Calcium-dependent protein kinases responsible for the phosphorylation of a bZIP transcription factor FD crucial for the florigen complex formation

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.

THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis

“…S9). It has recently been shown that CPK33 is present in the nucleus of shoot apical cells (Kawamoto et al, 2015); the inconsistence with the plasma membrane localization of CPK33 in the leaves and roots in our study may be due to the different tissues detected. When the CPK33-GFP transgene was cotransformed into Arabidopsis mesophyll protoplasts together with the THI1-RFP, the GFP and RFP signals both appeared at the plasma membrane (Fig.…”

StCDPK3 Phosphorylates In Vitro Two Transcription Factors Involved in GA and ABA Signaling in Potato: StRSG1 and StABF1

“…We also show the formation of a StCDPK3-StABF1-ABRE element interaction complex in vitro , reinforcing the hypothesis that these proteins are involved in the same molecular pathway, most likely associated with ABA signaling. Our findings are in accordance with the involvement of CDPKs in signaling pathways triggered by ABA and in the activation of bZIP TFs [60,62–64]. …”

Expression of a kinase-dead form of CPK33 involved in florigen complex formation causes delayed flowering

“…Taken our previous work. 12 and the dominant negative effect of CPK33KD in the present work together, we suggest that CPK33 is an important component of florigen complex formation via phosphorylation of FD T282. It is an important issue to demonstrate and reveal phosphorylation state of FD T282 in planta.…”

“…The assay was based on the mobility shift by phos-tag SDS-PAGE as previously described. 12 Phosphorylation-dependent mobility shift of C4 peptide was detected for wild-type CPK33 in the presence of calcium, but not for CPK33KD (Fig. 1A).…”

“…Delayed flowering phenotype was observed only under certain conditions in which only limited induction of florigen occurs (SD/4LD/SD). 12 Therefore, clearer evidences in support for the impact of CPK33 on promotion of flowering are needed. Here, we report that expression of a kinase-dead form of CPK33 caused a clear delayed-flowering phenotype by interfering with the endogenous CDPK activity including CPK33.…”