Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis

Abstract

Cytokinins are plant hormones with profound roles in growth and development, including control of leaf longevity. Although the cytokinin signal is known to be perceived by histidine kinase receptors, the underlying molecular mechanism and specificity of the receptors leading to delayed leaf senescence have not yet been elucidated. Here, we found that AHK3, one of the three cytokinin receptors in Arabidopsis, plays a major role in controlling cytokinin-mediated leaf longevity through a specific phosphorylation of a response regulator, ARR2. This result was obtained through identification of a gain-of-function Arabidopsis mutant that shows delayed leaf senescence because of a missense mutation in the extracellular domain of AHK3. A loss-of-function mutation in AHK3, but not of the other cytokinin receptors, conferred a reduced sensitivity to cytokinin in cytokinin-dependent delay of leaf senescence and abolished cytokinin-dependent phosphorylation of ARR2. Consistently, transgenic overexpression of wild-type, but not an unphosphorylatable mutant ARR2, led to delayed senescence of leaves.

Fig. 1.

The Arabidopsis ore12-1 mutant shows extended leaf longevity. (A) Phenotypes of whole wild-type (Col, Left) and ore12-1 (Right) plants at different ages. DAG, days after germination. (Scale bar, 1 cm.) (B) Extended life span of an ore12-1 mutant leaf. (Top) Age-dependent senescence phenotype of a wild-type and an ore12-1 leaf. (Middle) The chlorophyll content and the photochemical efficiency (Fv/Fm) in wild-type and ore12-1 leaves were examined at different ages starting at 12 days after leaf emergence when the leaf had just reached its full growth. Error bar indicates standard deviation (n = 30). (Bottom) Expression of the molecular-senescence markers CAB and SAG12 in wild-type and the ore12-1 leaves.

Fig. 2.

ORE12 is AHK3, a histidine kinase cytokinin receptor. (A) The structure of the ORE12/AHK3 gene and of its protein product. The open triangle and asterisk indicate the position of the ore12-1 mutation. TM, transmembrane domain; ED, extracellular domain; HKD, histidine kinase domain; RLD, receiver-like domain; RD, receiver domain. (B and C) Ectopic expression of either ore12-1 (B) or AHK3 (C) recapitulates the ore12-1 phenotype. The chlorophyll content was examined every 2 days during dark-induced senescence. Error bar indicates standard deviation (n = 12). (D) Up-regulation of cytokinin-inducible A-type ARR genes in the ore12-1 mutant. Total RNA extracted from wild-type (Col) and the ore12-1 leaves was used to examine the expression of the A-type ARR genes ARR4 –9 and 15 for RT-PCR.

Fig. 3.

AHK3 positively transmits cytokinin signals. (A and B) Senescence response of the loss-of-function ahk3 mutant. Detached leaves of wild-type and ahk3 plants were incubated in darkness with or without treatment with BA, a synthetic cytokinin. (C) Reduction of cytokinin-induced A-type ARR gene expression in the ahk3 mutant. RT-PCR was performed with total RNA extracted from wild-type or the ahk3 leaves before (0) and after treatment with (+CK) or without (–CK) t-zeatin for 1 h. (D) Inhibition of cytokinin-mediated hypocotyl growth in wild-type, ore12-1, ahk3, and an AHK3 overexpression ³⁵S::AHK3–40 line. Seedlings were grown in the presence of t-zeatin at the indicated concentrations. At least 15 hypocotyls were examined for each treatment.

Fig. 4.

Cytokinin induces phosphorylation of ARR2 in mature leaf cells, leading to increased leaf longevity. (A) Structure of ARR2. The conserved amino acid residues in the receiver domain are noted. D80 is the predicted target residue for phosphorylation and is mutated to Asn in ARR2^D80N. (B) Cytokinin-induced mobility shift of ARR2. Cells transfected with ARR2-HA were preincubated for 4 h (0h) before treatment for 1 h with (+) or without (–) t-zeatin (100 nM). Cytokinin-induced mobility shift is abolished by phosphatase treatment. Cells transfected with ARR2 were treated with (+CK) or without (–CK) t-zeatin for 1 h. Crude extracts of cytokinin-treated cells were incubated with (+CIP) or without (–CIP) calf intestine alkaline phosphatase. The proteins were detected with an anti-HA antibody. The Coomassie-brilliant-blue-stained RbcS protein (RBC) serves as a protein-loading control. (C) ARR2^D80N mutation reduced its capacity to transactivate the ARR6 promoter (Upper). Protoplasts were cotransfected with the ARR6-LUC reporter and an effector plasmid expressing ARR2, ARR2^D80N, or ARR2^D80E mutant proteins. Vector DNA was used as a control. A limited amount (2 μg) of an effector plasmid was transfected to avoid nonspecific activation by protein overexpression. Immunoblot with an anti-HA antibody shows protein level of ARR2, ARR2^D80N, and ARR2^D80E (Lower). (D) Ectopic expression of ARR2 (³⁵S::ARR2), but not that of ARR2^D80N (³⁵S::ARR2^D80N), resulted in delayed leaf senescence during dark incubation. Error bar indicates standard deviation (n = 30).

Fig. 5.

Cytokinin-induced phosphorylation of ARR2 is mediated by AHK3. (A) Impaired phosphorylation of ARR2 in the ahk3 mutant. (B) Restoration of impaired ARR2 phosphorylation in the ahk3 cells by AHK3 and ore12-1. ARR2 was transfected into the ahk3 cells with AHK3 (Upper) or ore12-1 (Lower). In the case of ore12-1 transfection, protein extract from the ahk3 cells transfected with ARR2 was included as a control. The transfected cells were treated with (+CK) or without (–CK) t-zeatin for 1 and 3 h. The proteins were detected with an anti-HA antibody. (C) AHK3-dependent action of ARR2 in inducing the ARR6-LUC expression. Wild-type or ahk3 mutant cells were transfected with the ARR6-LUC reporter alone (Control) or cotransfected with ARR6-LUC and the indicated plasmids. Transfected cells were treated with (+CK) or without (–CK) t-zeatin. Error bar indicates standard deviation (n = 3).

Fig. 6.

Specificity of AHK3 in cytokinin-mediated leaf senescence and phosphorylation of ARR2. (A and B) Senescence response of the loss-of-function ahk2 and ahk4 mutants with or without cytokinin. Error bar indicates standard deviation (n = 36). (C) Impaired ARR2 phosphorylation in the ahk3 cells but not in the ahk2 and ahk4 cells. Note that the mobility shift is observed in the ahk2 and ahk4 cells. (D) Senescence response of AHK2- and -4-overexpression transgenic lines. Error bar indicates standard deviation (n = 24). The chlorophyll content was examined every 2 days during dark-induced senescence.