1. Volume 16, Issue 11, Pages 1116-1122 (June 2006)
Cytokinins Regulate a Bidirectional Phosphorelay Network in Arabidopsis 
Ari Pekka Mähönen, Masayuki Higuchi, Kirsi Törmäkangas, Kaori Miyawaki, Melissa S. Pischke, Michael R. Sussman, Ykä Helariutta, Tatsuo Kakimoto 
Current Biology 
Volume 16, Issue 11, Pages (June 2006)
DOI: /j.cub
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2. Figure 1 wol Confers a Negative Activity on Cytokinin Signaling
(A) Seven-day-old seedlings. From left to right; Col wild-type (WT), wol, wol-sup10, cre1-2. The scale bar represents 3 mm.
(B–F) Sections of primary roots. The scale bar represents 10 μm. (B) Col WT. (C) wol. In (B) and (C), pericycle cells are marked with an asterisk, black arrows mark the xylem pole (B) or xylem (C), and red arrows mark the phloem. (D) wol-sup10. (E) cre1-2. (F)cre1-12 ahk2-2tk ahk3-3 triple mutant.
(G) Expression of ARR15 in root tips of plants treated without (blue) or with (red) 10 μM benzyladenine. 1, 2; WT Col. 3, 4; cre1-2. 5; cre1-12 ahk2-2tk ahk3-3 triple mutant. 6 and 7; wol. 8 and 9; wol-sup9. 10 and 11; wol-sup10. Expression levels are presented as relative values compared to the mean value of column 1 (WT Col without cytokinin). Vertical error bars represent standard deviations.
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3. Figure 2 Kinase and Phosphatase Activities of CRE1
(A–C) Autoradiograms showing phosphotransfer in vitro. Purified membrane fraction of SF9 insect cells expressing WT or mutant CRE1 and a purified recombinant HPt protein (AHP1, AHP2, AHP3, AHP5, or YPD1) were used. The top panels in (A) and (C) show the amounts of the CRE1 protein that was quantified with Western blotting. (A) CRE1-mediated phosphotransfer to AHP1. WT or a mutant CRE1, AHP1, and [γ32P]ATP were incubated in the presence or absence of the cytokinin thidiazuron (50 μM). The relative amounts of CRE1(T278I) and CRE1(F685L) protein were about 2-fold higher than the amounts of CRE1 and CRE1(D973N). (B) CRE1-mediated phosphotransfer to every HPt tested in the presence of 50 μM thidiazuron. (C) Dephosphorylation of AHP1 by CRE1. Phosphorylated AHP1 was purified and incubated with WT or a mutant CRE1 in the absence of cytokinin. Similar levels of WT and mutant CRE1 proteins were used.
(D) Schematic representations of kinase (left) and phosphatase (right) reactions mediated by CRE1. Note that phosphotransfer between an HPt protein and a response regulator is not experimentally demonstrated in this study. Green, cytokinin binding domain. Blue, histidine kinase domain. Red, receiver domain. Light blue, HPt domain protein.
(E) Kinase and phosphatase assays in yeast. Suppression of the lethality of sln1Δ in the absence of galactose represents histidine kinase activity of the introduced gene product. The lethality conferred by an introduced gene expressed in SLN1+ represents its phosphatase activity. Galactose induces PGAL1::PTP2 in the sln1Δ or SLN1 + yeast, which in turn allows yeast growth irrespective of the phosphorelay because PTP2 acts on a protein downstream of the phosphorelay system.
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4. Figure 3
Elimination of the Kinase Activity of CRE1 Results in Reduced Cytokinin Sensitivity and wol-Like Vasculature
(A, B, and D–H) Cross-sections of the primary root. (A) PCRE1::CRE1(T278I)-HA. (B) PCRE1::CRE1(T278I, H459Q, D973N)-HA. (D) PCRE1::CRE1(T278I, H459Q)-HA that exhibited an indeterminate root-growth phenotype. (E) PCRE1::CRE1(T278I, D973N)-HA. (F) PCRE1::CRE1(F685L). (G) PCRE1::CRE1. (H) PCRE1::AHK2. (G and H) Plants were grown in the presence of 10 nM benzyladenine. The scale bar represents 10 μm. (C) Western blotting with anti-HA. From left to right; PCRE1::CRE1(T278I)-HA, PCRE1::CRE1(T278I, H459Q, D973N)-HA, PCRE1::CRE1(T278I, H459Q)-HA, and PCRE1::CRE1(T278I, D973N)-HA. (A–H) The genetic background is cre1-2. (I) Expression of ARR15 in root tips of plants treated without (blue) or with (red) 10 μM benzyladenine. 1 and 2, Col WT. 3 and 4, cre and 6, wol. 7, PCRE1::CRE1-HA. 8 and 9, PCRE1::CRE1(T278I). 10 and 11, PCRE1::CRE1(T278I, H459Q, D973N)-HA. 12, PCRE1::CRE1(F685L). 13, PCRE1::AHK2. The genetic background of plants in columns 7–13 is cre1-2. Expression levels are presented as relative values compared to the mean value of column 1 (WT Col without cytokinin). Vertical error bars represent standard deviations. (J) Callus induction from hypocotyl segments. From top to bottom, WT Col, cre1-2, wol, cre1-2 carrying PCRE1::CRE1-HA, cre1-2 carrying PCRE1::AHK2, and wol carrying PCaMV35S::CRE1(T278I). Hypocotyl sections were cultured in the presence of 30 ng/ml of 2,4-dichlorophenoxyacetic acid (an auxin) plus various concentrations of kinetin (a cytokinin).
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5. Figure 4 A Model of the Phosphorelay Network in Arabidopsis
Cytokinin receptor histidine kinases are activated by cytokinins. Likewise, non-cytokinin receptor histidine kinases are presumably activated by other environmental signals. These various signals may initiate phosphorelays that converge on the AHPs, thus integrating responses to cytokinin and non-cytokinin signals. In the absence of cytokinins, CRE1 acts as a phosphatase on the phosphorelay network. In this situation, the phosphoryl group is likely transferred from an AHP to CRE1, then irreversibly to water. Similarly, it is possible that other histidine kinases have phosphatase activity, although this has not been experimentally demonstrated. Thus, the phospho-load of the network may be under positive and negative regulation by cytokinins and possibly additional, unknown signals.
Current Biology  , DOI: ( /j.cub )
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