Brassinazole Resistant 1 Activity Is Organ-Specific and Genotype-Dependent in Barley Seedlings

Abstract

Brassinosteroids (BRs) control many plant developmental processes by regulating different groups of transcription factors, and consequently gene expressions. The most known is BZR1, the main member of the BES1 family. However, to date, it is poorly characterized in crop species. The main goal of the presented study was to identify HvBZR1 and determine its activity in 5-day-old barley (the stage is related to one leaf on the main shoot and a few seminal roots) using two cultivars with different sensitivities to BRs. Using the anti-OsBZR1 antibody, we identified the forms of HvBZR1 transcription factor with different molecular weights, which can be related to different phosphorylated forms of serine/threonine residues. Two phosphorylated forms in the shoots and one dephosphorylated form in the roots were determined. A minor amount of the dephosphorylated form of the HvBZR1 in the Haruna Nijo shoots was also found. The phosphorylated forms gave a higher band intensity for Golden Promise than Haruna Nijo. The bands were similar in their intensity, when two different phosphorylated forms were compared in Golden Promise, while a reduced intensity was detected for the phosphorylated form with a lower molecular weight for Haruna Nijo. Degradation of the phosphorylated forms in the shoots (complete degradation in Golden Promise and significant but not complete in Haruna Nijo) and the presence of the dephosphorylated form in the roots were proven for the etiolated barley. In the case of Haruna Nijo, a wider range of the regulators of the BR biosynthesis and signaling pathways induced the expected effects, 24-EBL (0.001 µM) and bikinin (10 and 50 µM) caused low amount of the phosphorylated forms, and at the same time, a tiny band of dephosphorylated form was detected. However, the expression of genes related to the BR biosynthesis and signaling pathways was not a determinant for the protein amount.

Keywords: 24-epibrassinolide; BZR1; GSK3; Haruna Nijo; bikinin; brassinazole; glycogen synthase kinase 3; golden promise; root; shoot.

Figure 1

Phenotypic and molecular traits of 5-day-old shoots and roots measured for the Golden Promise and Haruna Nijo cultivars. (a) Representative example of plant phenotype in four biological replicates, scale bar = 10 cm; (b) immunodetection of HvBZR1 and HvGSK2.1, as well as a membrane staining of protein bands with a molecular weight in range from 25 to 35 kDa using the Ponceau S; * indicates a nonspecific product detected by the anti-OsBZR1 antibody; pBZR1 indicates the phosphorylated HvBZR1 forms; BZR1 indicates the dephosphorylated HvBZR1 form; (c–n) parameters of shoot growth (c–f), root growth (g–j), and shoot-to-root ratio (k–n); (o) immunodetection of HvGSK2.1 kinase and α-tubulin as a control; (p,q) expression profile of the genes related to the BR biosynthesis and signaling pathways. The results present the mean with a standard error of the mean (phenotypic traits n = 6, (c–n); expression profile n = 3, (p,q). The asterisks indicate significant differences, revealed by the Student’s t-test for p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 2

Phenotypic and molecular changes in 5-day-old shoots and roots of the Golden Promise and Haruna Nijo plants treated with 24-EBL (0.001 and 1 µM), bikinin (10 and 50 µM), and Brz (10 and 50 µM). EtOH (0.7%) was used as a control of solvent solutions for 24-EBL, while DMSO (0.7%) was the control of solvent solutions for BK and Brz. Mock represents the plants treated with the respective solvent solutions, i.e., EtOH and DMSO assumed as 1.00. (a) A phenotype of plant photography represents three biological replicates for each treatment, scale bar = 10 cm; (b) immunodetection of HvBZR1, the Roti-Blue presents proteins after staining as a loading control; pBZR1 indicates the phosphorylated forms of the HvBZR1; BZR1 indicates the dephosphorylated form of the HvBZR1; (c–f) growth parameters of shoots, (g–j) roots, and (k–n) shoot-to-root ratio. The results present the mean with a standard error (n = 6). The asterisks indicate significant differences, revealed by the Student’s t-test for p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).

Figure 3

Expression profiles of the genes encoding enzymes of BR biosynthesis and signaling pathways in two barley cultivars Golden Promise and Haruna Nijo. Shoots and roots were treated with 24-EBL (0.001 and 1 µM), bikinin (10 and 50 µM), and Brz (10 and 50 µM), mock represents the plants treated with the respective solvent solutions, i.e., EtOH and DMSO assumed as 1.00. The results present the mean with a standard error (n = 3). The asterisks indicate significant differences, revealed by the Student’s t-test for p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***).