| Brassinosteroids (BR) are a newly recognized class of phytohormones that are structurally very similar to the steroid hormones found in animals and insects. While a large number of physiological studies pointed to the importance of BRs, the analysis of BR-related mutants in Arabidopsis and pea provided convincing genetic evidence for an essential role of these steroids in plant growth and development. BR-mutants are of two types: BR-deficient mutants which are rescued by exogenous application of BR and are mainly due to lesions in genes involved in the BR biosynthetic pathway; and BR-insensitive mutants which fail to perceive exogenous BRs and are likely due to aberrations in genes involved in a signal-transduction pathway.;We found that a dwarf tomato (Lycopersicon esculentum Mill.) mutant, dumpy (dpy), was BR-deficient and was also rescued by application of all C-23-hydroxylated, 6-deoxo intermediates of brassinolide biosynthesis, as well as castasterone and brassinolide. However, 6-deoxocathasterone and all other upstream pathway precursors failed to rescue the mutant, suggesting that the dpy mutation is affected in the conversion of 6-deoxocathasterone to 6-deoxoteasterone, similar to the Arabidopsis constitutive photomorphogenesis and dwarfism (cpd) mutant. Measurements of endogenous levels of BR intermediates were consistent with this hypothesis. However, transformation of dpy with Arabidopsis CPD did not complement the mutation. Western analysis also showed elevated levels of CPD protein in dpy leaves compared to wild-type leaves. These observations suggest that there might be another gene product involved in the conversion of 6-deoxocathasterone to 6-deoxoteasterone.;Another approach to understanding the mode of action of BR is to isolate BR-regulated genes and determine the mechanism of gene regulation by BR. We exploited the dpy mutant to isolate BR-regulated genes by subtractive hybridization since the expression levels of these genes in the untreated mutant is likely to be low due to BR-deficiency. In our studies we focused on isolation of early responsive genes (within 4 h after BR-treatment) and used poly A+ RNA from BR-treated or untreated meristem and young expanding leaf tissues for subtraction. We report here the isolation and characterization of two BR-regulated genes LeBR1, a novel xyloglucan endotransglycosylase (XET) and LeBR2, a novel serine/threonine kinase. Further expression analysis of LeBR1 demonstrated that the mRNA levels of this gene are high in BR-treated samples at all the time-points tested (2--24 h). The endogenous levels of this gene were also found to be lower in dpy when compared to the wildtype. Analysis of LeBR2 also showed that mRNA levels of this gene are reduced in the dpy background when compared to the wildtype plants. Further analysis of endogenous levels of LeBR2 mRNA in different wild-type tissues showed that both young and mature leaves had high expression levels, but that some LeBR2 expression was present in all the tissue types examined. Computer (PSORT) analysis of the LeBR2 sequence suggests that the protein is likely localized to the inner mitochondrial membrane or transported into this organelle, as it contains a possible intra-mitochondrial signal and a putative transmembrane domain. |
