The subcellular distribution of the Arabidopsis histidine phosphotransfer proteins is independent of cytokinin signaling

Abstract

SUMMARYCytokinins are a class of mitogenic plant hormones that play an important role in most aspects of plant development, including shoot and root growth, vascular and photomorphogenic development and leaf senescence. A model for cytokinin perception and signaling has emerged that is similar to bacterial twocomponent phosphorelays. In this model, binding of cytokinin to the extracellular domain of the Arabidopsis histidine kinase (AHKs) receptors induces autophosphorylation within the intracellular histidine-kinase domain. The phosphoryl group is subsequently transferred to cytosolic Arabidopsis histidine phosphotransfer proteins (AHPs), which have been suggested to translocate to the nucleus in response to cytokinin treatment, where they then transfer the phosphoryl group to nuclear-localized response regulators (Type-A and Type-B ARRs). We examined the effects of cytokinin on AHP subcellular localization in Arabidopsis and, contrary to expectations, the AHPs maintained a constant nuclear/cytosolic distribution following cytokinin treatment. Furthermore, mutation of the conserved phosphoacceptor histidine residue of the AHP, as well as disruption of multiple cytokinin signaling elements, did not affect the subcellular localization of the AHP proteins. Finally, we present data indicating that AHPs maintain a nuclear/cytosolic distribution by balancing active transport into and out of the nucleus. Our findings suggest that the current models indicating relocalization of AHP protein into the nucleus in response to cytokinin are incorrect. Rather, AHPs actively maintain a consistent nuclear/cytosolic distribution regardless of the status of the cytokinin response pathway.

The Cytokinin Receptors of Arabidopsis Are Located Mainly to the Endoplasmic Reticulum

“…The membrane topology of cytokinin receptors is also highly relevant but currently unknown. The ligandbinding CHASE domain will most likely be on the extracytosolic side and the His kinase domain will most likely be oriented toward the cytoplasm and/or nuclear compartment, enabling them to interact with the AHP proteins (Suzuki et al, 2001b;Dortay et al, 2006), which are exclusively located in the cytoplasm and the nucleus (Hwang and Sheen, 2001;Punwani et al, 2010).…”

“…Arabidopsis (Arabidopsis thaliana) encodes three cytokinin receptors, ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1 (CRE1)/ AHK4 (Inoue et al, 2001;Suzuki et al, 2001a;Ueguchi et al, 2001;Yamada et al, 2001), which differ in their biological functions and biochemical properties (for review, see Heyl et al, 2011). The cytoplasmic part of all three receptors interact redundantly with His phosphotransfer proteins (AHP), which transmit the signal to the nucleus (Hwang and Sheen, 2001;Punwani et al, 2010), where type B response regulators, a class of Myb transcription factors, are activated. These mediate the majority, if not all, of the transcriptional responses to cytokinin (Argyros et al, 2008;Heyl et al, 2008;Ishida et al, 2008).…”

CAST AWAY, a Membrane-Associated Receptor-Like Kinase, Inhibits Organ Abscission in Arabidopsis

“…Using site-directed mutagenesis, 5#-GCCCCCTTCACCATGGCTGCTTGTATTTCGTTC-3# and 5#-GAACG-AAATACAAGCAGCCATGGTGAAGGGGGC-3# were used to generate pENTR::CST G2A , 5#-CCCCCTTCACCATGGGTGCTTCTATTTCGTTCTT-TTCTTCTTCC-3# and 5#-GGAAGAAGAAAAGAACGAAATAGAAGCA-CCCATGGTGAAGGGGG-3# were used to generate pENTR::CST C4S , and 5#-CCCCCTTCACCATGGCTGCTTCTATTTCGTTCTTTTCTTCTTCC-3# and 5#-GGAAGAAGAAAAGAACGAAATAGAAGCAGCCATGGTGAAGGGGG-3# were used to generate pENTR::CST G2A C4S . Recombination with pHBT-gw-GFP, pUC-gw-SPYNE, and pUC-gw-SPYCE destination vectors (Punwani et al, 2010) using Gateway technology (Life Technologies) was used to generate C-terminal fusions of each RLK to GFP, YFPn, and YFPc, respectively. The pUC-SPYCE plasmid was used as a YFPc control (Walter et al, 2004).…”

ArabidopsisHistidine Kinase CKI1 Acts Upstream of HISTIDINE PHOSPHOTRANSFER PROTEINS to Regulate Female Gametophyte Development and Vegetative Growth

“…AHP proteins were suggested to localize in the cytoplasm in the absence of cytokinin and, upon treatment with cytokinin, to translocate into the nucleus (Hwang and Sheen, 2001). However, a recent study demonstrated that AHP proteins maintained a constant nuclear/cytosolic distribution by balancing active transport and that this subcellular pattern was independent of cytokinin signaling (Punwani et al, 2010). Nevertheless, the nuclearlocalized and phosphorylated AHPs presumably activate a class of MYB transcription factors, designated as type-B Arabidopsis response regulators (ARR).…”

“…Nevertheless, the nuclearlocalized and phosphorylated AHPs presumably activate a class of MYB transcription factors, designated as type-B Arabidopsis response regulators (ARR). These type-B ARRs directly activate expression of type-A ARR genes that, in turn, negatively regulate type-B ARRs (Hwang et al, 2002;Heyl and Schmü lling, 2003;Kakimoto, 2003;Ferreira and Kieber, 2005;Mü ller and Sheen, 2007;Punwani et al, 2010). CRE1 is a bifunctional protein exerting kinase or phosphatase activities on AHPs in the presence or absence of cytokinin, respectively (Mä hö nen et al, 2006a).…”