Involvement of the MADS-box gene ZMM4 in floral induction and inflorescence development in maize

Abstract

The switch from vegetative to reproductive growth is marked by the termination of vegetative development and the adoption of floral identity by the shoot apical meristem (SAM). This process is called the floral transition. To elucidate the molecular determinants involved in this process, we performed genome-wide RNA expression profiling on maize (Zea mays) shoot apices at vegetative and early reproductive stages using massively parallel signature sequencing technology. Profiling revealed significant up-regulation of two maize MADS-box (ZMM) genes, ZMM4 and ZMM15, after the floral transition. ZMM4 and ZMM15 map to duplicated regions on chromosomes 1 and 5 and are linked to neighboring MADS-box genes ZMM24 and ZMM31, respectively. This gene order is syntenic with the vernalization1 locus responsible for floral induction in winter wheat (Triticum monococcum) and similar loci in other cereals. Analyses of temporal and spatial expression patterns indicated that the duplicated pairs ZMM4-ZMM24 and ZMM15-ZMM31 are coordinately activated after the floral transition in early developing inflorescences. More detailed analyses revealed ZMM4 expression initiates in leaf primordia of vegetative shoot apices and later increases within elongating meristems acquiring inflorescence identity. Expression analysis in late flowering mutants positioned all four genes downstream of the floral activators indeterminate1 (id1) and delayed flowering1 (dlf1). Overexpression of ZMM4 leads to early flowering in transgenic maize and suppresses the late flowering phenotype of both the id1 and dlf1 mutations. Our results suggest ZMM4 may play roles in both floral induction and inflorescence development.

Figure 1.

Expression profiling of shoot apices before and after the floral transition. A, Representative image of maize shoot apices collected from V4-stage plants before (left) and V6-stage plants after (right) the floral transition. The vegetative meristem (left) is more symmetrical in shape, while the reproductive meristem (right) is much taller than it is wide. Suppressed bract primordia (arrowheads) and BMs (arrows) are evident on the flanks of the reproductive meristem. Scale bar = 1 mm. B, Scatter plot comparison of the abundance of 9,698 unique 17-mer sequence tags that matched maize EST sequences and accumulated to 25 ppm in at least one of the two apex samples detected by MPSS. Transcript abundance in parts per million is compared for each tag from the vegetative apex sample (y axis) to its level in the reproductive apex sample (x axis). Tags showing a z-score cutoff of 2.92 defining the top 1% expression differences are colored red. The expression levels of the two MADS-box genes, ZMM4 and ZMM15, only detected in the reproductive stage apex are red with a black center and lie near the x axis.

Figure 2.

The genomic organization of the paired MADS-box loci in the grasses and gene structure in maize. A and B, Genome organization of the syntenic locus from rice (A) and wheat (B; Yan et al., 2003). C and D, Genome organization of the ZMM24-ZMM4 locus (C) and ZMM31-ZMM15 locus (D) from maize. E, Gene structure of each of the four MADS-box genes. Black boxes denote exons, thin black lines indicate introns, and the start codons (ATG) and stop codons (TAG or TGA) are marked. Large introns are reduced for clarity with their sizes indicated above each gene while overall coding sequence size is noted below each gene.

Figure 3.

Transcript abundance of the four MADS-box genes in different maize tissues. A, Transcript abundance in various tissues. B, Transcript abundance in vegetative and reproductive stages of the shoot apex and lateral shoot. Abundance was measured by MPSS and is reported in parts per million. Bars for ZMM4 are red, for ZMM15 are green, for ZMM31 are orange, and for ZMM24 are blue. Imm, Immature; SAM, vegetative SAM; SAM trans, reproductive SAM; LBM, lateral branch meristem; earT, ear tip (2–3 mm section); earB, ear base (2–3 mm section). Lengths listed refer to the total length of the structure at the stage sampled.

Figure 4.

Transcript localization of ZMM4 and ZMM15. A to C and E to G, In situ hybridization with a ZMM4-specific 3′ UTR antisense probe. I to K, In situ hybridization with a ZMM15-specific 3′ UTR antisense probe. D, H, and L, Representative sense control hybridizations of ZMM4 (D and H) and ZMM15 (L). Hybridizations were to longitudinal sections of V3/4 vegetative shoot apex (A), V5 late transitional shoot apex (B), V5/6 early reproductive shoot apex (C), V5 shoot apex (D), V8 primary ear (E), V9 primary ear (F), V10 primary ear (G, H, K, and L), V5 vegetative shoot apex (I), and V4/5 transitional shoot apex (J). In all images, the black scale bar is 500 μm and the red scale bar is 400 μm. The initiating BMs are marked by the black oval in C, the inner and outer glume primordia are marked by the black arrows in G, and ZMM15 expression in the youngest leaf primordium is marked by the black arrowheads in I.

Figure 5.

GUS staining of Pro_ZMM4:GUS TG plant inflorescences at growth stages V4 to V15. A, V4 vegetative SAM. B, V5 transitional elongating SAM. C, V5 late transitional SAM (white diamonds mark the position of suppressed bract primordia). D, Late V5 early inflorescence (white diamonds mark bract primordia and white triangles mark initiating BMs); E, V6 tassel primordium (white arrows mark the lateral shoots). F, V8 developing tassel. G, V10 tassel. H, V8 penultimate lateral shoot. I, V8 ultimate lateral shoot (black diamonds mark bract primordia). J, V10 developing ear primordia from the fifth lateral position (left) to the uppermost lateral position (right). K, Longitudinal section of V15 ear. L, Transverse section of V15 ear. M, Close-up of basal part of ear in K. Scale bars are 200 microns in A to D, 1 mm in E, 5 mm in F and G, 500 microns in H and I, and 1 mm in J to M.

Figure 6.

Quantitative expression levels of ZMM4, ZMM24, ZMM15, and ZMM31 in shoot apices before, during, and after the floral transition in three genotypes. Quantitative expression of the four MADS-box genes is shown as a ratio in arbitrary units (y axis) at different days after planting (x axis) for each graph. Ratios are the average of three technical reps and the error bars are sds. Expression in normal flowering B73 is indicated by circles, in dlf1-N2461A mild late flowering by squares, and in id1-m1 very late flowering by triangles. ZMM4 and ZMM15 are unfilled, whereas ZMM24 and ZMM31 are filled black. All graphs are at the same scale for ease of comparison across genes and genotypes. The time of the first appearance of BM initiation, marking the onset of reproductive growth, is denoted by the gray vertical bar for each genotype.