Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility

Abstract

The barley inflorescence (spike) comprises a multi-noded central stalk (rachis) with tri-partite clusters of uni-floretted spikelets attached alternately along its length. Relative fertility of lateral spikelets within each cluster leads to spikes with two or six rows of grain, or an intermediate morphology. Understanding the mechanisms controlling this key developmental step could provide novel solutions to enhanced grain yield. Classical genetic studies identified five major SIX-ROWED SPIKE (VRS) genes, with four now known to encode transcription factors. Here we identify and characterise the remaining major VRS gene, VRS3, as encoding a putative Jumonji C-type H3K9me2/me3 demethylase, a regulator of chromatin state. Exploring the expression network modulated by VRS3 reveals specific interactions, both with other VRS genes and genes involved in stress, hormone and sugar metabolism. We show that combining a vrs3 mutant allele with natural six-rowed alleles of VRS1 and VRS5 leads to increased lateral grain size and greater grain uniformity.The VRS genes of barley control the fertility of the lateral spikelets on the barley inflorescence. Here, Bull et al. show that VRS3 encodes a putative Jumonji C-type histone demethylase that regulates expression of other VRS genes, and genes involved in stress, hormone and sugar metabolism.

Fig. 1

The vrs3 phenotype. a Spike of the two-rowed cv. Bowman. b Spike of the vrs3 NIL BW419(int-a.1). Scale bar in a applies to b, 1 cm. c Bowman spikelet triplet with fertile central and infertile lateral spikelets. d–h Spikelet triplets from a homozygous vrs3 individual from the BW419(int-a.1)*Bowman F2 population. d Six-rowed spikelet triplet. e Spikelet triplet with fertile central spikelet and infertile, pointed lateral spikelets. f–h Spikelet triplet with additional fertile spikelets and awned palea. g Adaxial and h abaxial views of the same spikelet triplet showing the formation of additional florets within the central spikelet. Scale bar in c applies to d–h, 1 cm. i, j Scanning electron microscopy of developing spikes of i Bowman and j vrs3 NIL BW902(vrs3.f). Developing lemmas are highlighted in pink, anthers in green and glumes in yellow. Scale bar in i applies to j, 500 µm

Fig. 2

Identification of VRS3. a The refined genetic mapping interval of the vrs3 locus showing the number of recombinants between each marker. b Conserved synteny between orthologous regions on the physical maps of rice chromosome 10 and barley chromosome 1H within the genetic mapping interval. Rice gene models are shown in dark blue and barley in red. The yellow square represents the VRS3 gene candidate. c VRS3 gene model with regions encoding three putative functional domains JmjN (pink), JmjC (turquoise) and C5HC2 zinc-finger (orange). The 5′ and 3′untranslated regions are shown in pale grey. Positions of the mutant alleles are relative to the start codon (ATG). Allele colours represent the respective genetic background of the induced mutation: turquoise (Bonus), green (Hakata 2), dark blue (Hege), red (Foma), and purple (Kristina). Types of mutation are coded as: > non-synonymous substitution, *premature stop, FS, frameshift, SS, splice site

Fig. 3

The allelic diversity of VRS3 across wild, landrace and cultivated barley germplasm. a Frequency of Vrs3.w and Vrs3.x alleles across the major classification divisions (winter-sown, spring-sown, two-rowed and six-rowed) within cultivated barley. b Median joining network illustrating the occurrence and relationships between different Vrs3 haplotypes across wild species (purple), two-rowed landrace (green) and six-rowed landrace (grey) germplasm. Tick marks across the connecting bars represent the number of polymorphisms distinguishing haplotype groups. The size of the circle is proportional to the number of representatives within the allelic class. Vrs3.w and Vrs3.x are renamed haplotype 45 and 46 because three SNPs at the 5′ and 3′ end of the exome capture alignments were missing from the alignment

Fig. 4

Expression analyses. a–f In situ RNA hybridization of VRS3 in two-rowed barley cv. Bowman at lemma primordium (LP) (spike length 2 mm) and awn primordium (AP) stage (spike length 5 mm). a–c Longitudinal sections hybridised with antisense VRS3 probe (a) at LP stage showing VRS3 signal in the developing glumes (gl) of the lateral spikelet (ls, in middle plane) and potential signal (*) from the glume of the adjacent central spikelet (CS) and at (b) AP stage showing expression in the rachis (r, yellow arrowheads) and (c) at higher magnification. d–f Equivalent longitudinal sections hybridised with sense probe. Scale bars, 100 µm (a, b), 200 µm (c, d), 50 µm (e, f). g Heat map of differentially expressed (DE) genes implicated in regulating spike development and meristem identity. Legend indicates gene expression abundances (Z scores) across the different developmental stages AP (5 mm) and white anther (WA) (10 mm) in BW902(vrs3.f) and Bowman (n = 8 bioreps per genotype). h Venn diagram showing the overlap of DE genes between Bowman (WT) and BW902(vrs3.f) in AP (5 mm) and WA (10 mm) spikes. Numbers represent the DE genes genetically located outside of the vrs3.f introgression in BW902(vrs3.f). i–m Average gene transcript levels determined by quantitative RT-PCR in Bowman WT (black) and BW902(vrs3.f) (red) in AP and WA spikes; i VRS1; j VRS2; k VRS3; l VRS4; m VRS5. HvActin was used for normalization. x-axis shows the inflorescence developmental stages in which the RNA-seq experiment was performed. y-axis shows the relative expression level based on ∆Ct (cycle threshold) calculation. Mean ± S.E of three biological replicates is shown. P values were calculated based on Student’s t tests (two-tailed)

Fig. 5

Impact of vrs3 on grain size and uniformity in the six-rowed spike. a A typical six-rowed barley spikelet triplet (vrs1.a, Int-c.a, Vrs3) showing the central grain (Cen) flanked by two lateral grain (Lat). Scale bar, 1 cm. b, c Six-rowed barley spikes from b the current commercial six-rowed genotype model: vrs1.a, Int-c.a, Vrs3.w (662) and c with the introduction of vrs3: vrs1.a, Int-c.a, vrs3 (666), respectively; Scale bar, 1 cm. d, e Distributions of d central and e lateral grain width fractions within the 662 genotypes. f, g Distributions of f central and g lateral grain width fractions within the 666 genotypes. h–j comparison of the mean grain width, grain area and lateral to central grain area ratio between the 662 (n = 6) and 666 (n = 13) genotype combinations; black bars: 662 genotype, grey bars: 666 genotype. Error bars are ± S.E.D