A Comparison of a gross morphology of a severe dwarf mutant brd1-m1 (on the right) and a wild type maize plant (on the left). B Physical location of the mapped gene relative to the molecular markers used for mapping. M1 through M8 are markers tightly linked to the gene (). Approximate genetic map distances between the outside markers and the mutation are shown. The star designates the location of the mutation in brd1-m1 mapped between markers M4 and M5. Each line in the lower panel designates a particular recombinant event, where Mo17 alleles are shown in blue and B73 alleles are shown in red. C Predicted genes in the region between markers M4 and M5. The star indicates the location of the only mutation found in the coding regions of these predicted genes. D The predicted protein sequence encoded by maize brd1 (GRMZM2G103773) gene. A nonsense mutation found in brd1-m1 leads to the synthesis of a truncated protein of only 165 amino acids long. A cyp450 domain is shown in gray.

A Alignment of a portion of maize BRD1 with brC-6 oxidases from other plants. B Phylogenetic relationships between a maize BRD1protein and other P450 proteins in maize and other plants. An unrooted tree was constructed using the neighbor-joining method. Accession numbers are as follows: rice brd1 (AB084385), OsDwarf4 (Q5CCK3), At Dwarf (AB035868), AtBRC-6 ox2 (NP_566852), Tomato Dwarf (U54770), At CPD (X87367), At Rot3 (AB008097), At Dwarf4 (AF044216), ZmDWF1 (AAS90832), ZmDwarf3 (U32579), barley dwarf3 (AF326277), At KAO1 (AF318500), At KAO2 (AF318501), ZmDWF4 (GRMZM2G065635), CPD Vigna (AF279252).

Plants were germinated and grown in the darkness on MS medium with or without 10⁻⁶ M brassinolide. At 12 days after planting the seedlings were genotyped and wild type (+/+) and mutant (−/−) plants were identified. A brd1-m1 plants grown on media supplemented with brassinolide show epicotyl elongation, while mutant plants grown without brassinolide fail to show etiolation response. Arrows indicate the positions of the internodes. B Epicotyl length was measured in 12 day old seedlings germinated and grown in the darkness. The results are presented as mean values +/− standard deviation from four to eight plants. All of the groups exhibit statistically significant differences at p<0.01 or less (t-test). Number of plants in each category is shown above each of the columns. C qRT-PCR analysis of the brd1 expression level. The expression level of brd1 was normalized to the expression of the house-keeping mez2 gene and shown relative to the expression of the wild type plants grown without brassinolide. No RT controls were all negative (data not shown). The difference between the brd1 expression in mutant plants grown without brassinolide supplement and all other growth conditions is statistically significant at p<0.001 (t-test). The data are presented as mean values +/− standard deviation from three samples for each growth condition.

A Relative expression levels of maize brd1 based on qRT-PCR analysis. cDNA was synthesized from total RNA isolated from 14 days after pollination embryo (Emb) and endosperm (endo), immature ear (Imm ear), leaf (14 day old seedling), shoot apical meristem-enriched tissue (SAM), and root (14 day old seedling) tissue. The expression level of brd1 was normalized to the expression of the house-keeping mez2 gene and shown relative to the expression of the wild type plants grown without brassinolide. Bars represent standard deviation values. B Expression levels of Zmbrd1 in various plant tissues based on Maize Expression Atlas .

A Gross morphology of brd1-m1 mutant plants. B brd1-m1 mutants exhibit severe leaf structure abnormalities. An adult leaf from the mutant plant (bottom) is compared to the adult leaf of the wild type plant (top). C Variability in morphology of brd1-m1 mutant plants. D brd1-m1 mutants develop feminized tassels. E–F brd1-m1 mutants (F) exhibit abnormalities in auricle/ligular region of the leaf compared to the wild type plants (E). Triangle arrows indicate the border between an auricle and a blade. Adult leaves of the brd1-m1 mutant plants lack a border between an auricle and a blade (F). G–H Root growth in brd1-m1 mutants (G) is significantly impaired with almost no lateral root growth present. Three brd1-m1 mutant plants (G) are compared to two wild type seedlings (H). Seedlings segregating for brd1-m1 were germinated and grown in the darkness for 14 days. Scale bars on all of the pictures are 5 cm.

A–F Median longitudinal histological sections of juvenile (14-day-old) wild-type B73 (A, C, E) and brd1-m1 (B, D, F) plants. A–B Shoot apical meristem. C–F Cells in a stem underneath shoot apical meristem. Nodes separating divisional zones are designated by arrowheads in C. E–F Close-up of cell files in divisional zones shown in C–D. G–H Transverse sections of wild-type (G) and brd1-m1 (H) plants above meristem showing leaf growth. I–J Close-up of wild-type (I) and brd1-m1 (J) longitudinal sections of juvenile leaves. K–L Adult longitudinal sheath sections in wild-type (K) and brd1-m1 (L). M–N Adaxial leaf blade epidermal surface of wild-type (M) and brd1-m1 (N). Scale bars are 100 um.