Abstract
Appropriate timing of flowering is critical for reproductive success and necessarily involves complex genetic regulatory networks. A mobile floral signal, called florigen, is a key molecule in this process, and FLOWERING LOCUS T (FT) protein is its major component in Arabidopsis. FT is produced in leaves, but promotes the floral transition in the shoot apex, where it forms a complex with a basic region/leucine-zipper (bZIP) transcription factor, FD. Formation of the florigen complex depends on the supposed phosphorylation of FD; hitherto, however, the responsible protein kinase(s) have not been identified. In this study, we prepared protein extracts from shoot apices of plants around the floral transition, and detected a protein kinase activity that phosphorylates a threonine residue at position 282 of FD (FD T282), which is a crucial residue for the complex formation with FT via 14-3-3. The kinase activity was calcium-dependent. Subsequent biochemical, cellular, and genetic analyses showed that three calcium-dependent protein kinases (CDPKs) efficiently phosphorylate FD T282. Two of them (CPK6 and CPK33) are expressed in shoot apical meristem and directly interact with FD, suggesting they have redundant functions. The loss of function of one CDPK (CPK33) resulted in a weak but significant late-flowering phenotype.
Highlights
Calcium-dependent protein kinases responsible for the phosphorylation of a basic region/leucine-zipper (bZIP) transcription factor FD crucial for the florigen complex formation
The loss of CPK33 function results in a weak but significant lateflowering phenotype. These results indicate that calcium-dependent protein kinases (CDPKs), including CPK6 and CPK33, are responsible for the phosphorylation of FD, which is crucial for the florigen complex formation
Threonine-to-alanine substitutions of either T282 alone (T282A) or both threonine residues at 276 (T276) and T282 (T276A, T282A) abolished phosphorylation, while T276 alone (T276A) did not (Supplementary Fig. S1b). These results indicate that T282, but not T276, was phosphorylated by the protein kinase activity in the extract
Summary
Phosphorylation of FD and its requirement for interaction with FT and 14-3-3. To identify the FD kinases responsible for phosphorylation of T282, we first performed an in-gel kinase assay using FD as a substrate and protein extracts from shoot apices and whole aerial parts of wild-type plants (Columbia-0 (Col)) as kinase sources. Wild-type FD, phospho-mimic T282E mFD, and non-phosphorylatable T282A mFD expressed as fusion proteins with enhanced yellow fluorescent protein (EYFP) were localized in the nucleus (Supplementary Fig. S4a) These results indicate that FD is constitutively localized in the nucleus in a phosphorylation-independent manner. FD was expressed in the shoot apical meristem and was localized in the nucleus, when examined with a similar construct to express FD fused to enhanced green fluorescent protein (EGFP) (pFD::EGFP:FD; fd-1) (Fig. 5) These results suggest that FD and two candidate CDPKs are all present in the nucleus of shoot apical cells. As anticipated from the weak phenotype and functional redundancy, neither single nor double mutants showed a significant decrease in kinase activity for FD T282 in protein extracts from shoot apices (Supplementary Fig. S10b). As expected from functional redundancy, observed enhancement was weaker than that of fd-1 (Fig. 8)
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