Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley

Abstract

Inflorescence architecture of barley (Hordeum vulgare L.) is common among the Triticeae species, which bear one to three single-flowered spikelets at each rachis internode. Triple spikelet meristem is one of the unique features of barley spikes, in which three spikelets (one central and two lateral spikelets) are produced at each rachis internode. Fertility of the lateral spikelets at triple spikelet meristem gives row-type identity to barley spikes. Six-rowed spikes show fertile lateral spikelets and produce increased grain yield per spike, compared with two-rowed spikes with sterile lateral spikelets. Thus, far, two loci governing the row-type phenotype were isolated in barley that include Six-rowed spike1 (Vrs1) and Intermedium-C. In the present study, we isolated Six-rowed spike4 (Vrs4), a barley ortholog of the maize (Zea mays L.) inflorescence architecture gene RAMOSA2 (RA2). Eighteen coding mutations in barley RA2 (HvRA2) were specifically associated with lateral spikelet fertility and loss of spikelet determinacy. Expression analyses through mRNA in situ hybridization and microarray showed that Vrs4 (HvRA2) controls the row-type pathway through Vrs1 (HvHox1), a negative regulator of lateral spikelet fertility in barley. Moreover, Vrs4 may also regulate transcripts of barley SISTER OF RAMOSA3 (HvSRA), a putative trehalose-6-phosphate phosphatase involved in trehalose-6-phosphate homeostasis implicated to control spikelet determinacy. Our expression data illustrated that, although RA2 is conserved among different grass species, its down-stream target genes appear to be modified in barley and possibly other species of tribe Triticeae.

Keywords: EGG APPARATUS1; cytokinin; grain number; yield potential.

Fig. 1.

Spike morphology of different row-type loci and vrs4 phenotype. (A) Two-rowed spike (Vrs1): fertile central spikelets (CS) and sterile lateral spikelets (LS). (B) Six-rowed spike1 (vrs1): completely fertile CS and LS. (C) Six-rowed spike2 (vrs2): LS fertility observed at the spike base with occasional additional spikelets [additional spikelet (AS) fertile or sterile, AS in light green color]; along the spike, LS are occasionally enlarged and set seed, (D) Six-rowed spike3 (vrs3): spike base appears two-rowed, and the remaining portion appears six-rowed. (E) Six-rowed spike4 (vrs4): spike similar to vrs1, with frequent awn bearing AS (light green colored). (F) Intermedium-C (int-c): LS are enlarged, set seed, which are usually smaller than in vrs1, LS without awns. (G) Two-rowed wild-type spike (Piroline). vrs4 mutant spikes: (H) vrs4.l, (I) BW-NIL(mul1.a). (J) BW-NIL(vrs4.k): six-rowed-like appearance and formation of AS and additional florets (AF). (K) Spikelet triplet at one rachis internode. (L and M) BW-NIL(vrs4.k) (L) and vrs4.l (M) showing AS and AF; initial triplet: CS, LS with lemma (L) and palea (P). AF developed on the rachilla of LS. (N) Seed formation involving AS and AF. (O) Classes of axillary structures produced by vrs4 mutant vrs4.l and wild-type Piroline spikes. Total number of fertile or sterile axillary structures from autumn grown plants is shown. Data were recorded on five plants with on average two spikes per plant. (see SI Dataset S1A for SE and significance of t test).

Fig. 2.

Scanning electron microscopy and transcript localization of Vrs4 mRNA in immature barley spikes. (A) Wild-type inflorescence at lemma primordium (LP) stage showing inflorescence meristem (IM) forming double ridge (DR), upper ridge containing triple spikelet meristem (TSM), and lower leaf ridge (LR). TSM forms a triple mound (TM), which transitions into one central spikelet meristem (CSM) and two lateral spikelet meristems (LSMs). A pair of glume primordia (GP) and a single floral meristem (FM) are produced by each SM. (B) vrs4 at LP initiating additional spikelet meristems (ASMs) (red arrows). CSMs develop into branch-like inflorescence meristem (BIM). Occasionally, CSMs initiate additional florets on rachilla (asterisk). (C) vrs4 at stamen primordium (SP) stage showing BIM at DR and a developing additional floret meristem (AFM). (D) vrs4 at awn primordium (AP) stage showing BIM at TM. (E–H) In situ RNA hybridization of HvRA2 in two-rowed barley cv. Bonus. Transverse sections at DR (E), TM (F), GP (G), and SP (H). cs, central spikelet; ls, lateral spikelet; gl, glume. (I) Transcript levels of HvRA2 determined by quantitative RT-PCR in cv. Bonus. Constitutively expressed HvActin was used for normalization. X-axis represents spike developmental stages. Mean ± SE of three biological replicates. WA, white anther; GA, green anther. (J) Vrs1 expression in BW-NIL(vrs4.k) and wild-type MFB104. Mean ± SE of three biological replicates. Expression values are given at the bottom of the graph. (K) Vrs1 (yellow) and Vrs4 (green) are expressed in overlapping domains of lateral spikelets. (L) Spikes of cv. Montcalm carrying vrs1.a1 allele and vrs4 mutant allele mul1.a. Additional spikelets in mul1.a are indicated by red triangles. (Scale bars: 200 µm in A and B; 333 µm in C and D; and 100 µm in E–H.)

Fig. 3.

High-resolution linkage and physical map of vrs4 and analysis of vrs4 mutants. (A–C) Rice (A) and Brachypodium (B) chromosomal regions syntenic with barley chromosome 3H (C) in the vrs4 region. Predicted genes in rice, Brachypodium and barley chromosomes are indicated by ovals (Gene names start with Os01g (rice) or Bradi2g (Brachypodium) followed by five digit number). Solid black ovals mark the genes from which markers were derived for mapping in the vrs4 region. Numbers of recombinants are indicated between mapped markers on the barley chromosome. (D) Single BAC contig sequenced in the vrs4 region (22 overlapping BAC clones covering 1,073 kb) (see SI Appendix, Table S5 for corresponding BAC names). Predicted genes identified from the BAC sequences are indicated as circles. (E) HvRA2 gene structure showing a distinct grass-specific domain (red box), and RA2 as well as the LOB domains. Lesions in 18 ORF-mutant alleles are indicated below the gene structure (see also SI Appendix, Fig. S5). :, Nucleotide substitution leading to premature stop codon; *, INDELS leading to frame shift; >, nonsynonymous SNP in the coding region.

Fig. 4.

Models of Vrs4 interactions showing HvRA2 putative targets. (A) Functional Vrs4 suppresses additional spikelet formation and activates Vrs1 transcription. (B) Mutant vrs4 cannot control determinacy of the TSM; thus, additional spikelets are formed. Transcriptional activation of Vrs1 expression is omitted (red cross) resulting in lateral spikelet fertility. (C) HvRA2 may regulate transcripts of T6P synthase (T6PS) and HvSRA, a putative T6P phosphatase, thereby maintaining T6P homeostasis and spikelet determinacy. In Vrs4, transcript levels of PIN1-LIKE are maintained at constant level but are up-regulated in vrs4. In wild-type plants, HvRA2 functions upstream of HvHox1, which in turn may down-regulate transcripts of EA1-LIKE resulting in abortion of lateral spikelets, possibly producing a two-rowed spike (hypothetical). However, up-regulation of EA1-LIKE in vrs4 mutants may likely be due to enhanced meristematic activity related to the six-rowed spike phenotype and additional spikelets/florets. Transcript levels of genes involved in meristematic activity [e.g., cytokinin oxidase (CKX2), LONELY GUY-LIKE, and KNOX genes] are also regulated by HvRA2.