Functional Analysis Of AtBRX In Regulation Of Cytokinin-mediated Inhibition Of Lateral Root Initiation

Plant hormone cytokinin (CTK) has been implicated in many central developmental processes of plant growth. The high exogenous CTK inhibit primary root elongation and lateral root development. We isolated an Arabidopsis mutant insensitive to exogenous CTK in term of lateral root growth. Map-based cloning results showed that the gene, BRX, was point-mutated; therefore the mutant is named as brx-2. Physiological and molecular characterizations of brx-2 mutant give the following conclusions:1. The length of primary root of brx-2 is shorter and less sensitive to CTK inducedinhibition on lateral root growth compared with the wild type. By analyzing transgenic wild type and brx-2 mutant harboring CYCB1;1::GUS, we found that cell division activity of primary root meristems in brx-2 seedings was distinctly weaker than in that of WT plants in all CTK conditions. No GUS staining was detected in and around the pericycle cells of the BA-treated WT seedlings compared with the control. However, CYCB1;1::GUS staining in the pericycle cells in the BA-treated brx-2 seedlings was almost as intense as that in BA-free medium.2. Map base cloning showed that the mutantion was caused by a point mutantion of the BRX gene, encoding a protein of unknown biochemical function. Complemention results affirmed the conclusion.3. Embryonic brassinolide treatment can fully rescue the CTK-induced inhibition onLR growth phenotype of brx-2, while post-embryonic brassinolide treatment can not. These results indicate that CTK-mediated inhibition of LR initiation is not directly dependent on brassinosteroid level. It seems that the phenotype of brx-2 in presence of CTK may be indirectly caused by the aberrant primary root growth resulting from the embryonic brassinosteroid shortage.4. The brx-2 mutant showed insensitivity to CTK-induced inhibition on lateral root initiation, but overall classic CTK responses such as chlorophyll retention under senescence, and anthocyanin inducted by CTK were not changed. The expression of CTK signaling responsive genes are also normal. These results suggest that the overall upstream two-component systems of CTK signal pathway is not impaired in the brx-2 mutant.5. Treated with CTK, the wild type showed a loss of the first anticlinal division and local auxin response in pericycle cells. On the contrary, local GUS staining accumulated in the pericycle cells before the first short initial cells are formed by anticlinal division in brx-2 treated with CTK. These results indicate that CTK-induced arrest of LR formation is due to loss of local auxin response in the presumptive founder cells.Taken together, the decreased brassinosteroid level caused by loss function ofBRX is vital to primary root growth and consequently influence the local auxinresponse in pericycle cells and show less sensitive to CTK induced inhibition onlateral root growth.