Functional characterization of brassinosteroid mutants in maize

Brassinosteroids (BRs) are plant hormones that share structural similarities with animal steroids, which are decisive factors of sex determination. BRs are known to regulate morphogenesis and environmental stress responses, but their involvement in sex determination has been speculative. To date, no BR mutants have been characterized in plants with unisexual flowers, which represent a critical gap in our understanding of BR function. To elucidate the function of BRs in the monoecious maize, an approach, designated ICOMPASS (Integrated Characterization Of Mutants by Pharmacologically Assisted Selection Screening) was developed. The success of ICOMPASS does not solely depend on the hypothesis of functional conservation between species. ICOMPASS combines pharmacological studies, designed to define a potential mutant phenotype in a species of interest, with a multifunctional strategy to allow targeted and effective integration of database analysis and physiological experimental results. ICOMPASS was used to isolate twenty-one BR candidate mutants with a concomitant drastic reduction of false positives. Functional characterization of two candidate lines revealed that in maize BRs have both conserved and novel functions. Cloning and characterization of nana plant1 (na1), a classical dwarf mutant which also has feminized male flowers, revealed that BRs control sex determination in maize. na1 carries a loss-of-function mutation in a DET2 homolog – a gene of the BR biosynthesis. The mutant accumulates the DET2 substrate (24R)-24-methylcholest-4-en-3-one with a concomitant decrease of downstream BR metabolites. Treatment of wild-type maize with BR biosynthesis inhibitors mimicked both dwarf and tasselseed phenotypes of na1. Tissue-specific na1 expression in developing anthers also indicates the promotion of masculinity in male inflorescences by BRs. Characterization of a second BR gene candidate, designated brachytic crinkled leaf1 (bcr1) indicated that BRs, in addition to height and sex determination, control leaf angle and leaf morphology in maize. The most striking features of bcr1 mutants include semi-dwarf stature and increased leaf inclination angle. Cloning efforts identified a candidate gene, Bcr1-c1, linked to the bcr1 phenotype. Synteny mapping revealed the rice ortholog of the Bcr1-c1; the GRAS transcription factor Dwarf and Low-Tillering ( Dlt), whose mutant shares striking similarities to the bcr1 phenotype. Phylogenetic analyses indicated that BCR1-C1 and its monocot orthologs shared highly conserved N- and C-termini (GRAS domain)