The rice F-box protein KISS ME DEADLY2 functions as a negative regulator of cytokinin signalling

Abstract

Cytokinins are plant hormones that play critical roles in growth and development. We recently determined that the transcriptional response to cytokinin of Arabidopsis is modulated by the KISS ME DEADLY (KMD) family of F-box proteins. Here we demonstrate a conserved function for a member of the rice KMD family. Ectopic overexpression of OsKMD2 in Arabidopsis results in decreased cytokinin sensitivity based on a hypocotyl growth response assay, the decrease in sensitivity correlating with a decrease in the levels of the transcriptional regulator AtARR12. Furthermore, OsKMD2 directly interacts with AtARR12 based on yeast two-hybrid and co-immunoprecipitation assays. These results indicate that both monocots and dicots employ a similar KMD-dependent mechanism to regulate the transcriptional response to cytokinin.

Cytokinins are plant hormones that regulate diverse growth and development processes, such as cell division, metabolism, chloroplast development and maintenance, and senescence.^1,2 Cytokinins also regulate responses to abiotic factors such as cold and salt stress, and biotic factors such as the response to pathogens and symbiotic partners.^3,4 Plant cells perceive cytokinins through a conserved, multistep histidine-to-aspartate phosphorelay system, evolutionarily related to the two-component signalling systems of prokaryotes.⁵ The initial cytokinin signalling pathway incorporates 3 types of signalling elements—receptors, histidine-containing phosphotransfer proteins, and type-B response regulators—with a regulatory phosphate passed sequentially from one signalling element to the next to control their activity.^1,2 The type-B response regulators function as transcription factors to control the expression of cytokinin-regulated genes. This model of cytokinin signal transduction is primarily derived from studies in the dicot Arabidopsis, but a similar cohort of signalling elements is present in other plant species, including the monocot rice (Oryza sativa) and the moss Physcomitrella patens, supporting a common pathway for the transmission of the cytokinin signal in land plants.^6,7

The transcriptional output of plant hormone signalling pathways is often controlled by protein degradation. For example, key transcriptional regulators for the phytohormones auxin, jasmonate, gibberellin, and ethylene are all targeted for degradation by the ubiquitin-proteasome pathway.⁸ Interestingly, in each case stability is controlled by the S-PHASE KINASE-ASSOCIATED PROTEIN1 (SKP1)/Cullin/F-box (SCF) E3-ubiquitin ligase complex. Specificity for the target to be degraded is dependent on the type of F-box protein present in the SCF complex. The Arabidopsis genome encodes almost 700 functional F-box proteins.⁹ We recently determined that the type-B ARRs of Arabidopsis (AtARRs), which control the transcriptional output for cytokinin, are also targets for degradation by an SCF complex, with the specificity being determined by the 4-member family of KISS ME DEADLY (KMD) F-box proteins.¹⁰ The AtKMDs directly interact with multiple type-B AtARRs, with the strongest interaction occurring with AtARR1 and AtARR12 based on yeast two-hybrid analysis,¹⁰ which are the type-B AtARRs that contribute most substantially to the Arabidopsis cytokinin response.^11,12 Ectopic overexpression of the AtKMDs results in cytokinin insensitivity, coincident with decreased levels of type-B AtARRs, and consistent with the AtKMDs functioning as negative regulators of cytokinin signalling.¹⁰

Phylogenetic analysis indicates that homologues to the AtKMDs exist in other plant species. As shown in Figure 1, the monocot rice, like the dicot Arabidopsis, contains 4 KMD homologues. Furthermore, BLAST searches employing the AtKMDs identify 6 putative homologues in the moss P. patens and 1 in the moss Selagenella moellendorffii. The S. moellendorffii homologue clearly clades within the KMD families of rice and Arabidopsis, being as closely related as AtKMD3 and AtKMD4 to the other KMDs of rice and Arabidopsis. Phylogenetic analysis thus suggests that the KMD-based regulatory mechanism is broadly conserved in land plants, being present in the same organisms that contain the complete set of cytokinin-signalling elements, including the targeted type-B response regulators.^6,7 However, we could not detect a clear homologue to the KMD F-box proteins in the green algae Chlamydomonas reinhardtii, where ancestral forms of the type-B response regulators have been identified, although not integrated into a cytokinin signalling pathway.⁶

Figure 1. Phylogenetic analysis of KMD F-box proteins. The phylogenetic tree was constructed using the phylogeny.fr pipeline.¹⁴ Representative F-box proteins from the dicot Arabidopsis thaliana (At), the monocot Oryza sativa (Os), and the mosses Physcomitrella patens (Pp) and Selaginella moellendorffii (Sm) were analyzed. Branches with support values of less than 50% support were collapsed. The clades are those designated in Schumann et al. (2011).

To test conservation of function between the monocot and dicot KMD genes, a cDNA clone of OsKMD2, driven by the CaMV 35S promoter and fused to a GFP reporter, was stably transformed into wild-type Arabidopsis. Multiple independent lines were generated in which the transgene was ectopically expressed to varying levels based on detection of the OsKMD2-GFP fusion by immunoblot analysis (Fig. 2A). To examine the effect of OsKMD2 on cytokinin responsiveness, these overexpression lines were examined for their inhibition of hypocotyl elongation in response to exogenous cytokinin (Fig. 2B). In dark-grown, wild-type seedlings, increasing concentrations of the cytokinin t-zeatin result in an inhibition of hypocotyl elongation. All 3 transgenic lines exhibited a reduced cytokinin response in this growth assay compared to wild-type seedlings, the differences being most pronounced at 0.01 and 0.1 μM t-zeatin. The 35S::OsKMD2-3 line, which produced the highest level of the OsKMD2-GFP fusion protein (Fig. 2A), exhibited the strongest effect on cytokinin responsiveness (Fig. 2B). The effect of the OsKMD2 gene on this growth response to cytokinin is similar to what is observed when members of the Arabidopsis KMD family are ectopically expressed.¹⁰ These results indicate that OsKMD2 acts as a negative regulator of cytokinin responses and support functional conservation among this gene family of monocots and dicots.

Figure 2. Functional analysis of OsKMD2. (A) Overexpression of OsKMD2 based on immunoblot analysis. For OsKMD2-GFP overexpression lines, cDNAs covering the complete coding sequence were amplified with 5’-AGATCTATGG GTTACAACGA GCTGATTCCG-3’ and 5’-AGGCCTGATT TCTAGAAGGC AGGCGGCC-3’, cloned into pCR8/GW/TOPO (Invitrogen), and recombined into pEarleyGate103. The GFP-tagged OsKMD2 protein was detected by immunoblotting using an HRP-conjugated anti-GFP antibody. (B) Effect of cytokinin on hypocotyl growth of wild-type and OsKMD2 overexpression lines. The analysis of hypocotyl length was performed as described.¹⁰ (C) Yeast two-hybrid analysis of the interaction of OsKMD2 and AtARR12. AtAHP2 was used as a positive control. (D) Coimmunoprecipitation of OsKMD2 with AtARR12. Arabidopsis protoplasts were co-transfected with a plasmid expressing AtARR12-HA together with either OsKMD2-GFP or a GFP control. Immunoprecipitation and immunoblot analyses were performed as described.¹⁰ (E) Overexpression of OsKMD2 results in reduced protein levels of AtARR12. Protein levels of the indicated fusion proteins were determined by immunoblot analysis with anti-Myc and anti-GFP antibodies, with α-tubulin as the loading control.

The Arabidopsis KMDs directly interact with type-B ARRs to control their degradation.¹⁰ To determine if the action of OsKMD2 in Arabidopsis also involved direct interaction with type-B ARRs, we examined the ability of OsKMD2 to interact with the response regulator AtARR12 using yeast two-hybrid and co-immunoprecipitation assays (Fig. 2C, D). Yeast two-hybrid analysis demonstrated a clear interaction between OsKMD2 and AtARR12 (Fig. 2C), similar to that found with the phosphotransfer protein AtAHP2, a known interactor of type-B AtARRs.¹³ For co-immunoprecipitation analysis, ARR12-HA was co-transfected with either OsKMD2-GFP or GFP into Arabidopsis protoplasts. Immunoprecipitation with an anti-GFP antibody resulted in co-immunopreciption of ARR12-HA with OsKMD2-GFP, but not with the negative control GFP, confirming the ability of OsKMD2 to directly interact with ARR12 (Fig. 2D). To determine if the interaction of OsKMD2 with ARR12 affected the stability of ARR12 in planta, we crossed a transgenic line expressing 35S::ARR12-Myc with wild-type or 35S::OsKMD2-GFP plants, and analyzed the level of the ARR12-Myc protein in the F₁ seedlings. Elevated expression of OsKMD2 resulted in a decrease in the level of ARR12 protein (Fig. 2E), consistent with what we previously observed with the Arabidopsis KMD F-box proteins.¹⁰

These results support a conserved function for the rice F-box protein OsKMD2 with the KMD family of F-box proteins of Arabidopsis, based on their shared ability to: (1) decrease cytokinin sensitivity when overexpressed; (2) directly interact with the type-B response regulator AtARR12; and (3) reduce the protein level of AtARR12 when overexpressed. Based on these functional analyses, coupled with their phylogenetic relationship, we propose that both rice and Arabidopsis employ 4-member families of KMD F-box proteins, which serve to regulate the transcriptional output from the cytokinin signalling pathway by targeting the type-B response regulators for degradation.

10.4161/psb.26434