Figure 7.

Colocalization of Active MKKs with Mitochondria and Correlation of Subcellular Localization with Activity in Stomatal Assays.

(A) Confocal image of a mature guard cell expressing CAMKK7-YFP (green), which localizes to punctate structures.

(B) and (C) Confocal image of mature guard cells expressing WTMKK7-YFP (B) or a catalytically inactive (KI)-CAMKK7 (C). These MKK7 variants also localize to punctate structures.

(D) Confocal image of a mature guard cell expressing CAMKK7∆D. This MKK7 variant localizes to larger structures not consistent with mitochondria.

(E) and (F) Confocal images of a pair of guard cells expressing CAMKK7-YFP that have been treated with MitoTracker Orange. The punctuate structures described in (A) and shown in (E) stain positively with MitoTracker Orange (F).

(G) Overlay of MitoTracker Orange and YFP signals described in (E) and (F). Arrow indicates enlargement of the region measured (yellow) to show colocalization of fluorescent signal. Merging of signals suggests CAMKK7 colocalizes with mitochondria. Bar = 15 μm.

(H) Graph depicting fluorescence signals measured along yellow line (G) for YFP (E), MitoTracker (F), and overlaid signals (G). The pattern of fluorescence intensities suggests that CAMKK7-YFP and MitoTracker Orange signals colocalize. Note that CAMKK7∆D in (D) colocalizes with large cytosolic structures and not mitochondria-like structures. The CAMKK7∆D construct fails to induce CAMKK7-like stomatal clusters. All images were collected at 400× magnification and are of 5-DPG abaxial cotyledons.