TCP transcription factor, BRANCH ANGLE DEFECTIVE 1 (BAD1), is required for normal tassel branch angle formation in maize

Abstract

In grass inflorescences, a structure called the "pulvinus" is found between the inflorescence main stem and lateral branches. The size of the pulvinus affects the angle of the lateral branches that emerge from the main axis and therefore has a large impact on inflorescence architecture. Through EMS mutagenesis we have identified three complementation groups of recessive mutants in maize having defects in pulvinus formation. All mutants showed extremely acute tassel branch angles accompanied by a significant reduction in the size of the pulvinus compared with normal plants. Two of the complementation groups correspond to mutations in the previously identified genes, RAMOSA2 (RA2) and LIGULELESS1 (LG1). Mutants corresponding to a third group were cloned using mapped-based approaches and found to encode a new member of the plant-specific TCP (TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR) family of DNA-binding proteins, BRANCH ANGLE DEFECTIVE 1 (BAD1). BAD1 is expressed in the developing pulvinus as well as in other developing tissues, including the tassels and juvenile leaves. Both molecular and genetics studies show that RA2 is upstream of BAD1, whereas LG1 may function in a separate pathway. Our findings demonstrate that BAD1 is a TCP class II gene that functions to promote cell proliferation in a lateral organ, the pulvinus, and influences inflorescence architecture by impacting the angle of lateral branch emergence.

Fig. 1.

Phenotypic characteristics of the tassel angle mutant bad1. (A) Normal tassel. (B) Tassel lateral branch angle mutant bad1-2. (C) Analysis of first and second lateral branch angle in mutants compared with normal tassels. (D) Photo of the junction between the main spike and the first lateral branch in normal (D) and bad1-2 (E) tassels. The arc in D delimits the pulvinus. (F) Transverse sections through the first basal branch of normal plants and (G) bad1-2 mutant stained with 1% aqueous safranin O and 0.5% fast green in 95% ethanol. Hand section of fresh pulvinus tissues through first basal lateral branch of a normal plant (H) and a bad1-1 mutant (I) stained with 18% (wt/vol) phloroglucinol in HCl. Black arrows in F–I denote tissue composing the pulvinus.

Fig. 2.

Positional cloning of BAD1 and phylogenetic tree. (A) Syntenous physical regions (not to scale) of sorghum chromosome 2 (Top), maize chromosome 2 (Middle), and rice chromosome 9 (Bottom). Positions of conserved markers/genes are indicated. (B) Graphic representation of the BAD1 gene and the bad1 mutant alleles. The arrowheads indicate the mutation (threonine-to-methionine) in bad1-1 and in bad1-2 (serine-to-phenylalanine). Red indicates wild-type sequence and green indicates mutant sequence. BAD1 protein features include a predicted TCP conserved domain. (C) ClustalW alignment of TCP domains from BAD1 and TB1 from maize; OsTB1 and REP1/OsTCP24 from rice; CYC4 and DICH from Antirrhinum; and BRC1/AtTCP18, BRC2/AtTCP12, and AtTCP1 proteins from Arabidopsis. Amino acids in a gray background indicate conserved amino acids in the TCP family; amino acids in red indicate conserved residues in proteins belonging to the same TB1/CYC clade; amino acids in yellow indicate the conserved residues in BAD1, TB1, REP1/OsTCP24, OsTB1, BRC1/AtTCP18, and BRC2/AtTCP12; and amino acids in green indicate the conserved residues in CYC4, DICH, and AtTCP1.

Fig. 3.

Expression profile of BAD1 RNA and protein in inbred A619. (A) Expression of BAD1 in different maize tissues by RT-PCR. (B–H) RNA in situ hybridization targeting the 3′ UTR of BAD1 (dark brown) in developing inflorescences including a longitudinal section (B) and a transverse section (C) through a 0.5-cm developing tassel and longitudinal (E and F) and (G) transverse sections through a developing ear. Sense control in a developing tassel (D) and ear (H). (I–L) Immunolocalization in longitudinal section through the first lateral branch of 5-wk-old tassel to 7-wk-old tassels. (I) Immunolocalization of BAD1 in 5-wk-old tassel before pulvinus initiation. (J) BAD1 in the pulvinus of 7-wk-old tassels, when the pulvinus has begun to initiate development. (K) Negative control with empty secondary antibody. (L) Positive control labeled with actin antibody. The Lower grayscale images of I–L correspond to the Upper fluorescence images. White arrows in J highlight the localized expression within the developing pulvinus at 7 wk. AM: axillary meristem; LT: leaf tip; IM: inflorescence meristem; SPM: spikelet pair meristem; SM: spikelet meristem.

Fig. 4.

Interaction among BAD1, RA2, and LG1 and the model for pulvinus development. (A) BAD1 expression in the developing tassel of A619 inbred lines, lg1 mutant, and ra2 mutant by qRT-PCR. Values for the y axis are arbitrary units of expression level relative to actin. Error bars indicate SD of at least three replicates. (B) Western blot of BAD1 in developing tassels and ears of A619 inbred lines, lg1 mutants in an unknown background, and ra2 mutants in A619 background. Tassel phenotype in A619 tassel (C), bad1 mutant in A619 background (D), ra2 mutant in A619 background (E), and bad1/bad1;ra2/ra2 double mutant in A619 background (F). Quantification of the number of lateral branches (G) and first (H) and second (I) lateral branch angle from base of tassel in A619 tassel, bad1 mutant, ra2 mutant, and bad1/bad1;ra2/ra2 double mutant. (J) Model showing how the size of the pulvinus influences the lateral branch angle in the tassel. When the pulvinus is large, the lateral branch angle is large, and when the pulvinus is missing or reduced in size, the lateral branch angle is acute, which results in an upright tassel branch phenotype. The relationship between BAD1 and RA2 is indicated. The question marks indicate the uncertainty of placement of LG1, REL2, and BAF1 that are also known to influence tassel branch angle.