HOMEOBOX2, the paralog of SIX-ROWED SPIKE1/HOMEOBOX1, is dispensable for barley spikelet development

Abstract

The HD-ZIP class I transcription factor Homeobox 1 (HvHOX1), also known as Vulgare Row-type Spike 1 (VRS1) or Six-rowed Spike 1, regulates lateral spikelet fertility in barley (Hordeum vulgare L.). It was shown that HvHOX1 has a high expression only in lateral spikelets, while its paralog HvHOX2 was found to be expressed in different plant organs. Yet, the mechanistic functions of HvHOX1 and HvHOX2 during spikelet development are still fragmentary. Here, we show that compared with HvHOX1, HvHOX2 is more highly conserved across different barley genotypes and Hordeum species, hinting at a possibly vital but still unclarified biological role. Using bimolecular fluorescence complementation, DNA-binding, and transactivation assays, we validate that HvHOX1 and HvHOX2 are bona fide transcriptional activators that may potentially heterodimerize. Accordingly, both genes exhibit similar spatiotemporal expression patterns during spike development and growth, albeit their mRNA levels differ quantitatively. We show that HvHOX1 delays the lateral spikelet meristem differentiation and affects fertility by aborting the reproductive organs. Interestingly, the ancestral relationship of the two genes inferred from their co-expressed gene networks suggested that HvHOX1 and HvHOX2 might play a similar role during barley spikelet development. However, CRISPR-derived mutants of HvHOX1 and HvHOX2 demonstrated the suppressive role of HvHOX1 on lateral spikelets, while the loss of HvHOX2 does not influence spikelet development. Collectively, our study shows that through the suppression of reproductive organs, lateral spikelet fertility is regulated by HvHOX1, whereas HvHOX2 is dispensable for spikelet development in barley.

Keywords: Anther and pistil development; CRISPR; HD-ZIP class I transcription factors; barley (Hordeum vulgare); gene duplication; inflorescence architecture; nucleotide diversity; spikelet fertility.

Fig. 1.

HvHOX1 and HvHOX2 are functional HD-ZIP class I transcription factors. (A) Bimolecular fluorescence complementation (BiFC) assay for HvHOX1 and HvHOX2 proteins. The signal in the yellow fluorescent protein (YFP) panel indicates the dimer formation, and the cyan fluorescent protein (CFP) panel discloses the nucleus (blue, dark spot). The last overlay panel exhibits the localization of protein dimers in the nucleus. Scale bar: 10 µm. (B) Western blot for HvHOX1 and HvHOX2 proteins used for BiFC assay is shown. EV, empty vector; HA, hemagglutinin; c-myc, avian myelocytomatosis virus oncogene cellular homolog; 25 and 55 denote protein size in kilodaltons. (C) The specific binding of HvHOX1 and HvHOX2 proteins on HD-Zip I cis-element (BS, binding sequence) indicated with a star is shown by an EMSA. Dihydrofolate reductase (DHFR), negative control. Different numbers denote the protein volume in µl. (D) The transactivation property of HvHOX1 and HvHOX2 proteins is shown by the GUS activity relative to LUC. Data shown are means ±SE (n=3); letters above the bar indicate that the mean values are significantly different at the 1% probability level by one-way ANOVA with Newman–Keuls multiple comparison test. EV, empty vector; GUS, β-glucuronidase; LUC, luciferase.

Fig. 2.

Two-rowed spikes have delayed and reduced lateral spikelet development compared with its central and six-rowed lateral spikelets. (A–F) A series of early spike developmental stages of two-rowed cv. Bowman is shown: (A) Double ridge (DR); (B) triple mound (TM); (C) glume primordium (GP); (D) lemma primordium (LP); (E) stamen primordium (SP); and (F) awn primordium (AP). (G, H) A comparison of the Waddington (W) 5.5-stage spike of the two-rowed Bowman and its six-rowed mutant BW-NIL(vrs1.a) shows that the lateral spikelet organs of Bowman (G) are reduced and delayed compared with the mutant (H). Scale bar in (A): whole spike, 500 µm; magnified three nodes, 100 µm; (B, C): 500 µm and 200 µm; (D) 500 µm and 100 µm; (E, F) 200 µm and 100 µm; (G, H) whole spike, 800 µm; magnified three nodes, 200 µm.

Fig. 3.

HvHOX1 and HvHOX2 expression patterns during spike development. (A–C) The expression patterns of HvHOX1 and HvHOX2 genes in the whole spikes (A), central spikelets (B), and lateral spikelets (C) during early spike developmental stages. (D–I) Expression variation of HvHOX1 and HvHOX2 genes in the whole spike (D, G), central spikelet (E, H), and lateral spikelet (F, I) of Bowman and BW-NIL(vrs1.a) during later spike developmental stages, respectively. Mean values are compared with multiple Student’s t-tests; *P<0.05, **P<0.01, ***P<0.001, mean values are significantly different; ns, not significantly different.

Fig. 4.

Spatiotemporal expression patterns of HvHOX1 and HvHOX2 and their network of co-expressed genes during spike growth and development. (A–C) HvHOX1 promoter activity (GFP expression) in central spikelets’ stamen and pistil (A), tapetum and filament of stamen (B), and lateral spikelet’s stamen (C) at Waddington 8.5 stage. (D–F) Similarly, HvHOX2 promoter activity in the central spikelet’s stamen and pistil (D), tapetum of an anther (E), and lateral spikelets’ stamen (F). Green color, GFP fluorescence; red color, chlorophyll autofluorescence. Scale bar: 100 µm. Modules of the co-expressed genes were assigned colors, shown by the horizontal bars below the dendrogram. (G) Merged modules are shown under the dynamic module profile. (H, I) The expression (log₂ normalized value) heat map of the red module (H) and the co-expressed gene clusters (I) of HvHOX1 and HvHOX2.

Fig. 5.

The HvHOX2 is dispensable for barley spikelet development. (A) The guide sequences used to generate the mutants and the mutated nucleotide sequences of two mutants of HvHOX1 and HvHOX2 genes by CRISPR are shown. (B–I) Images representative of E07 mutants. A comparison of immature (B–E) and mature (F–I) spikes reveals that the wild-type (null transgenic) (B) and Hvhox2 (C) immature spikes have a much delayed lateral spikelet development compared with the Hvhox1 single (D) and Hvhox1–Hvhox2 double (E) mutants. (F–I) Similarly, the wild-type (null transgenic) (F) and Hvhox2 (G) mature spikes have sterile lateral spikelets (without grain formation), while the Hvhox1 single (H) and Hvhox1–Hvhox2 double (I) mutants’ spikes possess fertile lateral spikelets or grains. Scale bar: (B–E) whole spikes, 500 µm; magnified three nodes, 200 µm. HD, homeodomain; LZ, leucine zipper. (J–O) Grain or spikelets’ area, width, and length of central (J–L) and lateral (M–O) of wild-type, single and double mutants of HvHOX1 and HvHOX2. Mean values (J–O) are compared with one-way ANOVA with Tukey’s multiple comparison test; different letters denote the values are significantly different at 5% probability levels, and the same letter shows that they are not significantly different.