The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors

Abstract

SUMMARYArabidopsis thaliana has three membrane-located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand-binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/ AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans-zeatin (tZ) with an apparent K D of 1.4 and 4.0 nM, respectively. Real-time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin-sensitive P ARR5 :GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter-swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.

Cytokinin Activity of cis-Zeatin and Phenotypic Alterations Induced by Overexpression of Putative cis-Zeatin-O-glucosyltransferase in Rice

“…Based on studies with Arabidopsis, cZ has generally been thought to be a cytokinin derivative with no or low activity (Inoue et al, 2001;Spíchal et al, 2004;Romanov et al, 2006;Stolz et al, 2011). Although recent studies with nonmodel plants suggested possible roles for cZ (Vyroubalová et al, 2009;Goggin et al, 2010;Lomin et al, 2011), its physiological significance was yet to be clarified.…”

“…Thus, contrary to widespread assumptions, cZ plays a role as an active cytokinin in several aspects of rice growth and development. Previous biochemical studies on HK cytokinin receptors showed that the affinity to cZ was similar to that to tZ in maize HK (ZmHK1; Lomin et al, 2011), while cZ affinities were much lower than tZ affinities in Arabidopsis HKs (AHK2, AHK3, and CRE1/AHK4; Romanov et al, 2006;Stolz et al, 2011). Four cytokinin receptors have been identified in rice (OsHK3, OsHK4, OsHK5, and OsHK6; Ito and Kurata, 2006;Pareek et al, 2006;Du et al, 2007;Schaller et al, 2007).…”

Cytokinins - recent news and views of evolutionally old molecules

“…Individual receptors of Arabidopsis thaliana are most sensitive to the bases of the isoprenoid-type CKs and thidiazuron, but differ in their recognition of CK metabolites. In contrast to CRE1/AHK4 and AHK2, which effectively discriminate between CK basis and their conjugates, AHK3 has broader specificity and, alongside ciszeatin and dihydrozeatin (DZ) (Spíchal et al 2004;Stolz et al 2011) noticeably recognises CK ribosides and nucleotides (Spíchal et al 2004). AHK3 shows a stronger preference towards trans-zeatin-type CKs than iP-type CKs, whereas CRE1/AHK4 recognises both trans-zeatin and iP similarly (Spíchal et al 2004;Romanov et al 2006).…”

“…Based on the opposite predominant occurrences of trans-zeatin-and iP-type CKs in xylem and phloem sap, respectively, and the fact that AHK3 is predominantly expressed in the shoot but CRE1/AHK4 is expressed in the root (Ueguchi et al 2001b;Higuchi et al 2004), the specific function of AHK3 of responding to a longdistance signal coming from the roots was suggested (Romanov et al 2006). A recent study by Stolz et al (2011) showed that AHK2 is highly similar to CRE1/AHK4 in ligand preference and function, as proved by the promoter-swap experiments. Interestingly, in contrast to AHKs, all maize His kinases have very high affinity to both isoprenoid and aromatic CKs and are effectively activated by cis-zeatin (Yonekura- .…”

“…Expression of CRE1/AHK4, AHK3 and AHK2-CHASE-TM (AHK2-CHASE including the two adjacent transmembrane domains) in Escherichia coli DrcsC and cps::lacZ mutant strains producing b-galactosidase in reaction to CK , and direct receptor binding assays (Romanov et al 2005) were used for molecular and biochemical characterisation of the receptors Yamada et al 2001;Spíchal et al 2004;Romanov et al 2006). It has been shown that the receptors bind isoprenoid and aromatic CKs, and also thidiazuron (a derivative of phenylurea), in a highly specific manner with K d in the nanomolar range Romanov et al 2005;Romanov et al 2006;Lomin et al 2011;Stolz et al 2011). Individual receptors of Arabidopsis thaliana are most sensitive to the bases of the isoprenoid-type CKs and thidiazuron, but differ in their recognition of CK metabolites.…”