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Dynamic evolution of small signalling peptide compensation in plant stem cell control

Abstract

Gene duplications are a hallmark of plant genome evolution and a foundation for genetic interactions that shape phenotypic diversity1,2,3,4,5. Compensation is a major form of paralogue interaction6,7,8 but how compensation relationships change as allelic variation accumulates is unknown. Here we leveraged genomics and genome editing across the Solanaceae family to capture the evolution of compensating paralogues. Mutations in the stem cell regulator CLV3 cause floral organs to overproliferate in many plants9,10,11. In tomato, this phenotype is partially suppressed by transcriptional upregulation of a closely related paralogue12. Tobacco lost this paralogue, resulting in no compensation and extreme clv3 phenotypes. Strikingly, the paralogues of petunia and groundcherry nearly completely suppress clv3, indicating a potent ancestral state of compensation. Cross-species transgenic complementation analyses show that this potent compensation partially degenerated in tomato due to a single amino acid change in the paralogue and cis-regulatory variation that limits its transcriptional upregulation. Our findings show how genetic interactions are remodelled following duplications and suggest that dynamic paralogue evolution is widespread over short time scales and impacts phenotypic variation from natural and engineered mutations.

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Fig. 1: Loss of the tobacco SlCLE9 orthologue abolished compensation.
Fig. 2: Weak fasciation of phclv3 mutants in petunia indicates more potent compensation.
Fig. 3: A highly conserved dodecapeptide amino acid is associated with potent compensation in groundcherry.
Fig. 4: Variation in Solanaceae compensation is due to changes in both the SlCLE9 orthologue dodecapeptide and its expression.

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Data availability

Raw data and information for CRISPR-generated alleles, all quantifications, synteny analysis and exact P values (one-way ANOVA and Tukey test) are in Supplementary Data 17. The raw Sanger sequence traces for edited sequences are in Supplementary Data 8. The groundcherry and petunia BioProject accession numbers are PRJNA704671 and PRJNA750419, respectively.

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Acknowledgements

We thank members of the Lippman laboratory for comments and discussions and also critical friends Y. Eshed and M. Bartlett. We thank S. Soyk for discussions and initial peptide sequence analysis in tomato and J. Kim and A. Krainer for technical support. We thank A. Horowitz Doyle, K. Swartwood, M. Tjahjadi, L. Randall and P. Keen from the Van Eck laboratory for performing tobacco, groundcherry and tomato transformations. We thank T. Mulligan, K. Schlecht, A. Krainer and S. Qiao for assistance with plant care. This research was supported by National Natural Science Foundation of China (grant nos. 31972423 and 31991183) and Chinese Academy of Sciences (grant no. 153E11KYSB20180019) to C.X., the Howard Hughes Medical Institute, an Agriculture and Food Research Initiative competitive grant from the USDA National Institute of Food and Agriculture (grant no. 2016-67013-24452) and the National Science Foundation Plant Genome Research Program (grant nos. IOS-1732253 and IOS-1546837) to Z.B.L.

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Authors and Affiliations

Authors

Contributions

C.-T.K. designed the research and conducted the experiments, prepared the figures and wrote the manuscript. L.T. performed the petunia CRISPR experiments, tomato transgenic complementation tests, genetic, RNA-seq and phenotypic analyses. X.W. performed the groundcherry RNA-seq, phenotypic analyses and wrote the manuscript. I.G performed the tobacco genetic and phenotypic analyses. A.H. performed CLE family analyses. G.R. characterized CRISPR mutations. J.V.E generated transgenic plants and CRISPR lines. C.X. supervised and led the petunia CRISPR experiments and tomato transgenic complementation tests, genetic, RNA-seq and phenotypic analyses, contributed ideas and edited the manuscript. Z.B.L. conceived and led the research, supervised and performed the experiments, prepared the figures and wrote the manuscript. All authors read, edited and approved the manuscript.

Corresponding authors

Correspondence to Cao Xu or Zachary B. Lippman.

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Extended data

Extended Data Fig. 1 CRISPR-generated mutations of the tobacco NbCLV3a and NbCLV3b genes.

a, CRISPR-generated sequences of nbclv3a mutant alleles. b, CRISPR-generated sequences of nbclv3b mutant alleles. Guide RNA and PAM sequences are highlighted in red and bold underlined, respectively. Blue letters and dashes indicate insertions and deletions, respectively. Numbers in parentheses represent gap lengths. c, Shoots and inflorescences of nbclv3a/b T0 plants. Three strong lines (nbclv3a/bCR-3-T0, nbclv3a/bCR-4-T0 and nbclv3a/bCR-5-T0) show similar phenotypes compared to null nbclv3a/b mutants in Fig. 1g. Weak (nbclv3a/bCR-6-T0) and moderate (nbclv3a/bCR-7-T0) lines show regular shoot architecture but fasciated floral organs. White arrowheads indicate flowers. d, Sepal number of weak and moderate nbclv3a/b T0 plants. Box plots, 25th-75th percentile; centre line, median; whiskers, full data range. The letters indicate the significance groups at P < 0.01 (One-way ANOVA and Tukey test). Different letters between genotypes indicate significance (See Supplementary Data 7 for specific P values). The exact sample sizes (n) are shown as discrete numbers. At least twice experiments were repeated independently with similar results.

Extended Data Fig. 2 Conserved non-coding sequence (CNS) analysis of the promoter regions of SlCLV3 and SlCLE9 orthologues in the Solanaceae family.

a, Conservatory analysis of Solanaceae CLV3 and CLE9 promoters. Purple boxes define highly similar regions of each gene’s orthologues in the indicated species, and dark purple boxes define highly similar regions of the paralogous gene (for example CLV3B or CLE9B) in the indicated species. Green boxes define Solanaceae CNSs. b, Multiple alignment and logo sequences of SlCLV3 dodecapeptide orthologues in the Solanaceae family. c, Multiple alignment and logo sequences of SlCLE9 dodecapeptide orthologues in the Solanaceae family.

Extended Data Fig. 3 CRISPR-generated mutations of groundcherry PgCLV3 and PgCLE9.

a, Gene structure and sequences of pgclv3 CRISPR mutants. b, Flowers and fruits of pgclv3. White arrowheads mark petals or locules. Percentages indicate the proportions of flower and fruit phenotypes. Scale bar, 1 cm. c, Gene structure and sequences of pgcle9 CRISPR mutants. The orange rectangles in the gene structures indicate the regions of the CLE dodecapeptides in a and c. Guide RNA and PAM sequences are highlighted in red and bold underlined, respectively, in a and c. Blue letters and dashes indicate insertions and deletions, respectively, in a and c. Numbers in parentheses represent gap lengths in a and c. d, Shoot and an extremely fasciated primary flower of the pgclv3 pgcle9 double mutant. e, Development of extra shoots (S) from the primary shoot and apex of a pgclv3 pgcle9 double mutant. L, leaf petioles. At least twice experiments were repeated independently with similar results.

Extended Data Fig. 4 Sequence alignments of CLV1 receptor homologues.

a, Alignment of the Solanaceae CLV1 protein sequences. Red letters indicate the two ultra-conserved amino acids involved in the physical binding of CLE dodecapeptides. b, Alignment of CLV1 homologues in angiosperms. All the sequences are from the Phytozome v12.1 database (phytozome.jgi.doe.gov). Yellow highlights mark the conserved Asp and Phe. Detailed sequence information is shown in Supplementary Data 10.

Extended Data Fig. 5 Groundcherry pgclv1 pgclv3 and petunia phclv1 phclv3 double mutants are severely fasciated like tomato slclv1 slclv3 double mutants.

a, Gene structure and sequences of two phclv1 CRISPR mutants. Guide RNA and PAM sequences are highlighted in red and bold underlined, respectively. Blue letters and dashes indicate insertions and deletions, respectively. Numbers in parentheses represent gap lengths. b, Flowers, fruits/pods, and meristems from pgclv1, phclv1, and slclv1 single mutants. White arrowheads mark petals or locules. 7L, 7th leaf primordium, 8L, 8th leaf primordium. 22L, 22th leaf primordium. C, Side and top-down views of a pgclv1 pgclv3 double mutant shoot, inflorescence/floral meristem, and primary inflorescence. 6L, 6th leaf primordium. D, Side and top-down views of a phclv1 phclv3 double mutant shoot and primary flower. E, Side and top-down views of a slclv1 slclv3 double mutant shoot, flower, vegetative meristem and primary inflorescence. Fasciated flowers and vegetative meristems are shown in insets of c and e. f, g, Petal (f) and locule (g) numbers of groundcherry WT, pgclv1, pgclv1 pgclv3, pgclv1 pgcle9, and petunia WT, phclv1, and tomato WT, slclv1, slclv1 slclv3, and slclv1 slcle9. Not that all three Solanaceae clv1 single mutant fasciation phenotypes are similarly weak. Box plots, 25th-75th percentile; centre line, median; whiskers, full data range in d and e. The letters indicate the significance groups at P < 0.01 (One-way ANOVA and Tukey test) in f and g. Different letters between genotypes indicate significance in f and g (See Supplementary Data 7 for specific P values). P values (two-tailed, two-sample t-test) in f and g. Exact sample sizes (n) are shown in f and g. At least twice experiments were repeated independently with similar results.

Extended Data Fig. 6 Transgenic complementation tests of tomato slclv3 single and slclv3 slcle9 double mutants.

a, b, Complementation tests of tomato slclv3 single mutants. Inflorescence images (a) and petal number quantifications (b) of WT and slclv3 compared to the T1 transgenic plants gSlCLV3SlCLV3, gPgCLE9PgCLE9, gSlCLE9PgCLE9, gSlCLV3PgCLE9, and gSlCLV3SlCLE9. c, Diagrams of the constructs used for complementation tests of slclv3 slcle9 double mutants. gSlCLV3SlCLV3 (SlCLV3 genomic DNA). gSlCLV3SlCLE9 (SlCLV3 genomic DNA including the sequence for SlCLE9 dodecapeptide). gSlCLE9SlCLE9 (SlCLE9 genomic DNA). gSlCLE9SlCLE9S6G (SlCLE9 genomic DNA including the sequence for PgCLE9 dodecapeptide). Black and orange rectangles mark the coding sequences and the dodecapeptide sequences, respectively. The numbers with minus (-) and plus (+) signs indicate the positions of the upstream sequences and the downstream sequences from the adenines of start codons, respectively. d, Carpel number quantifications of WT, slclv3, slclv3 slcle9 mutants compared to the T1 transgenic plants gSlCLV3SlCLV3-2, gSlCLV3SlCLE9-2, gSlCLE9SlCLE9-2, and gSlCLE9 SlCLE9S6G. Data are based on at least three independent transgenic lines for each construct. Box plots, 25th-75th percentile; centre line, median; whiskers, full data range in b and d. The letters indicate the significance groups at P < 0.01 (One-way ANOVA and Tukey test) in b and d. Different letters between genotypes indicate significance in b and d (See Supplementary Data 7 for specific P values). Exact sample sizes (n) are shown in b and d. At least twice experiments were repeated independently with similar results.

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1, CLE dodecapeptide sequences of SlCLV3 and SlCLE9 homologues. Supplementary Table 2, Differentially expressed genes between petunia WT and phclv3 from mRNA-seq. For the statistical test, Wald test was performed and adjustments were made for multiple comparison. Significant differential expression was identified using padj ≤ 0.05 cut-offs and |log2 ratio| ≥ 1 (Methods). Supplementary Table 3, Differentially expressed genes between groundcherry WT and pgclv3 from mRNA-seq. For the statistical test, Wald test was performed and adjustments were made for multiple comparison. Significant differential expression was identified using q ≤ 0.01 cut-offs (Methods). Supplementary Table 4, Primers used in this study.

Supplementary Data 1–7

Supplementary Data 1, CRISPR-generated mutations in this study. Supplementary Data 2, Quantification data for organ numbers in this study. Supplementary Data 3, Quantification data for meristem size from Figs. 1, 2 and 3. Supplementary Data 4, Normalized counts from mRNA-seq for Figs. 2 and 3. Supplementary Data 5, Syntenic region of SlCLV3 homologues, defined by Conservatory orthogroups. Supplementary Data 6, Syntenic region of SlCLE9 homologues, defined by Conservatory orthogroups. Supplementary Data 7, Exact P values in this study (one-way ANOVA with Tukey test).

Supplementary Data 8–10

Supplementary Data 8, Sequencing trace files. Supplementary Data 9, Petunia PhCLE9 sequence. Supplementary Data 10, CLV1 homologue sequences.

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Kwon, CT., Tang, L., Wang, X. et al. Dynamic evolution of small signalling peptide compensation in plant stem cell control. Nat. Plants 8, 346–355 (2022). https://doi.org/10.1038/s41477-022-01118-w

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