Unraveling the Molecular Mechanisms of Maize brown midrib1 (bm1) Mutation

Brown midrib1 (bm1) maize plants offer significant advantages when used in silage and biofuel applications because they have reduced lignin contents.  Maize Cinnamyl Alcohol Dehydrogenase 2 (CAD2) has been implicated as the underlying gene for bm1phenotypes but has not been conclusively proved.  We have cloned cad2 alleles from a Dow AgroSciences (DAS) bm1 (bm1-das1) and the public reference bm1 (bm1-ref) mutants and showed that they contain frame-shift mutations.  In bm1-das1, the frame shift is caused by a 3444-bp transposon insertion in the first intron and chimeric RNA splicing. In bm1-ref, it is the result of a two-nucleotide (AC) insertion in the third exon.  These mutations are predicted to produce truncated, non-functional CAD2 proteins of 48 amino acids (AA) for bm1-das1 and 147 AA for bm1-ref, compared to 367 AA for the wild type (WT).  Consistently, significant reductions in cad2 RNA expression and in total lignin contents are observed in these mutants.  High throughput molecular assays capable of differentiating mutant and WT alleles have been developed and used to demonstrate that these mutations in CAD2 are the molecular bases for bm1 phenotypes.