(A) Steady-state levels of AHK5 transcript in different tissues and developmental stages of Arabidopsis detected by semi-quantitative RT-PCR using different cycle numbers. For the detection of AHK5, up to 40 cycles of PCR were performed using primers 3 and 10 (left panel) or primers 8 and 9 and 40 cycles of PCR (right panel). GC, cDNA from guard cell-enriched non-treated leaf sample; GC+H, cDNA from guard cell-enriched leaf samples treated with 0.5 mM H₂O₂ for 2 h; WL, cDNA from whole-leaf sample. ACTIN and EF1 were used as controls. Primer numbers indicated in panel C. (B) Expression of a P_AHK5-GFP-AHK5genomic construct in transiently transformed tobacco leaf cells. Agrobacteria carrying the P_AHK5-GFP-AHK5 construct were infiltrated into the abaxial side of the leaf and the GFP fluorescence analysed by CLSM 2 days later. Top panels = GFP images, lower panels = GFP image overlaying bright field image. The scale bar represents 20 μm. Images shown from repeat experiments. (C) AHK5 gene structure, position of primers used for genomic and RT-PCR and T-DNA insertions in ahk5-1 (Col-0) and ahk5-3 (Ws4). (D) Analysis of AHK5 expression in seedlings of wild type, ahk5-1 and ahk5-3 mutant, P_35S-AHK5 complemented and AHK5 over-expressing plants using the indicated primer pairs (see Supplementary data for sequences).