Unlike animal steroids, which rely on intracellular steroid receptors to directly alter gene activities, plant steroids use transmembrane receptor kinases to initiate a phosphorylation-mediated signaling cascade to convey their signals into the nucleus. Recent studies have begun to unravel the biochemical details of individual steps of the brassinosteroid signal transduction pathway, including ligand binding and receptor dimerization at the cell surface, signal transmission across the cell membrane, the phosphorylation of cellular targets in the cytosol, and gene regulation inside the nucleus.
CHO-IR cells expressing human insulin receptor are grown to 80% confluence in Hamm’s F12 medium with 10% fetal bovine serum and without hypoxanthine. Trypsinized cells are seeded in 6-well plates at 1 × 106 cells/well in 2 ml of medium without fetal bovine serum. After 24 h, medium is replaced with 1 ml of serum-free medium containing GSK-3 inhibitor or control (final DMSO concentration <%) for 30 min at 37°C. Cells are lysed and centrifuged 15 min at 4°C/14000g. The activity ratio of GS is calculated as the GS activity in the absence of glucose-6-phosphate divided by the activity in the presence of 5 mmol/l glucose-6-phosphate, using the filter paper assay of Thomas et al.
Our laboratory has also established a long-term collaboration with Physicists at Dr. Marta Ibañes laboratory (Physics Faculty, Univ. Barcelona). We have combined our expertise to elaborate mathematical models that contribute to understand fundamental plant developmental biology questions (vascular patterning, root growth and telomere dynamics). We pioneered an interdisciplinary study to establish the BR contribution to vascular development in Arabidopsis shoots. Only a few labs in the world have the combined experimental and computational understanding to tackle these questions.