Evolution of buffering in a genetic circuit controlling plant stem cell proliferation


Precise control of plant stem cell proliferation is necessary for the continuous and reproducible development of plant organs1,2. The peptide ligand CLAVATA3 (CLV3) and its receptor protein kinase CLAVATA1 (CLV1) maintain stem cell homeostasis within a deeply conserved negative feedback circuit1,2. In Arabidopsis, CLV1 paralogs also contribute to homeostasis, by compensating for the loss of CLV1 through transcriptional upregulation3. Here, we show that compensation4,5 operates in diverse lineages for both ligands and receptors, but while the core CLV signaling module is conserved, compensation mechanisms have diversified. Transcriptional compensation between ligand paralogs operates in tomato, facilitated by an ancient gene duplication that impacted the domestication of fruit size. In contrast, we found little evidence for transcriptional compensation between ligands in Arabidopsis and maize, and receptor compensation differs between tomato and Arabidopsis. Our findings show that compensation among ligand and receptor paralogs is critical for stem cell homeostasis, but that diverse genetic mechanisms buffer conserved developmental programs.

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Fig. 1: Buffering of stem cell homeostasis in tomato depends on transcriptional compensation from SlCLE9.
Fig. 2: Arabidopsis stem cell homeostasis is controlled by multiple redundant CLE genes.
Fig. 3: SlCLE9 compensation acts primarily through the receptor kinase SlCLV1.
Fig. 4: Buffering-impacted tomato domestication alongside a dynamic evolution of active compensation in flowering plants.

Data availability

Raw data for all quantifications are included as Supplementary Tables. All RNA-seq data from tomato are available from the National Center for Biotechnology Information. The tomato Sequence Read Archive (SRA) project and BioProject accession nos. are SRP161864 and PRJNA491365, respectively. The maize SRA projects and BioProject accessions numbers are SRR7970748, SRR7970747, SRR7970749, SRR7970750 and PRJNA494874, respectively. RNA-seq data from Arabidopsis was obtained from Klepikova et al.54 and Mandel et al.55.


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We thank C. Brooks, A. Krainer and J. Dalrymple for their technical support; P. Keen for assistance with the tomato transformation; T. Mulligan, S. Vermylen and S. Qiao from the Cold Spring Harbor Laboratory, and staff from Cornell University’s Long Island Horticultural Research and Extension Center, for assistance with plant care; and H. Shinohara and Y. Matsubayashi from Nagoya University for the tomato peptide binding assays. Initial work from Z.L.N. was supported by funds from the Virginia Polytechnic Institute and State University. The research for this study was supported by: a PEW Latin American Fellowship (no. 29661) to D.R.L.; a NIGMS MIRA award from the National Institutes of Health to Z.L.N. (award no. R35GM119614-01); an Agriculture and Food Research Initiative competitive grant no. 2016-67013-24572 of the USDA National Institute of Food and Agriculture and the Next-Generation BioGreen 21 Program SSAC (grant no. PJ01322602) from the Rural Development Administration, Republic of Korea to D.J.; an Agriculture and Food Research Initiative competitive grant no. 2015-67013-22823 of the USDA National Institute of Food and Agriculture to Z.B.L.; and the National Science Foundation Plant Genome Research Program (grant no. IOS-1732253 to J.V.E. and Z.B.L., and grant no. IOS-1546837 to D.J., M.E.B., Z.L.N and Z.B.L.).

Author information




These authors contributed equally: C.-T.K., C.S., E.D.A. and J.M. D.R.L. performed the tomato CRISPR and Arabidopsis molecular and phenotypic analyses, prepared the figures and wrote the manuscript. C.X. performed the tomato CRISPR and complementation experiments, genetic and phenotypic analyses, prepared the figures and helped edit the manuscript. C.T.K. performed the tomato CRISPR experiments, and the genetic, RNA-seq and phenotypic analyses. C.S. generated the Arabidopsis CRISPR knockouts, performed the plant transformations and the genetic and phenotypic analyses. E.D.A. performed the maize CRISPR experiments and the genetic and phenotypic analyses. L.L. performed the maize RNA-seq analyses. J.M. performed the synteny and CLE clustering analyses. Z.H.L. performed the tomato RNA-seq analyses. D.S.J. performed the Arabidopsis genetic and phenotypic analyses. J.V.E. performed the tomato transformations. D.P.J. conceived and supervised the maize research and edited the manuscript. M.E.B. conceived, designed and guided the synteny and CLE clustering experiments and helped prepare the figures and write the manuscript. Z.L.N. conceived and supervised the Arabidopsis research and wrote and edited the manuscript. Z.B.L. conceived and led the research, supervised and performed the experiments, and wrote the manuscript.

Corresponding authors

Correspondence to David P. Jackson or Madelaine E. Bartlett or Zachary L. Nimchuk or Zachary B. Lippman.

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Integrated supplementary information

Supplementary Fig. 1 CRISPR–Cas9 targeting and genetic analysis of SlCLE3.

a, CRISPR/Cas9 design and sequencing of targeted regions in SlCLV3, SlCLE9 and SlCLE3. Black boxes indicate exons. Red arrows, single-guide RNAs (gRNAs; Target 1 and Target 2). Black arrows, forward (F) and reverse (R) primers for PCR genotyping and sequencing. Sequences of slclv3, slcle9 and slcle3 mutant alleles 1 and 2 (a1 and a2) identified from T0 plants. gRNA target sequences in red, and protospacer adjacent motif sequences in bold and underlined. Blue dashed lines, deletions. Space between each target region is indicated in parentheses. b, Representative inflorescences from axillary shoots in slclv3, slclv3 slcle9, slclv3 slcle9 slcle3 and slcle9 slcle3 mutants. Red arrowheads, branches. c, RT–qPCR of SlCLE3 from reproductive meristems of WT and slclv3 normalized to SlUBI. Mean ± s.e.m.; two biological and three technical replicates). d, Quantification of locule number in slcle9 and slcle9 slcle3, slclv3 slcle9 and slclv3 slcle9 slcle3 (n = 166, 31, 34 and 28). Boxplots, 25th–75th percentile; whiskers, full data range; center line, median. One-way ANOVA and Tukey; letters represent significance groups at P < 0.05 in d. Scale bar, 2 cm in c.

Supplementary Fig. 2 Expression analysis and CRISPR–Cas9 multiplex targeting of Arabidopsis CLE gene family members.

a, RNA-seq data of all CLE family members in WT meristems from 7 to 16 d after germination (DAG). Expression levels represented as total gene reads normalized by size factor for each CLE gene. Data are from Klepikova et al.54. b, RNA-seq data of all Arabidopsis CLE family members in single biological replicates at 8 and 10 DAG from WT and clv3. Expression levels are shown as reads per kilobase of transcript per million mapped reads. Data are from Mandel et al.55. c, CRISPR/Cas9 generated mutations in 11 Arabidopsis CLE genes in the clv3 background. In red, gRNA target sequences. Protospacer adjacent motif sequences are bold and underlined. Space between each target region is indicated in parenthesis. Blue dashed lines, deletions. Blue letters indicate nucleotide insertions compared to WT. Asteriks, in-frame mutations. The in-frame mutation within CLE11 removes the first three amino acids of the dodecapeptide and is therefore likely a null allele. The second mutation in CLE19 restores frame after an intervening mutant sequence of more than 25 amino acids.

Supplementary Fig. 3 Arabidopsis passive CLE compensation is mediated by CLV1 and BAM receptors.

a,b, Representative confocal micrographs and meristem quantifications (perimeter, height, width) of WT, clv1, clv3, clv1 clv3, bam1/2/3, clv3 bam1/2/3 and clv1 bam1/2/3 (n = 16, 15, 15, 15, 15, 15, 11, and 13). c, Carpel number quantifications of same genotypes as in b, (n = 150, 116, 150, 150, 150, 144, 77, 50). One-way ANOVA and Tukey test; letters represent significance groups at P < 0.05 in b and c. Scale bar, 50 µm in a.

Supplementary Fig. 4 CRISPR–Cas9 targeting and genetic analysis of the leucine‐rich repeat receptor genes in tomato.

a, CRISPR–Cas9 design and sequencing of targeted regions in SlCLV1, SlCLV2, SlBAM1 and SlBAM4. Black boxes, exons. Red arrows, gRNAs (Target 1 and Target 2). Black arrows, forward (F) and reverse (R) primers for PCR genotyping and sequencing. Sequences of T0 plants from CR-slclv1, CR-slclv2, CR-slbam1 and CR-slbam4 mutant alleles 1 and 2 (a1 and a2, respectively). gRNA target sequences in red, and protospacer-adjacent motif sequences in bold and underlined. Blue dashed lines, deletions. Space between each target region is indicated in parentheses. b, Representative inflorescences from single and higher-order mutants. Red arrowheads, branches. c, Fold change in expression of SlBAM1, SlBAM2, SlBAM3 and SlBAM4 relative to WT RNA-seq from single slclv1, slclv3 and double mutants slclv1 slclv3 and slcv3 slcle9 (two biological replicates, 4-fold change, 1 cpm cutoff, FDR < 0.10). d, Quantification of locule number from single and higher-order mutants (n = 104, 94, 92, 35, 43, 166, 77, 116, 58, 113, 38, 35, 63, 24 and 34). Blue dashed line, WT mean. Boxplots, 25th–75th percentile; whiskers, full data range; center line, median. One-way ANOVA and Tukey; letters represent significance groups at P < 0.05 in d. Scale bar, 2 cm in b.

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Rodriguez-Leal, D., Xu, C., Kwon, CT. et al. Evolution of buffering in a genetic circuit controlling plant stem cell proliferation. Nat Genet 51, 786–792 (2019). https://doi.org/10.1038/s41588-019-0389-8

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