Barren inflorescence1 functions in organogenesis during vegetative and inflorescence development in maize

Abstract

Maize (Zea mays) has a highly branched inflorescence due to the production of different types of axillary meristems. Characterization of the barren inflorescence class of mutants has led to the discovery of genes required for axillary meristem initiation in the inflorescence. Previous studies showed that barren inflorescence2 (bif2) encodes a serine/threonine protein kinase that regulates auxin transport, and barren stalk1 (ba1) encodes a basic helix-loop-helix transcription factor that acts downstream of auxin transport. Here, we characterize Barren inflorescence1 (Bif1), a classical semidominant mutation of maize. Developmental, histological, and genetic analyses show that Bif1 mutants are defective in the initiation of all axillary meristems in the inflorescence. Real time RT-PCR experiments show that both bif2 and ba1 are expressed at lower levels in Bif1 mutants. Double-mutant analyses demonstrate that Bif1 exhibits an epistatic interaction with ba1 and a synergistic interaction with bif2. The dramatic phenotypic enhancement observed in Bif1; bif2 double mutants implies that bif1 plays an overlapping role with bif2 in the initiation of lateral organs during vegetative development. The phenotypic resemblance of Bif1 to bif2 mutants and the reduction of auxin transport in Bif1 mutants suggest that bif1 functions as a regulator of auxin transport in maize.

Figure 1.—

Mature inflorescence phenotype of the Bif1 mutant. (A) Mature tassels of normal, Bif1/+, and Bif1/Bif1 plants. In the normal tassel, long branches are indicated at the base of the main spike. Spikelet pairs cover the branches and the main spike. In the Bif1 mutants, there are reduced numbers of branches and spikelets in the tassel. (B) Mature ears of normal, Bif1/+, and Bif1/Bif1 plants, showing fewer kernels and disorganized rows in Bif1 mutants. (C) Quantification of tassel branch number. (D) Quantification of tassel spikelet number. (E) Percentage of spikelets that occur singly vs. paired. Bars represent mean value and error bars represent standard error of the mean.

Figure 2.—

Scanning electron microscopy (SEM) images of developing Bif1 inflorescences. (A) Normal tassel, showing files of developing spikelet pair meristems (SPMs) on the flanks of the inflorescence meristem (IM). (B) Bif1/+ tassel, with reduced numbers of SPMs. (C) Bif1/Bif1 tassel with few SPMs. (D) Normal ear, showing organized rows of SPMs. (E) Bif1/Bif1 ear, with a fasciated inflorescence meristem and few SPMs. Bar, 100 μm.

Figure 3.—

Histology and RNA in situ hybridization with kn1 in developing Bif1 tassels. (A–C) Longitudinal sections of 5-week-old tassels stained with TBO, with SPMs visible as areas of intense staining. (A) Three developing SPMs on the flanks of the inflorescence meristem in a normal tassel. (B) Bif1/+, showing a single SPM in the same area as there are three SPMs in normal. (C) Bif1/Bif1 with a slight protrusion on the surface of the rachis but no evidence of developing SPM. (D–F) RNA in situ hybridization with kn1. (D) Meristematic cells and vasculature are indicated by kn1 expression in normal tassels. The absence of kn1 on the flanks of the inflorescence meristem (IM) indicates the formation of the suppressed bract primordia (BR) that subtend SPMs. (E) Bif1/+ inflorescences have fewer areas of kn1 expression on the flanks of the inflorescence. (F) Bif1/Bif1 inflorescence with kn1 expression only in the inflorescence meristem and in the vasculature. Bar, 100 μm.

Figure 4.—

Analysis of Bif1; ra1 double mutants. (A) Mature tassel phenotype showing all genetic classes from a segregating Bif1; ra1 family. (B) Ear phenotype of a segregating Bif1; ra1 family. (C–E) Higher-magnification images showing individual branches from ears. (C) Branch from a ra1/ra1 ear. (D) Branch from a Bif1/+; ra1/ra1 ear. (E) Bif1/Bif1; ra1/ra1 ear. (F–I) Quantitative analysis of Bif1; ra1 double mutants. For all charts, bars represent mean value of the data set, and error bars represent standard error of the mean. (F) Average number of branches per tassel. (G) Average number of spikelets per tassel. (H) Average number of spikelets per branch, measured on a branch at the base of the tassel. (I) Average number of spikelets in the top 2 cm of the tassel main spike.

Figure 5.—

Quantification of floret and floral organ numbers in Bif1 mutants. (A) Percentage of spikelets containing two, one, or zero florets per spikelet. (B) Quantification of lemma and palea number per floret. (C) Percentage of florets containing the indicated number of stamens per floret.

Figure 6.—

Real-time RT–PCR analysis of the expression of bif2 and ba1 in Bif1 mutants. (A) Expression level of bif2 in the immature tassel of Bif1 mutants relative to normal siblings. (B) Expression level of bif2 in the immature ears of Bif1 mutants relative to normal siblings. (C) Expression level of ba1 in the immature tassel of Bif1 mutants relative to normal siblings. (D) Expression level of ba1 in the immature ears of Bif1 mutants relative to normal siblings. Mean plus or minus SE is shown for one representative experiment using three biological and three technical replicates for each sample.

Figure 7.—

Analysis of Bif1; bif2 double mutants. (A) Mature tassel phenotype of a Bif1; bif2 segregating family. (B and C) Quantification of tassel characteristics in a Bif1; bif2 segregating family. (B) Average tassel branch number. (C) Average spikelet number per tassel. (D) Vegetative phenotype of a Bif1; bif2 family showing reduced plant height in Bif1; bif2 double mutants. (E and F) Quantification of vegetative phenotypes. (E) Average plant height in centimeters. (F) Average leaf number.

Figure 8.—

Analysis of Bif1; ba1 double mutants. (A) Mature tassel phenotype of Bif1; ba1 family. (B) Average number of spikelets per tassel. (C) Average number of ears per plant.

Figure 9.—

Measurement of auxin transport in normal and Bif1 inflorescences. Dark shading, without NPA; light shading, with NPA. (A) Measurement of basipetal (lanes 1 and 2) and acropetal (lanes 3 and 4) transport in normal ears. (B) Measurement of basipetal transport in immature ears of a family segregating for Bif1. Lanes 1 and 2, normal siblings; lanes 3 and 4, Bif1/+; lanes 5 and 6, Bif1/Bif1.