Investigation of Gene Expression, Growth Kinetics, and Wall Extensibility during Brassinosteroid-Regulated Stem Elongation

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Abstract
Brassinosteroids promote stem elongation in a variety of plants but little is known about the mechanism of action of these plant growth regulators. We investigated a number of physiological and molecular parameters associated with brassinosteroid-enhanced elongation. Continuous growth recordings of soybean (Glycine max L. cv Williams 82) epicotyls showed that there was a 45-min lag before 0.1 [mu]M brassinolide (BR) exerted a detectable effect on elongation. BR caused a marked increase in Instron-measured plastic extensibility, suggesting that BR may promote elongation in part by altering mechanical properties of the cell wall (wall loosening). Structure-function studies suggested that the dimensions of the brassinosteroid side chain were critical for promotion of elongation and expression of BRU1, a gene regulated specifically by active brassinosteroids. Auxin-BR interactions were examined by using small auxin up RNA (SAUR) gene probes and the auxin-insensitive diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.). We have shown that in wild-type tomato, which elongates in response to exogenous auxin, a transcript of identical size to the soybean SAUR 15A is strongly induced within 1 h by 50 [mu]M 2,4-dichlorophenoxyacetic acid or indoleacetic acid, whereas in the dgt mutant, which does not elongate in response to auxin, no transcript is expressed. Furthermore, BR promotes equal elongation of hypocotyls in both wild-type and dgt tomatoes but does not rapidly induce the SAUR 15A homolog in either genotype. BR does not cause rapid induction of SAUR 6B in elongating soybean epicotyls but does lead to increased expression after 18 h. This late BR activation of SAUR 6B is controlled, at least in part, at the transcriptional level and is not accompanied by an increase of free indoleacetic acid in the tissue. We conclude that although both BR and auxin affect wall relaxation processes, BR-promoted elongation in soybean and tomato stems acts via a mechanism that most likely does not proceed through the auxin signal transduction pathway.