HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley

Abstract

Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1, and propose causative gene sequences: a C2H2 zinc-finger gene HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels, resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data, we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement.

Keywords: Triticeae; B-class; Barley; C2H2; MADS-box; cereal; flower development; multiovary; transcription factors.

Fig. 1.

Florets, reproductive organs, and grains in the wild type (WT) and mov2.g. (A) Exposed WT and mov2.g florets. Palea or lemma have been removed to show internal floral organs. (B) Reproductive organs in WT florets consist of three stamens and one carpel. (C) Reproductive organs in mov2.g florets consist of supernumerary carpel-like structures (arrows). Scale bars: 1000 µm. (D) Manually cross-pollinated WT and mov2.g spikes. White arrows indicate multiple grains per floret. (E) Examples of multiple grains per floret produced by manually cross-pollinated mov2.g spikes. Scale bars: 2000 µm. Lemma (le), palea (pa), lodicule (lo), stamen (st), carpel (ca), supernumerary carpel-like structure (sc), pollen (po).

Fig. 2.

SEM of mov2.g developing inflorescences. In the wild type (WT), primordia giving rise to stamens are false-coloured in yellow, cells giving rise to the carpel in purple, and lodicules in green. In each panel from W3.5 onwards, the primordia are false-coloured in a single floret to enable comparison between genotypes. In mov2.g, the central carpel (purple) and lodicules (green) appear to be retained; cells giving rise to the additional carpel-like structures are false-coloured in blue (different shades). White arrows indicate ovule primordia. Waddington stage is indicated for each developmental time point; from stage W5.0, lemma and/or stamens have been removed to expose the carpels. Scale bars: 200 µm.

Fig. 3.

Spatial expression of HvSL1 during wild-type inflorescence development. Waddington stages correspond to lemma–floret primordia (W3.0), stamen primordia (W3.5), ovule primordium (W5.0), and stamen and carpel development (W7.0). Annotations and arrows indicate floral primordia (fp), lemma primordia (lp), stamen primordia (sp), lemma (le), lodicule (lo), stamen (st), carpel (ca), and ovule (ov). A sense HvSL1 probe was used as negative control to determine probe specificity. Scale bars: 100 µm.

Fig. 4.

Phenotype of CRISPR Hvsl1 knockout plants. All knockout plants contained two lodicules and supernumerary carpels. (A) HvSL1-1, (B) HvSL1-3, (C) HvSL1-5, (D) HvSL1-6, and (E) HvSL1-10. Partial stamen conversions (sc) are annotated. Scale bars: 100 µm.

Fig. 5.

Expression pattern as assessed by in situ hybridization for barley B-class genes. (A) HvMADS2, (B) HvMADS4, and (C) HvMADS16. Annotations indicate stamen primordia (sp), lemma (le), lodicule (lo), stamen (st), anther filament (af), carpel (ca), and ovule (ov). Scale bars: 100 µm.

Fig. 6.

Florets and floral organs in the wild type (WT) and mov1. (A) Exposed WT and mov1 florets. Palea or lemma have been removed to show internal floral organs. (B) The floral organs in a WT floret consist of two lodicules, three stamens, and one carpel. (C) In mov1 florets, the lodicules are converted into bract-like organs, and stamens are converted into additional carpels. Scale bars: 1000 µm. Lemma (le), palea (pa), lodicule (lo), stamen (st), carpel (ca), supernumerary carpel-like structure (sc), central carpel (cc), leaf-like organ (lf), pollen (po).

Fig. 7.

SEM of mov1 inflorescence development. In the wild type (WT), primordia giving rise to stamens are false-coloured in yellow, cells giving rise to the carpel in purple, and lodicules in green. In each panel from W3.5 onwards, the primordia are false-coloured in a single floret to enable comparison between genotypes. In mov1, the central carpel (purple) is retained while the stamens are converted into additional carpels. Cells giving rise to the additional carpels are false-coloured in different shades of red/orange. White arrows in mov1 (W3.0) indicate creases in the floral meristems. The white star at W5.0 indicates separation of a single floral meristem into two distinct florets; for greater detail, see Supplementary Fig. S11. Waddington stage is indicated for each developmental time point; from W5.0, lemma and/or stamens have been removed to expose the carpels. Scale bars: 200 µm.

Fig. 8.

Transcript abundance of floral genes assessed by qRT-PCR in mov2.g and mov1 developing inflorescences. Gene expression of B-class genes (HvMADS2, HvMADS4, HvMADS16), C-class genes (HvMADS3, HvMADS58), D-class gene HvMADS13, and carpel genes HvDL and HvSL1 was assayed in (A) mov2.g and (B) mov1 inflorescences at stages W2.0 (double ridge), W3.5 (stamen primordia), W4.5 (carpel primordium), and W6.0 (stamen and carpel development). Error bars represent ±SE. For each time point, two-tailed t-test P-values ≤0.05 (*), ≤0.005 (**), and ≤0.001 (***) are shown for differences between the wild type and mov1. For each sample, n=3 independent biological replicates.

Fig. 9.

Spatial expression of floral homeotic genes in wild-type (WT) and mov1 inflorescences. Expression patterns as detected by in situ hybridization are shown in WT and mov1 inflorescences at stage W6.0 for genes HvMADS16, HvMADS3, HvMADS13, and HvDL. Lemma (le), lodicule (lo), stamen (st), carpel (ca), ovule (ov), and carpel-like structure (cl). Scale bars: 200 µm.

Fig. 10.

Model for barley stamen specification. (A) As inflorescence development progresses in the wild type, genes of the ABC model combine in floral quartets to specify lodicules in whorl 2 and stamens in whorl 3, with putative repression of carpel and ovule-specific genes. The absence of (B) HvSL1 in mov2.g plants and (C) of HvMADS16 in mov1 plants affects the balance and composition of the floral quartets that form in these whorls, leading to an altered specification of the resulting floral organs. Within the floral quartets, B-class genes act as obligate GLO (HvMADS2 and HvMADS4)/DEF (HvMADS16) heterodimers. Dashed arrows indicate direct or indirect interaction, solid arrows indicate direct interactions found in this study, while dotted arrows indicate a process or consequence. For each mutant, the missing protein is indicated in grey, and the red symbol indicates the affected interaction.