FRIZZY PANICLE drives supernumerary spikelets in bread wheat

Abstract

Bread wheat (Triticum aestivum) inflorescences, or spikes, are characteristically unbranched and normally bear one spikelet per rachis node. Wheat mutants on which supernumerary spikelets (SSs) develop are particularly useful resources for work towards understanding the genetic mechanisms underlying wheat inflorescence architecture and, ultimately, yield components. Here, we report the characterization of genetically unrelated mutants leading to the identification of the wheat FRIZZY PANICLE (FZP) gene, encoding a member of the APETALA2/Ethylene Response Factor transcription factor family, which drives the SS trait in bread wheat. Structural and functional characterization of the three wheat FZP homoeologous genes (WFZP) revealed that coding mutations of WFZP-D cause the SS phenotype, with the most severe effect when WFZP-D lesions are combined with a frameshift mutation in WFZP-A. We provide WFZP-based resources that may be useful for genetic manipulations with the aim of improving bread wheat yield by increasing grain number.

Figure 1.

SS phenotypes in bread wheat. A, Schematic representations of a spike (left) and a spikelet (right) from bread wheat N67 and of a theoretical wild-type (WT) spikelet (boxed). B, Schematic illustration of various SS structures: a cluster of spikelets at a rachis node referred to as a MRS, three spikelets (triple spikelet), and two spikelets in horizontal positions at a rachis node referred to as HSs; additional sessile spikelets with lateral branch bearing spikelets at a node referred to as a RS; and a single spikelet at a node for the wild-type spike. C, Illustration of the spike structure in the line NIL-mrs1 harboring numerous SSs (left), a rachis node with additional spikelets (right) where SSs are indicated with red arrows, and schematic representation of a branch-like structure (b) bearing ectopic spikelets at a rachis node in the lower part of the NIL-mrs1 spike (boxed). D to G, Light microscopy images of the NIL-mrs1 inflorescence at several developmental stages. D, Illustration of the spikelet differentiation stage; green arrows indicate glume primordia. E and F, Early floret differentiation stage. E, Secondary AxMs that later produce ectopic spikelets are indicated with asterisks. F, Spikelet meristems of ectopic spikelets form glume primordia. G, Late floret differentiation stage when all floral organs of wild-type spikelets (ws) are differentiated in the upper part of the inflorescence. H, A branch-like structure dissected from a rachis node of the NIL-mrs1 inflorescence. White squares indicate the ectopic spikelet (es). I, Location of ectopic spikelets at rachis nodes of the NIL-mrs1 inflorescence. Bars = 0.25 mm. J to S, Scanning electron microscopy images of N67 and NIL-mrs1 inflorescences at various developmental stages. J and K, Spikelet differentiation stage in the wild type (J) and mrs1 (K). L, Early floret differentiation stages when the spikelet meristem produces the FM in the wild type. M and N, Differentiation of secondary AxMs (indicated by asterisks) in the mrs1 mutant. O, Early floret differentiation stage showing lemmas. P and Q, The development of glumes (gl* and indicated by white arrows), lemma, and FMs by secondary AxMs (indicated by asterisks) in the mrs1 mutant. R, Floret differentiation stage showing differentiated floral organs in a basal floret of the wild type. S, The development of ectopic spikelets in mrs1. Bars =100 μm. es^2nd, Ectopic spikelet of the second order, developing at the place of a floret of the first order ectopic spikelet; f , floret with floret organ primordia; f*, floret of an ectopic spikelet; Fm, floret meristem; Fm*, floret meristem of an ectopic spikelet; gl, glume; im, inflorescence meristem; l, lemma; l*, lemma of an ectopic spikelet; lo, lodicule; pa, palea; pi, pistil; ts, terminal spikelet; sm, spikelet meristem; sm*, spikelet meristem of an ectopic spikelet; st, stamen.

Figure 2.

WFZP gene characterization in bread wheat. A, Composite genetic map of the wheat chromosome 2D, including the mrs1 locus and flanking conserved orthologous set (COS) markers. B, Orthologous chromosomes in rice (Chr7), sorghum (Chr2), and Brachypodium distachyon (Brachy.; Chr1) are shown, with conserved genes linked with black lines. C, Annotated BAC clones are illustrated with conserved genes (black boxes) linked with black connecting lines. Copia (green), Gypsy (blue), uncharacterized (yellow), and class II (red) TEs are indicated. D, Comparison of WFZP homoeologous gene structures with red boxes highlighting the conserved AP2/ERF domain. Gray boxes indicate small deletions, light blue triangles indicate small insertions, and gray triangles indicate insertion of TEs. Red and blue lines indicate nonsynonymous and synonymous single nucleotide substitutions, respectively. Red arrows indicate the positions of primers designed from the B. distachyon (Bradi1g18580) gene sequence and that were used for BAC library screening.

Figure 3.

Structural and functional characterization of WFZP genes. A, Schematic representation of the WFZP-A and WFZP-D gene structures and the mutations identified. The light red box indicates the AP2/ERF domain, and gray boxes indicate deletions. B, WFZP haplotypes of SS (red) and standard spiked (normal spiked [NS], green) lines. C, Alignment of the amino acid sequences of the AP2/ERF domains of wild type, WFZP-D, and wfzp-D.1, wfzp-D.2 mutants. Black asterisks indicate amino acids that confer specificity to the GCC-box binding site. D, Relative abundance of WFZP homoeolog mRNAs as determined by quantitative reverse transcription (qRT)-PCR with samples from spikes of bread wheat ‘Chinese Spring’ at various developmental stages of the spike (see below) and from roots and leaves. The values shown correspond to the mean values for three biological and three technical replicates, normalized to the value for the Ta.304.3 gene used as a reference. Error bars represent ses between replicates. Spike developmental stages were assigned as follows: spikelet differentiation stage (SD; when spikelets are differentiated to form two opposite rows at the rachis and glume primordia are initiated), early floret differentiation stage 1 (EFD1; spikelet meristems initiate floral meristems and lemma primordia are apparent), early floret differentiation stage 2 (EFD2; floral organ primordia are apparent in basal florets), and late floret differentiation stage (LFD; floral organs are differentiated in each floret).

Figure 4.

Functional validation of FZP in B. distachyon. FZP mutant screening identified two alleles with independent mutations (Bd8202 and Bd8972) located in the AP2/ERF domain (A), both exhibiting SSs not present in the wild type (WT, Bd 21-3; B). B. distachyon fzp mutants and wild-type plants show similar growth and architecture (C, left). Spikes with different penetrance of the SS phenotype can be observed in mutants (C, right; stars indicate SSs). D, Spikes in fzp mutants show spikelets with a single pedicel as observed in the wild type but with dichotomous or trichotomous rachilla.

Figure 5.

Evolutionary and functional model of FZP in grasses. FZP genes are illustrated as black horizontal bars showing substitutions in the AP2/ERF domain (red boxes), frameshift mutations (red bars), and TE insertions in the promoter region (red triangle) leading to FZP nonfunctionality and driving SS phenotypes in B. distachyon and wheat. In wild-type (WT) bread wheat, FZP represses the formation of AxMs from spikelet meristems (SMs) and controls the establishing FM identity. By contrast, in SS mutants, AxM is not suppressed and leads to ectopic spikelet meristem (eSM) formation.