Members of a recently discovered subfamily of cytokinin receptors display differences and similarities to their classical counterparts

Abstract

Cytokinins represent a group of plant hormones that have been shown to be essential for plant growth and development. A recent large-scale phylogenetic analysis of components of the cytokinin signal transduction pathway revealed, among other findings, the existence of a second, previously unknown subfamily of cytokinin receptors. Here we report that the cytokinin binding domains of the members of the 2 subfamilies contain residues that are highly conserved in either or in both subfamilies. Experiments using fluorescence microscopy hint at an ER and a plasma membrane localization for 2 members of the newly identified subfamily. These data provide new insights in the conservation of sequence and localization properties among the 2 subfamilies.

Abbreviations

CHASE

cyclase histidine kinase associated sensory extracellular

CHK

CHASE domain containing histidine kinase

GFP

green fluorescent protein

Cytokinins are a group of phytohormones that are crucial for many developmental processes as well as for the response of plants to changes in their abiotic and biotic environment.¹ The cytokinin signal transduction is mediated via a multistep His-to-Asp phospho-relay system. Binding of cytokinin leads to an autophosphorylation of the histidine kinase domain of the receptor and is followed by an intramolecular phosphotransfer to the response regulator receiver domain. Subsequently downstream signaling components are activated by phosphorylation.² Binding of cytokinin is accomplished by the Cyclin Histidine kinase Associated Sensory Extracellular (CHASE) domain and thus histidine kinase receptors including such a domain are referred to as CHASE domain containing Histidine Kinases (CHKs³). The steadily rising number of complete genome sequences from different plant species has facilitated the phylogenetic analysis of single genes and of whole pathways. This is also true for the cytokinin regulatory pathway.^4-8 Recently, we have conducted an in-depth analysis of putative cytokinin signaling components comprising more than 40 species ranging from cyanobacteria to angiosperms.⁹ This analysis identified a subfamily of cytokinin receptors not described until now. Sequence analysis revealed that while the domain architecture of the members of this subfamily is identical to those of the classical cytokinin receptors, the CHASE domains of the 2 subfamilies are rather diverse.

Here we report the results of additional analyses, both bioinformatic and experimental. A global comparison of the CHASE domains showed moderate levels of similarity between the new and the classical cytokinin receptors (between 38% and 48% aa identity), while classical as well as the newly identified displayed a higher level of conservation of the CHASE domains among themselves, the latter with a wide variation (71–96% and 47–81%, respectively) (Fig. 1). A detailed comparison between members from both subfamilies revealed many regions within the CHASE domains of the classical receptors in which the amino acid sequences are identical or highly similar. In those areas only few residues are also highly conserved in the CHASE domains of the newly identified cytokinin receptors. Those amino acids which are conserved in both, quite divergent subfamilies are likely to be crucial for the function of cytokinin receptors and thus good candidates for further investigation. In this context it is surprising that residues which have been shown to be important for cytokinin binding in the classical cytokinin receptors^10,11 display a much lower level of conservation among members of the new subfamily (6.5% identity as compared to 50% in the classical receptors).⁹ However, despite these differences in key positions of the sequence of the CHASE domain, members from both subfamilies have been shown to bind trans-zeatin, the most active cytokinin in the binding assay.^9,11-13 Interestingly, we also observed a 19 aa stretch at the C-terminal end of the CHASE domains (positions 238 to 256 of the alignment) which is highly conserved in the classical receptor subfamily, but shows no conservation among the newly identified subclade of cytokinin receptors (Fig. 1). The biological function of these residues awaits further characterization.

Figure 1.

Conservation of amino acids among the CHASE domains of the newly identified and the classical cytokinin receptors. All CHASE domains were aligned in a single multiple sequence alignment with MAFFT.¹⁸ Conservation was inferred using Jalview 2.7¹⁹ for the 2 groups independently. Blue shows the conservation among the newly identified receptors and brown shows the conservation among the classical cytokinin receptors. Conservation score is given in numbers from 0 (lowest) to 10 (highest), the height of the bars (higher bar represent higher level of AA conservation), but also in color code where the lightest color represents strongest conservation and dark color represent low levels of conservation. The residues highlighted in green represent amino acids found to be important for cytokinin binding in the CHASE domain of AHK4.¹¹ * = marks highest conserved residues (10).

Two members of the newly identified subfamily of cytokinin receptors, MpCHK1 from Marchantia polymorpha, and PpCHK4 from Physcomitrella patens, have been functionally characterized.⁹ A cytokinin binding assay confirmed the ability of these 2 receptors to bind cytokinin and a bacterial complementation assay confirmed a dose-dependent translation of the cytokinin signal into a cellular response.⁹ In order to fulfill its biological role a protein needs to localize to the appropriate subcellular compartment. To investigate to which cellular compartment the 2 members of the newly identified subfamily localize, vector constructs encoding N-terminal GFP fusion proteins of MpCHK1, and PpCHK4, respectively, under the control of the 35S promoter were transiently transformed into Nicotiana benthamiana leaves and fluorescence was analyzed in epidermal cells. The GFP:MpCHK1 protein showed fluorescence signals in what seems to be the perinuclear regions and at least partial co-localization with the ER-maker mCherry-HDEL (Fig. 2 A1–3).¹⁴ To a lesser extent also a co-localization with the plasma membrane dye FM4-64 was detected (Fig. 2 B1–3). The fluorescence of GFP:PpCHK4, also localized to the same regions highlighted by the ER marker protein mCherrry-HDEL (Fig. 2 C1–3). In addition, also for this members of the newly identified subfamily of cytokinin receptors a signal was detected which co-localized with the plasma membrane. Thus for both members of the newly identified subfamily a localization to the subcellular compartments of the ER and the plasma membrane seem the most likely. This subcellular localization is similar to the one described for classical cytokinin receptors previously.^15-17

Figure 2.

Subcellular localization of the MpCHK1 and the PpCHK4 protein fused to GFP and expressed in leaves of N. benthamiana. A1-3; B1-3: GFP:MpCHK1; C1-3; D1-3: GFP:PpCHK4. Both receptor proteins were expressed transiently in tobacco leaves as fusion proteins with GFP under the control of the 35 CaMV promoter. Fluorescence was analyzed 7 days after infiltration in prepared leaf epidermal cells. A1, B1, C1, and D1 show the GFP fluorescence exited at 488 nm, emission detected at 509 nm; A2 and C2 show fluorescence at 625-665 nm of FM4-64 dye, staining the plasma membrane; B2 and D2 show the fluorescence exited at 587 nm, emission detected at 610 nm of mCherry-HDEL, staining the ER; A3, B3, C3 and D3 show an overlay from fluorescent channels. Scale bars, where shown, correspond to 25 μm.

In summary, the additional data presented here corroborate the previously published analyses and thus further emphasize the role of the members of the newly described subfamily as functional cytokinin receptors.⁹ However, their biological role is currently unclear. Since members from both subfamilies are encoded in the genome of P. patens, more in-depth information, such as the temporal and spatial expression patterns during plant development and in response to environmental stimuli or their ligand-binding preferences, will provide further insights into possible differences of their in planta functions. Such experiments and the results of the currently ongoing genome sequencing projects from early diverging land plants and charophyte algae will help to elucidate the evolution and functional divergence of cytokinin receptors.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

We are grateful for the financial support of the Volkswagen Foundation to N.G. (Az I/83477) and of the EU Dunia BEAM program to M.H.