CPL2 and CPL3 act redundantly in FLC activation and flowering time regulation in Arabidopsis

ABSTRACT Reproductive success of plants greatly depends on the proper timing of the floral transition, which is precisely controlled by a complex genetic network. FLOWERING LOCUS C (FLC), a central floral repressor, is transcriptionally activated by the FRIGIDA (FRI) activator complex including FLC EXPRESSOR (FLX) and FLX-LIKE 4 (FLX4). C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3) forms a protein complex with FLX and FLX4 to mediate the dephosphorylation of FLX4, thereby promoting FLC expression to repress flowering in both winter and summer annuals. Here, we show that CPL2 acts redundantly with CPL3 to mediate FLC activation and flowering time. Similar to CPL3, CPL2 inhibits the floral transition, and is required for basal FLC expression in summer annuals and FLC activation in winter annuals. CPL2 directly interacts with FLX which further bridges the interaction between CPL2 and FLX4. Our results suggest that CPL2 and CPL3 function redundantly in regulating FLC expression to prevent precocious flowering.

The transition from vegetative development to reproductive development in flowering plants, known as the floral transition, is precisely controlled to occur at the appropriate time for their reproductive success. In the model plant Arabidopsis thaliana, multiple flowering genetic pathways including the photoperiod, autonomous, vernalization, thermosensory, gibberellin, and aging pathways, integrate different environmental and developmental flowering signals to ensure the proper timing of the floral transition. [1][2][3] Among these pathways, the vernalization pathway senses a prolong period of winter cold exposure to repress the expression of a key flowering repressor, FLOWERING LOCUS C (FLC) to induce flowering. 4 Based on whether vernalization is needed for rapid flowering, Arabidopsis accessions can be classified into two groups: winter annuals and summer annuals. In winter annuals, FLC expression is activated by the FRIGIDA (FRI) activator complex (FRI-C) consisting of FRI, FLC EXPRESSOR (FLX), FLX-LIKE 4 (FLX4), SUPPRESSOR OF FRI 4 (SUF4), FRI ESSENTIAL 1 (FES1), and FRI-LIKE 1 (FRL1). [5][6][7][8] In contrast, basal levels of FLC expression is established by several regulators including FLX and FLX4 in rapid-cycling summer annuals like Columbia (Col) that contains a nonfunctional fri allele. 6 We have recently shown that activation of FLC expression in flowering time control requires the RNA polymerase II (Pol II) C-terminal domain phosphatase-like protein, C-TERMINAL DOMAIN PHOSPHATASE-LIKE 3 (CPL3), 9 which has also been shown to mediate the dephosphorylation of RNA Pol II to regulate plant immune response. 10,11 CPL3 physically interacts with and dephosphorylates FLX4 through their common interacting protein FLX to enable the binding of dephosphorylated FLX4 to the FLC locus, thereby promoting FLC expression to inhibit flowering in both winter and summer annuals. In winter annuals, disruption of either CPL3, FLX or FLX4 could greatly suppress the extremely late-flowering phenotype of FRI. 6,8,9 However, unlike flx or flx4 that fully suppresses the FRI phenotype, cpl3 mutant could not completely suppress the late flowering of FRI, 9 implying that other regulator(s) may act redundantly with CPL3 in mediating FRI-dependent FLC activation.
In the Arabidopsis genome, there are several other CTD phosphatase-like proteins, including CPL1/FIERY2, CPL2 and CPL4, 12,13 which potentially function redundantly with CPL3 in flowering time control. Like CPL3, CPL4 contains a catalytic domain and a BRCA1 C Terminus (BRCT) domain at its C-terminus, whereas CPL1 and CPL2 each contain a catalytic domain and 1-2 dsRNA-binding domains (Figure 1(a)). We examined the potential redundancy between CPL3 and these three CPL proteins by examining the flowering phenotype of their mutants or their interaction with FLX or FLX4. Yeast two-hybrid assay revealed that CPL4 did not interact with CPL3 or components in FRI-C including FLX, FLX4, SUF4, and FES1 ( Figure 1(b)), excluding the possibility of CPL4 as a redundant factor of CPL3. Moreover, CPL4 is essential for plant viability and knockdown of CPL4 results in strong growth defects in plant growth, 14,15 preventing us from examining the flowering phenotype of cpl4 mutants. To examine the effect of CPL1 and CPL2 on flowering time control, we obtained seeds of cpl1-8 (GK-165H09-013365) 16 and cpl2-2 (SALK_059753), 13  Interestingly, cpl2-2 showed accelerated flowering under long days (Figure 2(a)), which has been consistently shown in a previous study. 13 The early-flowering phenotypes of cpl2-2 prompted us to investigate whether it functions redundantly with CPL3 to regulate FLC activation. To this end, we first examined the expression levels of FLC in cpl2-2 mutants and found that FLC expression was significantly downregulated in cpl2-2 mutants (Figure 2(b)). FLC expression was consistently downregulated in the developing seedlings of cpl2-2 before, during and after the floral transition (Figure 2(c)). Next, we examined whether CPL2 is required for FRI-dependent FLC activation in winter annuals. We crossed cpl2-2 with FRI-Col, 18 and found that cpl2-2 partially suppressed the extremely late-flowering phenotype of FRI (Figure 2(a)).
Consistently, further expression analysis showed that FLC expression was reduced in FRI cpl2-2 as compared with FRI ( Figure 2(b)). These observations indicate that CPL2 promotes FLC expression to inhibit flowering in both summer annuals and winter annuals, and that CPL2 may act redundantly with CPL3 in FLC activation.
CPL3 forms a protein complex with FLX and FLX4. 9 Similar to CPL3, CPL2 interacted with FLX but not FLX4 in yeast two-hybrid assays (Figure 3(a)). Further yeast threehybrid assay revealed that CPL2 interacted with FLX4 in the presence of FLX (Figure 3(b)). These results suggest that CPL2 interacts with FLX4 through their scaffold protein  FLX like CPL3 does. Interestingly, we found that CPL2 also interacted with CPL3 in yeast (Figure 3(c)), indicating that these two proteins may form heterodimers.
Next, we crossed FRI cpl2-2 with cpl3-8 to generate the FRI cpl2-2 cpl3-8 triple mutants. Either cpl2-2 or cpl3-8 partially suppressed the extremely late-flowering phenotype of FRI, whereas mutations in both CPL2 and CPL3 almost completely suppressed the late-flowering phenotype of FRI (Figure 2(a)). Consistently, FLC expression was almost fully restored in FRI cpl2-2 cpl3-8 triple mutants to the levels in Col (Figure 2(b)). However, FLC expression was slightly higher in FRI cpl2-2 cpl3-8 than FRI flx4-2 or FRI flx-2 (Figure 2(b)), indicating the possible involvement of other regulator(s) functioning redundantly or in parallel with CPL2 and CPL3 in regulating FLC expression.
Overall, our results suggest that CPL2 acts redundantly with CPL3 in FLC activation and flowering time regulation. Both CPL2 and CPL3 inhibit flowering in Arabidopsis and are required for the basal FLC expression in summer annuals and FRI-dependent FLC upregulation in the winter annuals. Although either cpl2 or cpl3 mutants only partially suppress FRI flowering phenotype, cpl2 cpl3 double mutants almost fully suppress the late flowering of FRI. CPL2/3 forms a protein complex with FLX and FLX4, two components in the FRI-C transcriptional activator complex. Since CPL3 has been shown to dephosphorylate FLX4 protein 9 and CPL2 also possesses the phosphatase activity, 10 it is possible that CPL2 also modulates the phosphorylation status of FLX4 to regulate FLC expression, thus affecting flowering. Besides CPL2/3, there are many other CTD phosphatases in Arabidopsis, 19 our revealed CPL2/3-FLX-FLX4-FLC functional module sheds new lights on the biological roles of CTD phosphatases in mediating dephosphorylation of key developmental regulators to modulate gene expression in plants.

Plant materials and growth conditions
Seeds of Arabidopsis were placed on soil and stratified at 4°C in darkness for 3 days before they were grown under long days (16 hr light/8 hr dark) at 23 ± 2°C. Mutant seeds of cpl2-2 (SALK_059753) and cpl1-8 (GK-165H09-013365) were obtained from the Arabidopsis Information Resource, while seeds of flx-2 and FRI flx4-2 were kindly provided by Prof. Scott Michaels (Indiana University).

Expression analysis
Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) and reverse transcribed with the M-MLV Reverse Transcriptase (Promega) following manufacturers' instructions. Quantitative real-time PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) with three independently collected biological samples. The expression of TUBULLIN 2 (TUB2) was used as the internal control. The normalized expression of target genes is calculated with the difference between the cycle threshold (Ct) of target genes and the Ct of control.

Yeast two-hybrid and three-hybrid assays
The coding sequences of CPL2, CPL3, SUF4, FES1, FLX4 and FLX were cloned into pGBKT7 (BD) or pGADT7 (AD) vectors (Clontech). For yeast two-hybrid assay, various combinations of AD and BD vectors were co-transformed into Y2HGold yeast cells using the Yeastmaker Yeast Transformation System 2 according to the manufacturer's instructions (Clontech). After transformation, yeast cells were grown on SD-Leu/-Trp and SD-Ade/-His/-Leu/-Trp mediums. For yeast three-hybrid assay, the coding sequence of FLX was cloned into pQH05 vector with a HIS3 selection marker. Various combinations of AD and BD vectors with pQH05 or pQH05-FLX were co-transformed into Y2HGold yeast and grown on SD-His/-Leu/-Trp and SD-Ade/-His/-Leu/-Trp mediums.