Figure 5.

Cytokinin Regulates VB Formation via AHK2 and AHK3 Phosphorelay.

(A) Transverse sections of the inflorescence stems of wild-type (Columbia-0 [Col-0]) ([a], [f], and [k]), ahk2 ([b], [g], and [l]), ahk3 ([c], [h], and [m]), ahk2 and ahk3 ([d], [i], and [n]), and Pro35S:CKX ([e], [j], and [o]) lines. Note the reduction of procambial layers in ahk2 and ahk3 plants and particularly in ahk2 ahk3 double mutants. The overall reduction in VB size is apparent in ahk2 ahk3 and in Pro35S:CKX lines, suggesting positive regulation of cytokinin signaling via the AHK2/AHK3 pathway in VB development in Arabidopsis inflorescence stems. Staining of handmade sections ([a] to [j]) with toluidine blue and thin sections made from fixed and embedded material ([k] to [o]). c, cambium; ic, interfascicular cambium; pc, procambium; mx, metaxylem; p, phloem. Bars = 100 μm in (a) to (e) and (k) to (o) and 50 μm in (f) to (j).

(B) The dwarfism resulting from deletion of ahk2 ahk3 is rescued in the presence of CKI1. Three-week-old wild-type (Col-0) and transgenic plants expressing Pro35S:CKI1-HA or Pro35S:CKI1^H405Q-HA in the ahk2 ahk3 background were used for phenotypic analysis.

(C) Expression analysis of HA-tagged CKI1 and CKI1^H405Q proteins under the control of the Pro35S promoter in transgenic lines. Total proteins from 3-week-old plants of each designated line were subjected to 7.5% SDS-PAGE. Actin proteins detected by immunoblotting serve as input controls.

(D) and (E) Ectopic expression of CKI1 rescues the abnormal vasculature of the ahk2 ahk3 mutant. Microscopy images of transverse sections of the inflorescence stems of wild-type (a), ahk2 ahk3 (b), Pro35S:CKI1-HA/ ahk2 ahk3 (c), and Pro35S:CKI1^H405Q-HA/ ahk2 ahk3 (d) plants. The arrow indicates extensively reduced VB. Bars = 100 μm.