Identification of barley DWARF gene involved in brassinosteroid synthesis

Abstract

Brassinosteroids (BR) are plant steroid hormones regulating various aspects of morphogenesis, such as seed development and germination, cell division and elongation, differentiation of tracheary elements, development during growth in darkness (skotomorphogenesis), photosynthesis and response to environmental stress. Brassinosteroid synthesis has been studied to a great extent in the dicot species, Arabidopsis thaliana, resulting in the identification of genes participating in this process. Much less is known about BR synthesis in crops, including the monocots. The purpose of this study was to identify and characterize barley coding sequence HvDWARF involved in brassinosteroid synthesis. This sequence, encoding brassinosteroid-6-oxidase, was identified on the basis of barley ESTs. This sequence was screened for nucleotide substitutions in semi-dwarf, chemically-induced barley mutants exhibiting changes in etiolation. The responsiveness of these genotypes to exogenous brassinosteroids was determined with the use of leaf-blade segment unrolling tests. The semi-dwarf phenotype of the BR-deficient mutants was rescued by the application of 10−5 M 24-epi-brassinolide. Two missense mutations were identified within the HvDWARF sequence in BR-deficient mutants 522DK and 527DK from variety ‘Delisa’. These substitutions cause changes of amino acid residues located within the conserved fragments of the encoded polypeptide. The transcription profile of HvDWARF and HvBAK1/SERK3, involved in BR signaling, was determined during the early stages of seedling development in BR-deficient and BR-insensitive mutants using real-time quantitative PCR. This analysis indicated that HvDWARF displays a uniformly low level of this process, whereas the transcription level of HvBAK1 proved to be spatially and temporally regulated.