There is evidence for a feedback regulation pathway from active BR on the expression of the BR biosynthetic enzymes (Bancos et al. 2002). In Arabidopsis, expression of the mRNAs of CYP90B1, CYP90C1, CYP90D1, CYP85A1 and CYP95A2 is increased in the BR-deficient mutant cpd (blocked at the
CYP90A1-catalysed step of the BR biosynthesis pathway), and CYP90A1 expression is increased in the cbb3 mutant (cabbage3, an EMS mutant in CYP90A which does not affect CYP90A mRNA expression). In all cases addition of BR dramatically reduces the expression of the mRNA of all six genes. Similarly, addition of BR to wild-type plants also reduces the expression of the mRNAs of these six genes. The same regulatory controls also seem to be acting on the expression of the rice CYP90D2 and CYP724A1 genes, as both are down-regulated following BR application. This feedback regulation presumably maintains BR homeostasis by reducing synthesis after BR concentrations have increased. Expression of the mRNA of the BR catabolising P450, CYP72B1, is up-regulated in response to applied BR (Goda et al. 2002) suggesting induction to catabolise increased BR and maintain homeostasis. The mRNAs of enzymes prior to the P450-mediated steps do not appear to be down-regulated by BR (Goda et al. 2002). The data to date suggest a concerted regulation of the expression of all the P450 enzymes involved in BR biosynthesis and catabolism rather than a single controlling step.
The feedback regulation is probably mediated via the BR perception and signal transduction mechanism. A plasma membrane leucine-rich repeat receptor protein kinase, BRI1, is an important part of the BR receptor. Ara-bidopsis bril (brassinosteroid insensitive 1) mutants accumulate high levels of castasterone and brassinolide. The expression of BR biosynthetic P450 mRNAs are elevated in bril mutants but not down-regulated by application of BR. The accumulation of BRs and the loss of feedback control in the bril mutant provides strong evidence that perception of BR via BRI1 is crucial in the regulation of BR biosynthesis. The repression of CYP90A1 by BR application also requires protein synthesis (Mathur et al. 1998), perhaps of a signal transduction component.
One of the biological roles of BR is in the regulation of photomorphogenic pathways. Mutants deficient in BR biosynthesis often exhibit photomorpho-genesis when grown in the dark, including opening of the apical hook, cotyledon expansion, suppression of hypocotyl elongation and leaf development. One of the suggested roles of BRs is in the dark-grown development (skoto-morphogenic) and light-grown development (photomorphogenic) pathways. The evidence for this comes from BR-deficient mutants such as det2 (dee-tiolated2, blocked in a sterol biosynthesis step) and cpd where constitutive photomorphogenesis is observed in dark-grown seedlings. BR appears to be needed for the elongation of the hypocotyl in both the light and the dark. CYP72B1 (the BR-inactivating P450) transcript is down-regulated compared to the dark expression level by white, blue and red light but not by far-red light, suggesting that this enzyme may be used to alter BR levels in response to light signals. The reduction of BR concentrations in response to light leading to de-etiolation is consistent with the phenotypes of the BR-deficient det2 and cpd mutants, but BR is also required for photomorphogenesis as evidenced by the severe dwarf phenotypes of BR-deficient mutants. This re sponse is complex and probably involves changes in BR sensitivity as well as changes in BR concentrations (Turk et al. 2003).
Other evidence linking the regulation of the ER-located P450s in the BR pathway with light signalling comes from studies of the Pra2 protein in pea (Kang et al. 2001). Pra2 is a small G protein that is expressed in the dark and is down-regulated by light. A yeast two-hybrid screen with Pra2 as bait identified CYP92A6 as an interacting protein; both proteins localise to the ER as GFP or RFP fusion proteins in onion epidermal cells. Using activity assays of CYP92A6 with testosterone as substrate it was shown that Pra2 binding activates CYP92A6. This suggests a model where light signals regulate BR accumulation via the direct action of Pra2 on CYP92A6 in the ER. The P450s provide the major controls on the BR biosynthesis pathway, with a large body of evidence for elaborate transcriptional control emerging. The evidence from Pra2 also suggests that more direct controls are operating on the BR biosynthetic P450s in the ER in response to environmental signals.
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