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Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits

Nature Genetics volume 48, pages 785–791 (2016)Cite this article

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Abstract

Shoot apical meristems are stem cell niches that balance proliferation with the incorporation of daughter cells into organ primordia. This balance is maintained by CLAVATA–WUSCHEL feedback signaling between the stem cells at the tip of the meristem and the underlying organizing center. Signals that provide feedback from organ primordia to control the stem cell niche in plants have also been hypothesized, but their identities are unknown. Here we report FASCIATED EAR3 (FEA3), a leucine-rich-repeat receptor that functions in stem cell control and responds to a CLAVATA3/ESR-related (CLE) peptide expressed in organ primordia. We modeled our results to propose a regulatory system that transmits signals from differentiating cells in organ primordia back to the stem cell niche and that appears to function broadly in the plant kingdom. Furthermore, we demonstrate an application of this new signaling feedback, by showing that weak alleles of fea3 enhance hybrid maize yield traits.

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Figure 1: fea3 mutant phenotypes and gene identification.
Figure 2: Expression of FEA3 and ZmWUS1.
Figure 3: Interactions between FEA3 and its putative ligand, ZmFCP1.
Figure 4: Computational model and tests of FEA3 function.
Figure 5: Arabidopsis FEA3 RNAi mutants are fasciated.
Figure 6: Yield traits for weak fea3 alleles.

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Acknowledgements

The fea3-0 allele was kindly provided by V. Shcherbak (Krasnodar Research Institute of Agriculture). We also thank U. Hernandez for assistance with cloning, A. Masson for assistance with peptide assays, and members of the Jackson laboratory for comments on the manuscript. We acknowledge funding from a collaborative agreement with DuPont Pioneer and from NSF Plant Genome Research program grant IOS-1238202 and grant MCB-1027445 and from Agriculture and Food Research Initiative competitive grant 2016-67013-24572 of the USDA National Institute of Food and Agriculture. The study also received support from the Gatsby Charitable Foundation (GAT3395/PR4) and the Swedish Research Council (VR2013-4632) to H.J. and through the Next-Generation BioGreen 21 Program (SSAC; project PJ01184302) from the Rural Development Administration, Republic of Korea.

Author information

Author notes

  1. Andrea L Eveland

    Present address: Present address: Donald Danforth Plant Science Center, St. Louis, Missouri, USA.,

  2. Jeremy Gruel and Young Koung Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, New York, USA

    Byoung Il Je, Young Koung Lee, Peter Bommert, Edgar Demesa Arevalo, Andrea L Eveland, Qingyu Wu, Alexander Goldshmidt & David Jackson

  2. Sainsbury Laboratory, University of Cambridge, Cambridge, UK

    Jeremy Gruel & Henrik Jönsson

  3. Agricultural Biotechnology, DuPont Pioneer, Johnston, Iowa, USA

    Robert Meeley

  4. Department of Biology, University of Massachusetts, Amherst, Massachusetts, USA

    Madelaine Bartlett

  5. Agricultural Biotechnology, DuPont Pioneer, Wilmington, Delaware, USA

    Mai Komatsu & Hajime Sakai

  6. Computational Biology and Biological Physics Group, Lund University, Lund, Sweden

    Henrik Jönsson

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Contributions

B.I.J. performed all experimental procedures except for those listed below, prepared figures, and wrote the draft of the manuscript. Y.K.L. and P.B. mapped and cloned FEA3. J.G. performed computational modeling, supervised by H.J. E.D.A. analyzed transactivation lines, using constructs generated by Q.W. A.L.E. performed expression analyses. A.G. performed ZmWUS1 reporter line construction and characterization. R.M. provided the ZmFCP1 mutants. M.B. performed phylogenetic analyses. M.K. and H.S. provided field genetics and mapping support and analysis. D.J. supervised the research and co-wrote the manuscript.

Corresponding author

Correspondence to David Jackson.

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Competing interests

The authors (B.I.J., Y.K.L., D.J., M.K., and H.S., on behalf of Cold Spring Harbor Laboratory and DuPont Pioneer) have obtained patent US20150047071 A1 based in part on this work from the US Patent and Trademark Office.

Integrated supplementary information

Supplementary Figure 1 Phenotypic analysis of fea3 mutants.

(a) Wild-type (WT) or fea3 SAM longitudinal and transverse sections probed in situ for KNOTTED1 (KN1) mRNA. The fea3 SAM showed a wider expression domain but normal patterning. Transverse sections are shown in the right panels. Scale bars, 50 μm. (b) The height of fea3 plants (right) is similar to that of wild-type plants (left). (c) Wild-type male spikelets normally contain two flowers, each with three anthers, and fea3 plants show normal spikelet and floret architecture. Anthers are indicated by arrowheads. (d) A wild-type female spikelet shows a carpel (asterisk), one degenerated floret (red arrow), and three immature anthers (arrowheads), and the fea3 mutant shows normal female spikelet and floret architecture. The images in c and d are from scanning electron microscopy. Scale bars in c and d, 100 μm.

Supplementary Figure 2 Structure and expression analysis of FEA3.

(a) A partial retrotransposon insertion (red text) between 10-bp duplicated target sites (blue) in the fea3-0 allele introduced a premature stop codon (asterisk). (b) FEA3 has two exons; mutations in the fea3-0 and EMS alleles are marked. (c) qRT–PCR indicates that FEA3 expression was enriched in SAM and root apical meristem (RAM), similar to FEA2. RE1 and RE2, root elongation zones; L-RAM, RAM of lateral root; Ct, coleoptiles; LS, leaf sheath; LB, leaf blade. (d–f) In situ hybridization of FEA3. (d) A sketch of the expression pattern in the SAM. (e) Hybridization with control sense probe. (f) FEA3 is similarly expressed in the central region of ear spikelet meristems (arrows). Scale bars, 50 μm.

Source data

Supplementary Figure 3 Complementation assay and localization of FEA3.

(a) Constructs for the FEA3-RFP fusion proteins. Dark green boxes, FEA3 coding region; light green boxes, UTR. Fusions were near the N- terminus, after the signal peptide, or at the C terminus. (b) FEA3-RFP transgenes rescued the fasciation phenotypes (Non-t, non-transgenic) (left); the genotypes of the plants are shown (the red arrow indicates the FEA3-RFP transgene, and the blue arrows indicate spurious PCR products from annealing of wild-type and FEA3-RFP PCR products) (right). (c,d) FEA3-RFP is localized to the inner-layer cells of the SAM central zone (c) and becomes enriched at the cell periphery after 2 h of D15 treatment (d) (laser confocal microscopy images). Note that the signal in upper SAM layers is background autofluorescence (asterisk). (e) Aqueous two-phase partitioning shows an increase in plasma membrane FEA3 localization after treatment with D15 peptide. S, soluble fraction; PM, plasma membrane fraction; IM, internal membrane-enriched fraction; cPM, control plasma membrane. (f–j) FEA3-RFP localization in young tassel (enlarged on right) (f), tassel spikelet meristems (arrows) (g), young ear (h), root apical meristem (i), and leaf primordium (j). FEA3-RFP was detected in the RAM in presumptive vascular initials but not in the quiescent center (QC); arrow indicates QC cells. In i, the tissue was counterstained with calcofluor white (blue). Scale bars, 50 μm.

Source data

Supplementary Figure 4 CLE peptide assays in maize roots.

fea2 roots showed partial resistance to CLV3; however, fea3 roots did not show resistance. Root length measurements are shown on the right. Error bars, s.d. (n = 10 for each genotype). **P < 0.001, Student’s t test; NS, not significant.

Source data

Supplementary Figure 5 Phylogenetic relationships among select CLE and FEA3-like genes.

(a–c) Although neither clade was ever strongly supported, Bayesian phylogenetic analyses of 45 CLE genes repeatedly resolved two gene clades, shown in green and blue (a). These two clades were further analyzed in separate, smaller analyses (b,c). CLE peptide sequences and gene identification numbers are listed in Supplementary Table 3. (d) FEA3 falls in a strongly supported clade with three genes from Arabidopsis. Thickened branches in all panels represent posterior probability >0.95.

Supplementary Figure 6 CLE peptide assays.

(a) fea3 roots show resistance specifically to ZmFCP1. Error bars, s.d. (n = 10 for each genotype). **P < 0.001, Student’s t test; NS, not significant. (b) In fea3 fea2 double-mutant segregating families, fea3 roots again showed resistance only to ZmFCP1, whereas fea2 showed resistance to all peptides tested and fea2 fea3 double mutants showed additive resistance to ZmFCP1. Error bars, s.d. (n = 10 for each genotype). **P < 0.001, Student’s t test; NS, not significant.

Source data

Supplementary Figure 7 FEA3 and ZmFCP1 expression, and the tissue template used in computational modeling.

Supplementary Figure 8 Schematic of the model.

Gene expression domains are represented with uniform color intensity, while diffusing molecules are shown with a color gradient.

Supplementary Figure 9 Model behavior example.

Expression domains of ZmWUS1 (top) and ZmCLV3 (bottom) for different genotypes. From left to right, wild type, Zmclv3, fea2, fea3, fea3 fea2, fcp1, and fcp1 overexpression.

Supplementary Figure 10 Expression predictions from the model.

Variation in gene expression levels of ZmWUS1 (green) and ZmCLV3 (red) between wild type and single or double mutants. fcp1-ox= fcp1 over-expression.

Supplementary Figure 11 Rice-like model behavior example.

Expression domains of ZmWUS1 (top) and ZmCLV3 (bottom) for different genotypes. From left to right, wild type, Zmclv3, fea2, fea3, fea3 fea2, fcp1, and fcp1 overexpression.

Supplementary Figure 12 Expression predictions from the rice-like model.

Variation in gene expression levels of ZmWUS1 (green) and ZmCLV3 (red) between wild type and single or double mutants. fcp1-ox= fcp1 over-expression.

Supplementary Figure 13 RNA levels of a maize CLV3 ortholog, ZmCLE7 (GRMZM2G372364), were increased in the inflorescence meristem tips of fea3 mutants in comparison to wild type (WT).

RNA-seq reads from two biological replicates were normalized and are plotted as fragments per kilobase per million (FPKM) reads mapped. The difference is significant (Students t test, P < 0.015).

Source data

Supplementary Figure 14 Two-component transactivation of ZmFCP1.

(a) Construction of the two-component system. LhG4 expression is driven by the primordium-specific YABBY14 promoter (pYABBY), which activates cis and trans pOp promoters. (b) Cis-activated NLS-RFP expression was detected specifically in leaf primordia (P1, P2) using laser confocal microscopy. (c) Transactivated ZmFCP1 expression was increased about 15-fold (three biological replicates, ±3.2 s.d.) as compared with the pOp-ZmFCP1 single-component line.

Source data

Supplementary Figure 15 Artificial microRNA and RNAi of Arabidopsis FEA3 and expression of CLE27.

(a,b) AtFEA3 artificial miRNA transgenic plants show fasciation and split inflorescence stems (arrows). (c) qRT–PCR shows RNAi specificity to the Arabidopsis FEA3 orthologous gene, At3g25670; two close homologs were not significantly downregulated. (d,e) AtFEA3 RNAi plants showed normal flower and silique development. (f) Floral organ number does not show a significant difference. Error bars, s.d. (n = 200 for each genotype). P > 0.6, Student’s t test; NS, = not significant. (g) CLE27 is expressed in the SAM peripheral zone, where cells destined to differentiate are incorporated into primordia, but not in the SAM central zone. (FACS sorting data are from the Arabidopsis eFP browser57,58).

Source data

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Table 1 and Supplementary Note. (PDF 3612 kb)

Supplementary Table 2

List of primers and oligonucleotide sequences. (XLSX 14 kb)

Supplementary Table 3

List of peptides. (XLSX 12 kb)

Supplementary Data 1

Data corresponding to Supplementary Table 1. (XLSX 20 kb)

Supplementary Data 2

Genotype data. (PDF 61 kb)

Supplementary Data 3

CLE alignments 2+2. (PDF 54 kb)

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Je, B., Gruel, J., Lee, Y. et al. Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits. Nat Genet 48, 785–791 (2016). https://doi.org/10.1038/ng.3567

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