 ~ 7.4, T-N 0.17 ~ 26, organic mater , , exchangeable potassium and calcium and magnesium were 0.78 ~ 1.15, 3.9 ~ 10.0, . L5 line of A. gigas was occurred in bolting at all regions, but the bolting ratio was 60.0% in 700 m region with non-mulching treatment. Manchu of A. gigas was not occurred in bolting at all regions. The accumulation bolting ratio of L5 line by non-mulching was higher than that of mulching as 90.4% and 72.8% in 100 m region. The MADS-box transcription factor FLC is one of the well-known examples as a strong floral repressor. We decided to isolate FLC homologs from A. gigas as a starting point of flowering mechanism research of this plant. We have isolated two RT-PCR products which showed very high amino acid sequence homology to Arabidopsis FLC.