A new Arabidopsis gene, FLK, encodes an RNA binding protein with K homology motifs and regulates flowering time via FLOWERING LOCUS C

Abstract

Posttranscriptional RNA metabolism plays versatile roles in the regulation of gene expression during eukaryotic growth and development. It is mediated by a group of RNA binding proteins with distinct conserved motifs. In this study, an Arabidopsis (Arabidopsis thaliana) gene, designated FLK, was identified and shown to encode a putative RNA binding protein with K homology motifs. A mutant in which FLK was inactivated by T-DNA insertion exhibited a severe late flowering phenotype both in long and short days. The late flowering phenotype was reversed by gibberellin and vernalization treatments. The FLOWERING LOCUS C (FLC) transcription was greatly upregulated, whereas those of FLOWERING LOCUS T and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 decreased in the mutant. These observations demonstrate that FLK regulates the autonomous flowering pathway via FLC. It is now evident that a battery of different RNA binding proteins are involved in the posttranscriptional regulation of flowering time in Arabidopsis.

Figure 1.

Flowering Phenotype of Arabidopsis Plants Described in This Study. flk1 is SALK 112850 obtained from ABRC. It has a T-DNA insertion in the first intron of the FLK gene. flk x flk1 is a genetic cross between flk and flk1.

Figure 2.

FLK Gene Structure and Expression. (A) FLK gene structure. The FLK gene consists of six exons and is located on chromosome 3. The T-DNA insertion sites in each mutant are indicated by triangles. (B) FLK expression. The FLK transcript was not detected in flk. A tubulin gene was used as a control for constitutive expression.

Figure 3.

FLK Structure and Multiple Alignment of KH Motif Sequences. (A) FLK structure. FLK consists of 577 residues and contains three KH motifs. (B) Multiple alignment of KH motif sequences from FLK and related proteins. OsFLK, a putative FLK homolog in rice (AAL31692); hnRNP-E2, a heterogeneous nuclear ribonucleoprotein E2 in African clawed frog (CAB50743); alpha-CP2, a poly(rC/U) binding protein in human (NP_114366). The sequences were aligned using the CLUSTAL W version 1.7 (Thompson et al., 1994). Three KH motifs are boxed.

Figure 4.

Expression of Flowering-Related Genes in flk and Genetic Crosses. (A) Transcript level analyses. The transcript levels were analyzed by RT-PCR–based DNA gel blot analyses. Note that FLC (bold) is upregulated, whereas FT and SOC1 (bold) are downregulated in flk. The other genes are not affected by the flk mutation. (B) Genetic crosses. flk was genetically crossed with 35S∷CO and 35S∷FT, and homozygotic lines were obtained by segregation ratios and RT-PCR runs. 35S∷CO, a transgenic plant overexpressing CO; 35S∷FT, a transgenic plant overexpressing FT. (C) FLK transcription in the mutants in the autonomous pathway. Thirty micrograms of total RNA was loaded onto each lane. C, Col-0; L, Landsberg erecta. The blot was probed with digoxigenin-UTP–labeled 18S rDNA as an RNA quality control. (D) Comparison of FCA transcripts in flk and wild-type plants. Poly(A)⁺ RNA was isolated from total RNA, and 3 μg was loaded onto each lane.

Figure 5.

Daylength Effects on Flowering of flk. (A) Flowering of wild-type and flk plants. Plants were grown either in long days (LD, 16 h light and 8 h dark) or in short days (SD, 8 h light and 16 h dark). (B) Flowering time measurements. They were measured by days to bolting and by total leaf numbers at flowering. flk did not flower even at 125 DAG in SD, when some cauline leaves exhibited senescence. The experiment was stopped at this point in time.

Figure 6.

Effects of Vernalization and GA on Flowering Time. (A) Vernalization effects. Plants were germinated and grown at 4°C for 6 weeks and transferred to normal growth condition. (B) GA effects. A GA solution of 20 μM was sprayed twice a week on the plants until flowering. Thirty to fifty plants were measured and averaged for each measurement.

Figure 7.

Growth Stage–Dependent and Tissue-Specific FLK Expression. (A) Growth stage–dependent expression. Wild-type plants were grown in normal growth condition, and aerial parts were harvested at the indicated growth stages (in DAG) for total RNA isolation. The blot was probed with the FLK gene sequence labeled with digoxigenin-UTP (see Methods). The flk sample was used as a negative control. (B) Tissue-specific expression. Plants were grown until flowering, and plant parts were separately harvested for total RNA isolation.

Figure 8.

Subcellular Localization of FLK. The FLK-GFP gene fusion was transiently expressed in A. cepa epidermal cells, and the subcellular localization was examined by fluorescent microscopy. Note that the FLK-GFP fusion is predominantly localized in the nucleus. Bars = 20 μm.