Figure 4: Characteristics of BHP.

(a) Schematic representation of the BHP protein. BHP protein has an ankyrin repeat and a protein kinase domain. Scale bar represents 50 aa. (b) Phylogenetic relationships between BHP and homologous proteins from Arabidopsis thaliana and other plant species. The alignment was performed using the ClustalW program with amino acid sequences from the kinase domain from Arabidopsis thaliana (At), Oryza sativa (Os), Populus trichocarpa (Pt), Physcomitrella patens (Pp), Ricinus communis (Rc), Sorghum bicolor (Sb), Selaginella moellendorffii (Sm), and Vitis vinifera (Vv). The tree was created using MEGA6 software with the neighbor-joining method. The Arabidopsis BHP is shown in bold red letters. The numbers next to the branches are bootstrap values (1,000 replicates). The scale bar represents 0.1 substitutions per site. (c) Subcellular localization of GFP or BHP-GFP protein in guard cells. Fluorescent images from GFP (green) were obtained using a confocal laser microscope from transgenic plants expressing GFP or BHP-GFP under the control of the 35 S promoter. Chlorophyll autofluorescence (red) was obtained and shown. Scale bar represents 10 µm. (d) Expression of BHP determined by RT-PCR analysis. RNA was isolated from leaves, guard cell protoplasts (GCPs), mesophyll cell protoplasts (MCPs), stems, petioles, and roots of 5-week-old WT plants. TUB2 was used as an internal control. (e) Expression pattern of BHP determined using the promoter-GUS assay. The GUS reporter gene was expressed in transgenic plants under the control of the BHP promoter. (i) Whole plant, (ii) cotyledon, (iii) true leaf, (iv) close-up image of true leaf, (v) root, and (vi) primary root tip.