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The maize Gα gene COMPACT PLANT2 functions in CLAVATA signalling to control shoot meristem size

Nature volume 502, pages 555–558 (2013)Cite this article

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Abstract

Shoot growth depends on meristems, pools of stem cells that are maintained by a negative feedback loop between the CLAVATA pathway and the WUSCHEL homeobox gene1. CLAVATA signalling involves a secreted peptide, CLAVATA3 (CLV3)2, and its perception by cell surface leucine-rich repeat (LRR) receptors, including the CLV1 receptor kinase3 and a LRR receptor-like protein, CLV2 (ref. 4). However, the signalling mechanisms downstream of these receptors are poorly understood, especially for LRR receptor-like proteins, which lack a signalling domain5. Here we show that maize COMPACT PLANT2 (CT2) encodes the predicted α-subunit (Gα) of a heterotrimeric GTP binding protein. Maize ct2 phenotypes resemble Arabidopsis thaliana clavata mutants, and genetic, biochemical and functional assays indicate that CT2/Gα transmits a stem-cell-restrictive signal from a CLAVATA LRR receptor, suggesting a new function for Gα signalling in plants. Heterotrimeric GTP-binding proteins are membrane-associated molecular switches that are commonly activated by ligand binding to an associated seven-pass transmembrane (7TM) G-protein-coupled receptor (GPCR)6. Recent studies have questioned the idea that plant heterotrimeric G proteins interact with canonical GPCRs7, and our findings suggest that single pass transmembrane receptors act as GPCRs in plants, challenging the dogma that GPCRs are exclusively 7TM proteins.

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Figure 1: ct2 mutant phenotypes.
Figure 2: Cloning and expression of CT2.
Figure 3: Interactions between CT2 and CLAVATA signalling.

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References

  1. Stahl, Y. & Simon, R. Plant primary meristems: shared functions and regulatory mechanisms. Curr. Opin. Plant Biol. 13, 53–58 (2010)

    Article  CAS  Google Scholar 

  2. Fletcher, J. C., Brand, U., Running, M. P., Simon, R. & Meyerowitz, E. M. Signaling of cell fate decisions by CLAVATA3 in Arabidopsis shoot meristems. Science 283, 1911–1914 (1999)

    Article  ADS  CAS  Google Scholar 

  3. Clark, S. E., Williams, R. W. & Meyerowitz, E. M. The CLAVATA1 gene encodes a putative receptor kinase that controls shoot and floral meristem size in Arabidopsis. Cell 89, 575–585 (1997)

    Article  CAS  Google Scholar 

  4. Jeong, S., Trotochaud, A. E. & Clark, S. E. The Arabidopsis CLAVATA2 gene encodes a receptor-like protein required for the stability of the CLAVATA1 receptor-like kinase. Plant Cell 11, 1925–1934 (1999)

    Article  CAS  Google Scholar 

  5. Nimchuk, Z. L., Tarr, P. T. & Meyerowitz, E. M. An evolutionarily conserved pseudokinase mediates stem cell production in plants. Plant Cell 23, 851–854 (2011)

    Article  CAS  Google Scholar 

  6. Assmann, S. M. G proteins Go green: a plant G protein signaling FAQ sheet. Science 310, 71–73 (2005)

    Article  ADS  CAS  Google Scholar 

  7. Urano, D. et al. G protein activation without a GEF in the plant kingdom. PLoS Genet. 8, e1002756 (2012)

    Article  CAS  Google Scholar 

  8. Jackson, D., Veit, B. & Hake, S. Expression of maize KNOTTED1 related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Development 120, 405–413 (1994)

    CAS  Google Scholar 

  9. Bommert, P. et al. thick tassel dwarf1 encodes a putative maize ortholog of the Arabidopsis CLAVATA1 leucine-rich repeat receptor-like kinase. Development 132, 1235–1245 (2005)

    Article  CAS  Google Scholar 

  10. Taguchi-Shiobara, F., Yuan, Z., Hake, S. & Jackson, D. The fasciated ear2 gene encodes a leucine-rich repeat receptor-like protein that regulates shoot meristem proliferation in maize. Genes Dev. 15, 2755–2766 (2001)

    Article  CAS  Google Scholar 

  11. Ashikari, M., Wu, J., Yano, M., Sasaki, T. & Yoshimura, A. Rice gibberellin-insensitive dwarf mutant gene Dwarf 1 encodes the α-subunit of GTP-binding protein. Proc. Natl Acad. Sci. USA 96, 10284–10289 (1999)

    Article  ADS  CAS  Google Scholar 

  12. van der Hoorn, R. A. et al. Structure-function analysis of Cf-9, a receptor-like protein with extracytoplasmic leucine-rich repeats. Plant Cell 17, 1000–1015 (2005)

    Article  CAS  Google Scholar 

  13. Gallavotti, A., Yang, Y., Schmidt, R. J. & Jackson, D. The relationship between auxin transport and maize branching. Plant Physiol. 147, 1913–1923 (2008)

    Article  CAS  Google Scholar 

  14. Fiers, M. et al. The 14-amino acid CLV3, CLE19, and CLE40 peptides trigger consumption of the root meristem in Arabidopsis through a CLAVATA2-dependent pathway. Plant Cell 17, 2542–2553 (2005)

    Article  CAS  Google Scholar 

  15. Müller, R., Bleckmann, A. & Simon, R. The receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1. Plant Cell 20, 934–946 (2008)

    Article  Google Scholar 

  16. Ullah, H. et al. Modulation of cell proliferation by heterotrimeric G protein in Arabidopsis. Science 292, 2066–2069 (2001)

    Article  CAS  Google Scholar 

  17. Bommert, P., Nagasawa, N. S. & Jackson, D. Quantitative variation in maize kernel row number is controlled by the FASCIATED EAR2 locus. Nature Genet. 45, 334–337 (2013)

    Article  CAS  Google Scholar 

  18. Brown, P. J. et al. Distinct genetic architectures for male and female inflorescence traits of maize. PLoS Genet. 7, e1002383 (2011)

    Article  CAS  Google Scholar 

  19. Katritch, V., Cherezov, V. & Stevens, R. C. Structure-function of the G protein-coupled receptor superfamily. Annu. Rev. Pharmacol. Toxicol. 53, 531–556 (2013)

    Article  CAS  Google Scholar 

  20. Pandey, S., Nelson, D. C. & Assmann, S. M. Two novel GPCR-type G proteins are abscisic acid receptors in Arabidopsis. Cell 136, 136–148 (2009)

    Article  CAS  Google Scholar 

  21. Perfus-Barbeoch, L., Jones, A. M. & Assmann, S. M. Plant heterotrimeric G protein function: insights from Arabidopsis and rice mutants. Curr. Opin. Plant Biol. 7, 719–731 (2004)

    Article  CAS  Google Scholar 

  22. Lease, K. A. et al. A mutant Arabidopsis heterotrimeric G-protein β subunit affects leaf, flower, and fruit development. Plant Cell 13, 2631–2641 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Shpak, E. D., Berthiaume, C. T., Hill, E. J. & Torii, K. U. Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Development 131, 1491–1501 (2004)

    Article  CAS  Google Scholar 

  24. Shiu, S. H. & Bleecker, A. B. Plant receptor-like kinase gene family: diversity, function, and signaling. Sci. STKE 2001, re22 (2001)

    CAS  PubMed  Google Scholar 

  25. Iyer-Pascuzzi, A. S. et al. Imaging and analysis platform for automatic phenotyping and trait ranking of plant root systems. Plant Physiol. 152, 1148–1157 (2010)

    Article  CAS  Google Scholar 

  26. Freeling, M. & Walbot, V. The Maize Handbook. (Springer-Verlag, 1994)

    Book  Google Scholar 

  27. Mohanty, A. et al. Advancing cell biology and functional genomics in maize using fluorescent protein-tagged lines. Plant Physiol. 149, 601–605 (2009)

    Article  CAS  Google Scholar 

  28. Chen, J. G., Gao, Y. & Jones, A. M. Differential roles of Arabidopsis heterotrimeric G-protein subunits in modulating cell division in roots. Plant Physiol. 141, 887–897 (2006)

    Article  CAS  Google Scholar 

  29. Dennehey, B. K., Retersen, W. L., Ford-Santino, C., Pajeau, M. & Armstrong, C. L. Comparison of selective agents for use with the selectable marker gene bar in maize transformation. Plant Cell Tissue Organ Cult. 36, 1–7 (1994)

    Article  CAS  Google Scholar 

  30. Harlow, E. & Lane, D. Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, 1988)

    Google Scholar 

  31. Larsson, C. & Widell, S. in Aqueous Two-Phase Systems: Methods and Protocols Vol. 11 Methods in Biotechnology, (ed Hatti-Kaul, R. ) 159–166 (Humana Press, 2000)

    Book  Google Scholar 

  32. McDonald, W. H., Ohi, R., Miyamoto, D. T., Mitchison, T. J. & Yates. J. R. III Comparison of three directly coupled HPLC MS/MS strategies for identification of proteins from complex mixtures: single-dimension LC-MS/MS, 2-phase MudPIT, and 3-phase MudPIT. Int. J. Mass Spectrom. 219, 245–251 (2002)

    Article  CAS  Google Scholar 

  33. Taylor, P. et al. Automated 2D peptide separation on a 1D nano-LC-MS system. J. Proteome Res. 8, 1610–1616 (2009)

    Article  ADS  CAS  Google Scholar 

  34. Washburn, M. P., Wolters, D. & Yates, J. R. III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nature Biotechnol. 19, 242–247 (2001)

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank T. Mulligan for plant care; T. Rocheford for growing the ct2 targeted EMS screens; F. Albeanu for help with two-photon microscopy; P. Yin for assistance with genotyping; K. Rivera and D. Pappin for performing proteomic experiments; and T. Zadrozny, Q. Wu and W. Boss for assistance and advice with two-phase partitioning. P.B. was supported by DFG grant Bo 3012/1-1, A.G. was supported by the BARD Postdoctoral award no FI-431-2008, B.I.J was supported by Dupont-Pioneer (CSHL-Collaborative agreement), P.B. and D.J. were supported by the National Science Foundation Plant Genome Program, DBI-0604923.

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Author notes

  1. Alexander Goldshmidt

    Present address: Present address: Monsanto Company, 700 Chesterfield Parkway, Chesterfield, Missouri 63017, USA.,

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, 11724, New York, USA

    Peter Bommert, Byoung Il Je, Alexander Goldshmidt & David Jackson

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Contributions

P.B., B.I.J., A.G. and D.J. designed experiments and analysed data; P.B. performed all experiments, except peptide assays and contributions to imaging, phenotyping and biochemical assays (B.I.J.) and fluorescence imaging (A.G.); and P.B. and D.J. wrote the paper.

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Correspondence to David Jackson.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Phenotypic analysis of ct2 mutants.

a, Wild-type seedling. b, ct2 seedling showing reduced height. c, Cleared wild-type vegetative SAM. d, Cleared ct2 vegetative SAM showing increased diameter (± denotes s.d.; n = 10 for each genotype). e, Wild-type SAM longitudinal section probed in situ for KNOTTED1 (KN1) mRNA. f, ct2 SAM longitudinal section probed in situ for KN1 mRNA showing a wider expression domain but normal patterning. g, h, Transverse sections of wild-type and ct2 SAM probed in situ for KN1. i, SEM of wild-type ear primordium showing a regular shaped inflorescence meristem (IM) and regular arrangement of spikelet pair meristems (SPM) on the flanks. j, ct2 ear primordium showing fasciation of the inflorescence meristem and irregular arrangement of SPM. k, Wild-type tassel primordium. l, ct2 tassel primordium showing reduced length of main rachis and tassel branches and increased spikelet density. Scale bars represent 100 µm (e–h), 1 mm (i, j) and 2 mm (k, l).

Extended Data Figure 2 ct2 alleles and complementation by the CT2–YFP transgene.

a, Schematic of the ct2 locus with mutations marked and agarose gel electrophoresis of partial ct2-Ref (left) and ct2-258 (right) complementary DNA (cDNA) fragments compared to the corresponding wild type. cDNA fragments show increased size for the ct2-Ref allele owing to the insertion within exon 14, and reduced size for the ct2-258 allele owing to skipping of exons 12 and 13. Primer locations are indicated in a. All 3 EMS induced alleles had mutations that induced aberrant splicing, which led to the introduction of premature stop codons. ct2-258, had a G to A transition at the last nucleotide of exon 12, disrupting the 5′ splice site of exon 12. This mutation caused skipping of exons 12 and 13. The G to A transition at the last nucleotide of exon 4 of the ct2-2 allele disrupted the natural 5′ splice site, resulting in activation of a cryptic 5′ splice site five nucleotides downstream. Another G to A transition at the last nucleotide of intron 2 in the ct2-27 allele disrupted the natural 3′ splice site generating a frame-shifted mRNA missing the first seven nucleotides of exon 3. b, ct2 and CT2–YFP transgene genotyping. The genotyping scheme relies on an AccI site single nucleotide polymorphism that is lost in ct2-Ref and amplification of the YFP insertion in the transgene, as described in the Methods. c, Leaf length was indistinguishable between transgenic, homozygous ct2-Ref and wild-type plants, but significantly longer than non-transgenic ct2-Ref homozygous mutants (P value = 0.001, Student’s t test; n = 10 for each genotype; error bars represent s.d.), indicating complementation. d, Seedling phenotypes of transgenic, wild-type plants. e, Seedling phenotype of transgenic, homozygous ct2-Ref plants. f, Seedling phenotype of non-transgenic, homozygous ct2-Ref plants; plants are dwarfed and develop shorter leaves. Pictures d–f are shown at identical magnification.

Extended Data Figure 3 CT2–YFP expression.

a, CT2–YFP expression in median optical section of the inflorescence meristem (IM) of an ear primordium, note expression is enhanced in outer layers. b, CT2–YFP expression in a spikelet pair meristem (SPM), showing enhanced expression in the L1 (arrows). c, More centrally localized CT2–YFP expression within a developing spikelet meristem (SM). d, CT2–YFP expression during early stages of floral development, in stamen (st) and carpel (ca) primordia. In both organs, YFP signal is enhanced in the L1 layer (arrow). e, Expression during ovule development, in the ovule (ov) and in the outer cell layers of the stamen primordia (st). f, CT2–YFP expression in roots that were counterstained with propidium iodide is localized along the cell periphery. Image e is a three-dimensional reconstruction of a Z-stack series; scale bars, 100 μm.

Extended Data Figure 4 ct2 double mutant analysis.

Spikelet density of ct2-Ref; td1-Ref double mutants was significantly higher than either single mutant (***P value = 0.0001; Student’s t test). In contrast, spikelet density in ct2-Ref; fea2-0 double mutants was not significantly different (NS) from that of ct2 single mutants (P value = 0.42; Student’s t test), even though each single mutant was significantly higher than normal (P value = 0.001; Student’s t test). However, fea2 mutants were significantly different from ct2;fea2 double mutants (**P value 0.001; Student’s t test), suggesting that CT2 also signals independently of FEA2 in control of spikelet density; n ≥ 10 for each genotype; error bars represent s.d. This experiment was conducted with 2 independent biological replicates.

Extended Data Figure 5 Full fractionation western blots and loading controls and FEA2 glycosylation and localization.

a, Western blot probed with an anti-FEA2 peptide antibody and a secondary HRP-coupled antibody (GE); loading quantities shown on right side by 0.1% Ponceau S stain. The anti-FEA2 peptide antibody detects a band of ∼70 kDa in B73 membrane enriched fractions (lane 3). Specificity of the antiserum is evident by the lack of a cross-reacting band in the membrane-enriched fraction from fea2 ears (lane 4). b, FEA2 is glycosylated. It runs at ∼70 kDa in B73 membrane-enriched fractions (lane 1) and is reduced in size upon treatment with PNGaseF glycosidase (lane 2). c, Subcellular fractionation of FEA2 by aqueous two-phase partitioning. The lower (L) and upper (U) phase fractions from B73 inflorescences were resolved on a 10% SDS–PAGE gel, and probed with antisera against BiP lumenal binding protein, or plasma membrane H+ ATPase (Agrisera), or FEA2. Similar loading is shown by Ponceau staining. The endogenous FEA2 distribution is similar to the plasma membrane H+ ATPase, suggesting that it localizes predominantly to the plasma membrane. d, Western blot probed with an anti-GFP peptide antibody and a secondary HRP coupled antibody (GE), loading quantities shown on right side by 0.1% Ponceau S stain. e, Complete scan of the IP western blot shown in Fig. 3d, showing full fractionation and all lanes on same gel. The blot was probed with an anti-FEA2 peptide antibody and a secondary HRP coupled antibody (GE). MF, membrane fraction. This experiment was conducted with 3 biological replicates.

Extended Data Figure 6 Overlapping sizes of FEA2 and CT2 protein complexes.

In the lower panel, membrane enriched extracts from wild-type ears were fractionated by gel filtration, and fractions separated by SDS–PAGE and probed with the FEA2 antiserum. FEA2 was detected in complexes ranging from ∼350 to 550 kDa, with a peak around 400 kDa. The positions of the marker proteins are indicated. In the upper panel, the CT2–YFP fusion protein was detected by spectroscopy as it eluted from the column. In cytoplasmic extracts, CT2–YFP in the soluble fraction (CT2–YFP SOL) eluted at ∼100 kDa, suggesting an interaction with unknown cytoplasmic protein(s). In contrast, a membrane-enriched fraction (CT2–YFP MF) showed that CT2–YFP membrane complexes were a broad range of sizes that overlapped with the FEA2 complex size at ∼400 kDa (shaded area).

Extended Data Table 1 ct2 mutant leaves are shorter and wider but cell size is not significantly different.
Full size table

Supplementary information

Supplementary Table 1 - Mass spectroscopy analysis of CT2-YFP IPs

We performed 2 independent IP/ Mudpit mass spec runs using CT2-YFP, and we only considered hits with 2 or more significantly matching peptides. The list of potential CT2 interactors includes matches that were present in both runs, after protein matches that were found in control (non-transgenic) IPs were eliminated from the list. The IP mass spec analysis detected CT2 itself, the heterotrimeric G protein subunit (annotated as ZmAGB1), and three predicted LRR receptors, see file for additional information. (XLSX 53 kb)

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Bommert, P., Je, B., Goldshmidt, A. et al. The maize Gα gene COMPACT PLANT2 functions in CLAVATA signalling to control shoot meristem size. Nature 502, 555–558 (2013). https://doi.org/10.1038/nature12583

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