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VRS2 regulates hormone-mediated inflorescence patterning in barley

Nature Genetics volume 49, pages 157–161 (2017)Cite this article

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Abstract

Plant architecture has clear agronomic and economic implications for crops such as wheat and barley, as it is a critical factor for determining grain yield. Despite this, only limited molecular information is available about how grain-bearing inflorescences, called spikes, are formed and maintain their regular, distichous pattern. Here we elucidate the molecular and hormonal role of Six-rowed spike 2 (Vrs2), which encodes a SHORT INTERNODES (SHI) transcriptional regulator during barley inflorescence and shoot development. We show that Vrs2 is specifically involved in floral organ patterning and phase duration by maintaining hormonal homeostasis and gradients during normal spike development and similarly influences plant stature traits. Furthermore, we establish a link between the SHI protein family and sucrose metabolism during organ growth and development that may have implications for deeper molecular insights into inflorescence and plant architecture in crops.

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Figure 1: vrs2 spike morphology, and hormone and sucrose measurements.
Figure 2: Barley genetic and physical map of vrs2 and mutant analysis.
Figure 3: Vrs2 expression and GWAS analysis.
Figure 4: Low-resolution model showing the overall trend of Vrs2 expression in relation to auxin, CK and GA concentrations during early barley spike development.
Figure 5: Genes regulating auxin, CK and GA metabolism during barley spike development.

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Acknowledgements

We thank the US Department of Agriculture–Agricultural Research Service (USDA-ARS); the National Small Grains Collection, Aberdeen (ID); the Nordic Genetic Resources Center (NordGen), Alnarp, Sweden; and the IPK Genebank, Germany, for providing the mutants and germ plasm for haplotype analysis. We thank J. Thiel for help with the quantitative real-time PCR analyses; S. Beier and M. Mascher for bioinformatics support; the IBGSC for prepublication access to barley sequence information of chromosome 5H; and S. König, I. Walde, H. Ibrahim, C. Trautewig, M. Pürschel, K. Lipfert, H. Ernst, B. Kettig, D. Böhmert and Y.A.T. Moya for excellent technical support. We also would like to thank the anonymous reviewers for their constructive and supportive evaluations while improving this manuscript. This work was supported by grants from the Genomics for Agricultural Innovation Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan, grant TRS1002, to T.K. and the HEISENBERG Program of the German Research Foundation (DFG), grants SCHN 768/8-1 and SCHN 768/9-1, to T.S.

Author information

Authors and Affiliations

  1. Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Gatersleben, Germany

    Helmy M Youssef, Kai Eggert, Ravi Koppolu, Ahmad M Alqudah, Naser Poursarebani, Shun Sakuma, Twan Rutten, Geetha Govind, Andreas Graner, Nese Sreenivasulu & Thorsten Schnurbusch

  2. Faculty of Agriculture, Cairo University, Giza, Egypt

    Helmy M Youssef

  3. Department of Agronomy and Plant Breeding, Faculty of Agriculture, Ilam University, Ilam, Iran

    Arash Fazeli

  4. Kihara Institute for Biological Research, Yokohama City University, Yokohama, Japan

    Shun Sakuma

  5. National Institute of Agrobiological Sciences (NIAS), Tsukuba, Japan

    Akemi Tagiri & Takao Komatsuda

  6. Reliance R&D Centre, Reliance Corporate Park, Ghansoli, Navi Mumbai, India

    Geetha Govind

  7. Nordic Genetic Resource Center (NordGen), Alnarp, Sweden

    Udda Lundqvist

  8. International Rice Research Institute (IRRI), Grain Quality and Nutrition Center, Metro Manila, Philippines

    Nese Sreenivasulu

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  1. Helmy M Youssef
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Contributions

T.S. conceived the project. H.M.Y., N.S., T.K. and T.S. designed the research. H.M.Y., K.E., R.K., A.M.A., N.P., A.F., S.S., A.T., T.R., G.G. and U.L. performed experiments. H.M.Y., R.K., A.F., A.M.A., S.S., U.L., N.S., T.K. and T.S. analyzed data. A.G. provided germ plasm and genotypic resources for the GWAS analysis. H.M.Y. and T.S. wrote the manuscript with contributions from all coauthors.

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Correspondence to Helmy M Youssef or Thorsten Schnurbusch.

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Integrated supplementary information

Supplementary Figure 1 SEM analysis of wild-type (cv. Bowman) and BW-NIL(vrs2.e) mutant spikes.

Lemma primordium (LP), stamen primordium (SP), awn primordium (AP) and white anther (WA) stages show no visible differences between the genotypes until AP. Differences started during the WA stage, during which vrs2 mutants showed more development in the lateral spikelets (red arrowheads) than WT (blue arrowheads) and additional spikelets at the base of the mutant spike (red stars, close-up SEM images of spikelets). CS, central spikelet; LS, lateral spikelet; AD, additional spikelet; G, glume; L, lemma; S, stamen.

Supplementary Figure 2 Putative early-13-hydroxylation pathway of gibberellic acid biosynthesis.

(a–d) GA53 (a), GA44 (b), GA19 (c) and GA8 (d) concentrations (pg mg DW−1) in cv. Bowman and BW-NIL(vrs2.e) at awn primordium (AP), white anther (WA) and green anther (GA) stages. Significant differences were calculated based on Student’s t tests (two-tailed) n = 4: ns = no significant differences; *P < 0.05, **P < 0.01. Error bars, s.e.m. Bioactive gibberellins, such as GA1, GA3, GA4 and GA20, were measured in all samples but remained under the detection limit in both genotypes.

Source data

Supplementary Figure 3 Amino acid sequence alignment of the two domains among the grass-specific clade.

This alignment shows the double-CCCH motif (red stars), the nuclear localization signal (KRRER), the IGGH domain (isoleucine-glycine-glycine-histidine sequence) and the amino acid differences between LKS2 and Vrs2 (black circles).

Source data

Supplementary Figure 4 Phylogenetic analysis of SHORT INTERNODES (SHI) protein members in different species of higher plants.

A maximum-likelihood (ML) phylogenetic tree (condensed tree) of SHI proteins from 111 different protein accessions. From the phylogenetic tree, seven separate clades are evident; one clade, including VRS2, LKS2 and 19 other grass SHI proteins, is highlighted in green. The ML phylogenetic tree was constructed using the ML heuristic method Nearest Neighbor Interchange (NNI) implemented in MEGA 6. Bradi, Brachypodium distachyon; LOC_Os, Oryza sativa; GRMZM, Zea mays; Sb, Sorghum bicolor; Glyma, Glycine max; AT, Arabidopsis thaliana; Potri, Populus trichocarpa; Bra, Brassica rapa.

Source data

Supplementary Figure 5 Vrs2 nucleotide analysis in 520 barley accessions (302 intermedium types and 218 accessions from the worldwide GENOBAR collection).

Shown are four mutant alleles in comparison with natural sequence variation of 20-nt haplotypes.

Supplementary Figure 6 Network analysis of vrs2.

Shown are 520 barley accessions (302 intermedium types (blue) and 218 GENOBAR collection (orange)) producing 20 haplotypes in addition to the vrs2 mutant alleles (red).

Supplementary Figure 7 Manhattan plots of association signals for five traits.

(a–e) Results obtained for phase duration in thermal time (GDD) (a), tiller number (b), leaf number (c), leaf area (d) and plant height (e) in the spring barley GENOBAR collection using the iSelect 9K SNP platform in addition to Vrs2-derived markers (SNPs highlighted in blue). n = 209 accessions. AP, awn primordium stage; Tip, tipping stage; HD, heading; Ae, anther extrusion; Hrv, harvest.

Source data

Supplementary Figure 8 Box plot analysis of eight markers (M1–M8) derived from Vrs2 resequencing for two traits.

Phase duration in thermal time (GDD) (a) and tiller number (b) in the spring barley GENOBAR collection. n is the number of individuals per haplotype. The center value of each box plot is the median. AP, awn primordium stage; Tip, tipping stage; HD, heading; Ae, anther extrusion; Hrv, harvest.

Source data

Supplementary Figure 9 Box plot analysis of eight markers (M1–M8) derived from Vrs2 resequencing for three traits.

(c–e) Leaf number (c), leaf area (d) and plant height (e) in the spring barley GENOBAR collection. n is the number of individuals per haplotype. The center value of each box plot is the median. AP, awn primordium stage; Tip, tipping stage; HD, heading; Ae, anther extrusion; Hrv, harvest.

Source data

Supplementary Figure 10 Box plot analysis for four morphological traits in WT (cv. Bowman) and BW-NIL(vrs2.e) plants.

(a–d) Box plot diagrams showing the number of tillers (a), the number of non-productive tillers (b) and the number of spikes per plant (c) in addition to spike internode length (d) in Bowman and BW-NIL(vrs2.e) plants, n = 10; significance is based on Student’s t tests (two-tailed): n = 10, ns = no significant difference; **P < 0.01. Error bars in d, s.e.m. The center value of each box plot is the median.

Source data

Supplementary Figure 11 SEM analysis of wild-type (Bowman) immature spikes.

The four spike stages used for the micro array study at the triple-mound (TM), glume primordium (GP), stamen primordium (SP) and awn primordium (AP) stages are displayed.

Supplementary Figure 12 Gene expression and hormonal regulation affecting wild-type and vrs2 mutant plants.

Left, in wild-type plants, Vrs2 positively regulates auxin (IAA) biosynthesis through transcriptional activation of TSA1. Increased IAA and/or increased SHY2 negatively regulate cytokinins through suppression of IPT5. Vrs2 also indirectly (via IAA) negatively regulates the conversion of bioactive gibberellic acid forms (e.g., GA3, GA4, GA20) through the suppression of GA2ox (e.g., predominantly GA2ox2) thereby promoting flowering. On the right BM-NIL(vrs2.e) mutant. Black solid arrows indicate induced/increased gene expression/hormone biosynthesis. Red solid lines indicate inhibited/reduced gene expression/hormone biosynthesis, black dashed arrows indicate lower induced/increased gene expression/hormone biosynthesis, and red dashed lines indicate lower inhibited/reduced gene expression/hormone biosynthesis.

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Supplementary Figures 1–12, Supplementary Tables 1 and 2, and Supplementary Note. (PDF 4374 kb)

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Youssef, H., Eggert, K., Koppolu, R. et al. VRS2 regulates hormone-mediated inflorescence patterning in barley. Nat Genet 49, 157–161 (2017). https://doi.org/10.1038/ng.3717

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