The CLV3 Homolog in Setaria viridis Selectively Controls Inflorescence Meristem Size

Abstract

The CLAVATA pathway controls meristem size during inflorescence development in both eudicots and grasses, and is initiated by peptide ligands encoded by CLV3/ESR-related (CLE) genes. While CLV3 controls all shoot meristems in Arabidopsis, evidence from cereal grasses indicates that different meristem types are regulated by different CLE peptides. The rice peptide FON2 primarily controls the size of the floral meristem, whereas the orthologous peptides CLE7 and CLE14 in maize have their most dramatic effects on inflorescence and branch meristems, hinting at diversification among CLE responses in the grasses. Setaria viridis is more closely related to maize than to rice, so can be used to test whether the maize CLE network can be generalized to all members of subfamily Panicoideae. We used CRISPR-Cas9 in S. viridis to knock out the SvFON2 gene, the closest homolog to CLV3 and FON2. Svfon2 mutants developed larger inflorescence meristems, as in maize, but had normal floral meristems, unlike Osfon2, suggesting a panicoid-specific CLE network. Vegetative traits such as plant height, tiller number and leaf number were not significantly different between mutant and wild type plants, but time to heading was shorter in the mutants. In situ hybridization showed strong expression of Svfon2 in the inflorescence and branch meristems, consistent with the mutant phenotype. Using bioinformatic analysis, we predicted the co-expression network of SvFON2 and its signaling components, which included genes known to control inflorescence architecture in maize as well as genes of unknown function. The similarity between SvFON2 function in Setaria and maize suggests that its developmental specialization in inflorescence meristem control may be shared among panicoid grasses.

Keywords: CLE; CLV3/ESR-related; FON2; Setaria; grass; inflorescence development; meristem maintenance; spikelet.

Figure 1

(A) Diagram of the proteins involved in maintenance of meristem size in Arabidopsis, focusing on those discussed in this paper (modified from Figure 2 in Kitagawa and Jackson, 2019). CLV3 is expressed at the apex of the meristem, where it interacts with CLV1 and BAM1-3, or with CLV2 and CRN, via Gα, to trigger a signaling process that ultimately restricts the expression domain of WUS. FCP1 interacts with FEA3 to repress WUS from below. Magenta lettering, CLE peptides; blue lettering, receptors, and receptor-like proteins. (B) Names of orthologous proteins in Arabidopsis, rice, and maize.

Figure 2

SvFON2 is expressed in early inflorescence, branch, and spikelet meristems. SEM, showing structure of the inflorescence. Sectioned inflorescences from comparable stages showing SvFON2 expression in the inflorescence meristem, branch meristems, spikelet meristems. (A,D) SEM images from early development of A10; images by Matt Box. (B,C,E–G) Expression of SvFON2, in situ hybridization, ME034. (B) inflorescence with branch meristems, slightly more mature than that in (A). (C) branch meristems. (D–F) inflorescence with differentiated spikelets and bristles; ridges visible on spikelets are glumes. (F) close-up of area inside box in (E). (G) sense control. Scale bars: (A–E,G), 100 μm; (F), 30 μm. IM, inflorescence meristem, BM, branch meristem, SP, spikelet, BR, bristle.

Figure 3

S. viridis fon2 mutants and phenotypic characterization during vegetative development. (A) Gene structure of SvFON2 and gene editing in the mutants. Black, white, and green colored boxes represent exon, intron, and CLE domain, respectively, of the SvFON2 gene. Blue boxes and text show PAM sites. Black arrows indicate target sites for the two guide RNAs (gRNA1 and gRNA2). ATG and TGA indicate the start and stop codons of the gene. Sequence below the gene model shows sequence comparison among wildtype (WT) and two independently edited mutants (fon2-1 and fon2-2). Red highlighted bases indicate the 1 bp insertion in the gRNA2 target region. Representative set of 4-week old plants (B). Roots from 5 DAS WT and fon2 showing no significant growth defects (C). Plant height (D), leaf number (E), and tiller number (F) of WT (green bars) and fon2 mutants (fon2-1 and fon2-2; white bars). Toluidine blue stained median sections of shoot apical meristems (SAM) at 6 DAS for WT (G) and Svfon2 mutant (H). SAM height (I), width (J), and area (K) of WT and fon2 vegetative meristems at six DAS. Significance values by Welch's t-test: *p < 0.01. Numbers of replicates for each statistical comparison listed in Table 1. Error bars are ± one standard deviation. SAM, shoot apical meristem. Scale bar: (B), 10 cm; (C), 2 cm; (G,H), 50 μm.

Figure 4

Phenotypic characterization of fon2 during reproductive development. Days to heading (A), panicle length (B), primary branches/cm (C), spikelets/branch (D) of WT (green bars) and fon2 mutants (fon2-1 and fon2-2; white bars). (F–I) Panicle apices from WT (F,H) and Svfon2 (G,I). (H,I) Representative SEM images of 17 DAS WT (H) and Svfon2 mutant (I). White arrows show novel sheet-like structures in the mutant. Main panicles (J) from WT and mutants. White arrows show that the mutant panicle apices are larger and have more tips. (K–N) Toluidine blue stained median sections of inflorescence meristems at nine DAS (K,L) and 12 DAS (M,N). (K,M), WT; (L,N), Svfon2. Significance values by Welch's t-test: *p < 0.01; **p < 0.001; ***p < 0.0001; black dot, p < 0.05. Numbers of replicates for each statistical comparison listed in Table 1. Error bars are ± one standard deviation. IM, inflorescence meristem; BM, branch meristem; Sp, spikelet; Br, bristle (sterile branch). Scale bars: (F,G), 5 mm; (H,I), 100 μm; (J), 1 cm; (K–N), 100 μm.

Figure 5

Inflorescence meristems and early floral development of Svfon2 mutants. (A–F) Representative scanning electron microscope images of 12 DAS WT (A,D) and Svfon2 mutants (B,C,E,F). (F) corresponds to the area marked by a white box in (E). White arrows show enlarged primary branch meristems. (G,H) Representative scanning electron microscope images of 17 DAS WT (G) and Svfon2 mutant (H). (I–N) Florets of WT (I,K,M) and Svfon2 mutant (J,L,N) immediately before anthesis. WT in (I,M), A10; WT in (K), ME34. (I,J) Upper (fertile) floret, lemma, and palea, viewed from the adaxial (relative to the floral axis) side. (K,L) Glumes and lemmas removed, floret viewed from the abaxial (relative to the floral axis) side, with the floral parts lying on the palea, and partially enveloped by the hyaline margins of the palea. Two normally formed lodicules are clearly visible, and the golden brown anthers can be seen through palea margins. The feathery stigma is slightly exserted from the WT palea, whereas it is still enclosed in the Svfon2 image. (M,N) Floral organs, showing gynoecium with an ovary, two styles and two stigmas, and three anthers on the right. (O) Total seed weight in the main panicle of WT (green bar) and svfon2 (white bar). Significance values by Welch's t-test: *p < 0.01. Numbers of replicates for each statistical comparison listed in Table 1. Error bars are ± one standard deviation. IM, inflorescence meristem; BM, branch meristem; A, anther; G, gynoecium; Le, upper lemma; Lo, lodicule; O, ovary; Pa, upper palea; Sti, stigma; Sty, style. Scale bars: (A–H), 50 μm; (I–N), 0.5 mm.