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Dissection of floral transition by single-meristem transcriptomes at high temporal resolution

Nature Plants volume 7, pages 800–813 (2021)Cite this article

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Abstract

The vegetative-to-floral transition is a dramatic developmental change of the shoot apical meristem, promoted by the systemic florigen signal. However, poor molecular temporal resolution of this dynamic process has precluded characterization of how meristems respond to florigen induction. Here, we develop a technology that allows sensitive transcriptional profiling of individual shoot apical meristems. Computational ordering of hundreds of tomato samples reconstructed the floral transition process at fine temporal resolution and uncovered novel short-lived gene expression programs that are activated before flowering. These programs are annulled only when both florigen and a parallel signalling pathway are eliminated. Functional screening identified genes acting at the onset of pre-flowering programs that are involved in the regulation of meristem morphogenetic changes but dispensable for the timing of floral transition. Induced expression of these short-lived transition-state genes allowed us to determine their genetic hierarchies and to bypass the need for the main flowering pathways. Our findings illuminate how systemic and autonomous pathways are integrated to control a critical developmental switch.

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Fig. 1: Short-lived gene programs define the floral transition of tomato SAMs.
Fig. 2: Flowering is delayed but transition programs remain intact in florigen-impaired plants.
Fig. 3: The florigen-independent DST pathway regulates flowering time via FALSIFLORA.
Fig. 4: Ever-vegetative dst sft apices largely maintain a young vegetative shoot program.
Fig. 5: Genes activated at the early transition stage impact SAM and inflorescence development.
Fig. 6: UF is a prime regulator of SAM floral transition programs.
Fig. 7: Transient SAM programs can restore flowering to ever-vegetative shoots.

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Data availability

The raw and transformed data are available from the Gene Expression Omnibus (accession GSE166929). To view and explore this dataset, see the interactive website at https://tanaylab.weizmann.ac.il/SMT/.

Code availability

For the slanting vignette, R package and source code, see https://github.com/tanaylab/slanter. Code for the analysis performed in this work can be found on our group’s GitHub page at https://github.com/tanaylab/Meir_et_al_nat_plants_2021_SMT/.

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Acknowledgements

We thank Z. Lippman, I. Efroni and members of the Tanay and Eshed groups for their valuable input, and Z. Amsellem, E. Chomsky and A. Lifshitz for technical assistance. This work was supported by Israel Science Foundation research grants 1913/19 and 2731/16 to Y.E. and European Research Council grant 712749 (scAssembly) to A.T.

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Author notes

  1. These authors contributed equally: Zohar Meir, Iris Aviezer.

Authors and Affiliations

  1. Faculty of Mathematics and Computer Science and Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

    Zohar Meir, Oren Ben-Kiki, Zohar Mukamel & Amos Tanay

  2. Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel

    Zohar Meir, Iris Aviezer, Grace Lhaineikim Chongloi, Revital Bronstein, Lior Tal, Gili Shalev-Schlosser, Tom Hai Harel & Yuval Eshed

  3. Department of Immunology, Weizmann Institute of Science, Rehovot, Israel

    Hadas Keren-Shaul & Diego Jaitin

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Contributions

Z. Meir, I.A., A.T. and Y.E. designed the study. I.A., G.L.C., R.B., L.T., G.S.-S., T.H.H. and Y.E. performed the genetic experiments and phenotypic analyses. Z. Meir and I.A. developed the SMT protocol with input from Z. Mukamel, H.K.-S. and D.J. Z. Meir performed the computational analyses. O.B.-K. developed the matrix slanting algorithm. A.T. and Y.E. guided the computational and experimental work, respectively. Z. Meir, A.T. and Y.E. wrote the manuscript with input from all authors.

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Correspondence to Amos Tanay or Yuval Eshed.

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Peer review information Nature Plants thanks Junko Kyozuka, Markus Schmid and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Quality controls for single meristem transcriptome (SMT) analysis of WT apices.

a) Distribution of morphology-based scores during random sampling of plants sown together and scored at five consecutive days (10-14 Days after germination - DAG). Bars showing fractions of stages observed in each day, out of total plants examined (n10 = 33, n11 = 34, n12 = 43, n13 = 38, n14 = 18). SE computed based on binomial sampling. Sample means below each distribution demarcate the averaged DAG each stage was observed during this sampling window. b-e) Example for standard MARS-seq quality control27 of single wells in SMT plate. Panel b showing distribution of reads between wells and c provide the respective distribution of UMIs, after mapping and removal of duplicates. d is showing UMIs obtained per well from External RNA Control Consortium (ERCC) spike-in (x-axis) and gene-mapped UMIs (y-axis). Red X-sign indicate designated empty wells. e shows distribution of the number of reads covering each UMI, as indication for low degree of complexity saturation at this sequencing rate. f) Mappability in two consecutive SMT seasons (with two different RNA extraction strategies, as specified in Methods). Showing distributions of mapping fractions for first (light-grey, left side of each violin plot) and second (dark-grey, right side of each violin plot) collection seasons. Median values depicted as black horizontal lines. n1st = 298, n2d = 650. g) Distribution of total-reads per meristem (log-scaled, after aggregation of technical replicates) in SMT MARS-seq plates processed during two collection seasons. h) Same as g, showing UMIs-per-meristem. For both g and h, the number of meristems assayed in each plate depicted above each distribution. i) Comparison between fraction of morphology-based scores of WT apices collected during unbiased sampling (light bars) and out of meristems processed by SMT in two consecutive seasons (darker bars). Bars show fractions and SE is based on binomial sampling. nSMT = 505, nunbiased = 166.

Extended Data Fig. 2 KNN-normalization of batch effects and identification of stage-related gene expression variance.

a) Scaled expression of two batch-related patterns in 207 WT meristems. Meristems shown as columns, and annotation on top indicate their morphology-based score. Names of selected genes are shown on right side of matrix (all genes are listed in Supplementary Table 1). Dots below matrix mark five dissection days of WT apices for SMT (note the skewed distributions and see statistical tests in d). b) Example for K-nearest-neighbors (KNN) normalization scheme. X- and y-axes are show normalized expression (UMI per 100 K UMIs) coming from the two gene modules depicted in a, and individual WT meristems are shown as dots. Three blue dots indicate selected meristems and red dots exemplify their K nearest neighbors (see Methods for more details). c) Normalized variance in gene expression without (top panel) and with KNN-normalization (middle panel). Lower panel is showing slight increment in variance following a randomized KNN-normalization control. Red dots indicate the number of genes with normalized variance above a uniform threshold (x1.1 - x + 1.35) in the three datasets. d) log2-ratio of total normalized gene expression (UMIs per 100k UMIs) between two gene modules shown in a, stratified by date of dissection (left), morphology-based score (middle) and number of leaves produced by meristems (right). Two-sided Kolmogorov-Smirnov test (***, P < 0.001; **, P < 0.01; no significant difference for unlabeled comparisons). Note the significant impact of dissection day, suggesting condition dependent coordinated expression. D29/07–26/08 = 0.36, P29/07–26/08 = 0.003; D29/07–02/10 = 0.4, P29/07–02/10 = 5*10−4; D29/07–03/10 = 0.51, P29/07–03/10 = 5*10−5; D31/07–26/08 = 0.43, P31/07–26/08 = 9*10−4; D31/07–02/10 = 0.47, P31/07–02/10 = 1*10−4; D31/07–03/10 = 0.54, P31/07–03/10 = 7*10−5. e) Distribution of Spearman’s correlation between normalized expression of 719 out of 956 marker genes depicted in c (with ρ > 0.6 to at least one gene), and assigned (black line) or randomly assigned (grey line) morphology-based scores given to meristems. Dashed vertical lines indicate threshold with ρ > 0.3 or ρ < −0.3. f) A representative gene module that maintain intra-stage gene expression co-variance. Top panel is showing scaled expression in 207 WT meristems of a representative gene module found by clustering of 156 stage-related genes marked in e. Lower panel is showing Spearman’s gene-gene correlation of this module within intermediate stages. Note the residual expression co-variance within stages.

Extended Data Fig. 3 Ordering of WT meristems by slanting.

a-c) Stepwise selection of stage marker genes by identification of anchor markers and subsequent refinement of best marker set and best meristem order. a) Distributions of summed normalized expression (UMI per 100k UMIs) for 50 gene modules in meristems grouped by their morphology-based scores. Modules obtained by K-means clustering analysis of 719 variant genes (including only genes with Spearman’s correlation higher than 0.6 to at least one gene, out of 956 variable genes depicted in Extended Data Fig. 2c). Upper panel is showing 12 clusters with clear stage-associated variance. Median values displayed as white dots. nVM = 23, nTM0 = 41, nTM1 = 37, nTM2 = 23, nLTM = 44, nEFM = 39. b) Log2 expression fold-change for same gene modules depicted in a, in meristems pooled either by morphology-based scores (left) or by randomly assigned scores (right). Numbers to the right indicate gene module-ID as in a. Randomization of stages strongly reduced variance of 12 gene modules selected as stage anchors (upper panel). c) Ordering of WT meristems by slanting. Top panel is showing inter-meristem Spearman’s correlations in five ordering solutions, and lower panel compare ranks of specific meristems in two consecutive orders. Dots indicate single meristems, colored by one of six morphology-based scores. Refinement of feature genes in each iteration relied on loss of expression variance following KNN-normalization, when selecting the K neighboring meristems based only on the pseudo-order itself (see Methods). The number of feature genes used for computing correlations in each iteration is indicated above each matrix. d) Comparison of meristem ranks, showing overall changes in ordering following filtering of feature genes described in c.

Extended Data Fig. 4 Quality controls for SMT analysis of sft apices.

a) Representative images for six morphology-based developmental scores assigned for each sft meristem. Dashed white lines demarcate dissection region. Mean and SE of days after germination (DAG) per stage computed from distributions shown in b. Scale bar = 100 μm. b) Distributions of six morphology-based scores during random sampling of sft plants at seven consecutive days (15-21 DAGs). Bars showing fractions of stages observed in each day and SE based on binomial sampling. For comparison, dashed horizontal lines indicate the respective averaged DAG of each stage in wild-type plants. n15 = 56, n16 = 51, n17 = 57, n18 = 56, n19 = 69, n20 = 24, n21 = 20. c) Comparison between fraction of morphology-based scores of sft apices collected during unbiased sampling (light bars) and out of meristems processed by SMT (darker bars). Bars display fractions and SE computed based on binomial sampling. nSMT = 162, nunbiased = 333. d) Comparison of log2 total-UMIs obtained from one randomly selected half of the replicates of sft meristems (denoted as replicate #1, x-axis) to the other half (replicate #2, y-axis). Single meristems are shown as dots and colored by morphology-based score. X-signs indicate replicates of designated empty wells. e) Spearman’s correlations between normalized expression levels (UMI per 100 K UMIs) of selected genes in two parts comprised of randomly selected half of the libraries. 155 sft meristems (where both replicates covered by at least 100 K UMIs) are shown as dots and colored by morphology-based score.

Extended Data Fig. 5 Comparison of gene expression between sft and WT SAMs by temporal alignment.

a) Position of each sft meristem in its resolved order (x-axis, as in Fig. 2b), and the position of its assigned WT-sister meristems according to the wild-type order defined in Fig. 1e. For further details and on temporal alignment see Methods and schematics in Supplementary Fig. 3. b) Distribution of the number of occurrences each WT meristems was selected as a sister to sft meristem (showing only WT meristems selected at least once). c) Spearman’s gene-gene correlation of 288 feature genes within sft meristems (upper triangle) and within matching WT-sister meristems (lower triangle), showing conserved and synchronized expression patterns of most stage marker genes. d) Scaled expression of 27 out of 288 marker genes that showed low correlation between WT and sft (complementary to Fig. 2c). e) Differential expression comparisons between WT and sft meristems pooled by matching developmental phases (top panel showing earliest veg phase and bottom latest FI phase). Definition of phases in both genotypes are as denoted in Fig. 2b. UMI counts in pools were down-sampled prior to each comparison (to the lower sum of UMIs between both pools in each case). 20 UMIs were added to each gene as regularization constant. X-axis showing log2-ratio between down-sampled counts (positive ratio – higher expression in sft, negative ratio – higher in WT). Y-axis show average of log2 total-UMI counts of each gene in the two compared pools. Top- and lowest- 10 genes in each comparison are highlighted and annotated.

Extended Data Fig. 6 The DELAYED SYMPODIAL TERMINATION (DST) gene.

a) Images of wild-type (left) and ever-vegetative dst sft (right) plants. The latter did not show any evidence for flowering after growing in our greenhouses for more than one year and producing over 70 leaves. b) Rescue of flowering by grafting a p35::SFT donor on an ever-vegetative dst sft recipient stock. Site of grafting is marked by white rectangle and red arrow demarcates inflorescence in a florigen-recipient branch. c) The DST gene was identified by map-based cloning using an F2 population of a cross between dst-1 and Solanum pimpinellifolium (LA1589). After mapping the gene to a narrow region, sequences of candidate genes of WT were compared with mutant lines and multiple lesions were identified in Solyc03g006900. Top panel: A list of mutated DST alleles indicating (from left to right) allele ID, description of lesion location (G1954A – replacement of Guanine by Adenosine at nucleotide positioned +1954 from TSS), as well as expected impact on the DSTa form (see below). Lower panel: DST gene structure. Three forms of DST transcripts (denoted DSTa-c) were recovered. dst alleles are annotated and marked according to the top panel; red - nonsense and frame-shift, black – missense mutations. UTRs and exons displayed by white and black boxes, respectively. Lines indicate introns and numbers specify length (bp). d) Protein sequence and conserved domains in DSTa. A Myb/SANT-like DNA-binding domain is depicted in blue whereas a Protein kinase C (PKC) catalytic domain is depicted in red. Alleles listed in c are boxed. e-f) cDNA of DST was cloned and used to prepare antisense probe for RNA in-situ hybridization (region is shown in f and primers are listed in Supplementary Table 11). Signal appears as dark purple color in panel e for longitudinal (top) and transverse (bottom) sections of vegetative apices, it is confined to leaf and leaflet primordia and is missing from the SAM center. LP – leaf primordia. Asterisk – Shoot apical meristem. g) Upper panel is showing temporal expression patterns of the DST gene in RNA-seq dataset published by Park et al25 in young primordia (P3) and shoot apical meristems collected at different stages along with P1 and P2. Lower panel is showing DST expression within the temporal range collected for SMT analysis – spanning LVM and TM stages. EVM - early Vegetative, MVM - middle vegetative, LVM – late vegetative, SIM - Sympodial inflorescence and SYM – Sympodial meristem.

Extended Data Fig. 7 Quality controls for SMT analysis of dst apices.

a) Representative images for six morphology-based developmental scores assigned for each dst meristem. Dashed white lines demarcate dissection region. Mean and SE of days after germination (DAG) per stage computed from distributions shown in b. Scale bar = 100 μm. b) Distribution of six morphology-based scores during random sampling of dst plants at six consecutive days (15-20 DAGs). Bars showing fractions of stages observed in each day and SE of binomial sampling. For comparison, dashed horizontal lines indicate the respective averaged DAG of each stage in WT plants. n15 = 54, n16 = 59, n17 = 56, n18 = 59, n19 = 60, n20 = 58. c) Fractions of morphology-based scores of dst apices collected during unbiased sampling (light bars) and out of meristems processed by SMT (darker bars). Bars display fractions of each score and SE derived from binomial sampling. nSMT = 157, nunbiased = 346. d) Comparison of log2 total-UMIs obtained from one half of the replicates in each dst meristem (denoted as replicate #1, x-axis) to the other half (replicate #2, y-axis). Single meristems are shown as dots and colored by morphology-based score. X-signs indicate replicates of designated empty wells. e) Correlations between normalized expression levels (UMI per 100 K UMIs) of selected genes in two groups of technical replicates. 148 dst meristems (with both technical replicates are covered by at least 100 K UMIs) are shown as dots and colored by morphology-based score. Rho and ‘r’ specifies Spearman’s and Pearson’s correlation coefficients, respectively.

Extended Data Fig. 8 Quality controls for SMT, and age analysis of ever-vegetative dst sft apices.

a) Comparison of log2 total-UMIs obtained from one half of the replicates in 48 dst sft meristem (denoted as replicate #1, x-axis) to the other half (replicate #2, y-axis). Single meristems are shown as dots and colored by the vegetative morphology-based score assigned to all dst sft apices. X-signs indicate replicates of designated empty wells. Rho specifies Spearman’s correlation. b) Correlations between normalized expression levels (UMI per 100 K UMIs) of selected variable genes in two randomly selected groups of technical replicates. 44 dst sft meristems (with both replicates covered by at least 100 K UMIs) are shown as dots and colored by morphology-based score. Rho and ‘r’ specifies Spearman’s and Pearson’s correlation coefficients, respectively. c) Images of an age series of dst sft apices that produced of 6-34 leaves. Dashed horizontal grey line shows estimated distance between primordia bases as a crude estimation for meristem size. L - last leaf produced by the imaged meristem. Scale bar = 100 μm. d) Differential expression between pooled dst sft meristems and WT meristems at the vegetative phase. X-axis showing log2-ratio between down-sampled counts (positive ratio – higher expression in WT, negative ratio – higher in dst sft). Y-axis show average of log2 total-UMI counts of each gene in the two compared pools. Top- and lowest- 10 genes in each comparison are highlighted and annotated. Note the downregulation of the FUL1, FUL2 and MC MADS box genes as well as FA (LFY), and the higher expression of the CHR 4 gene cluster. e) Analysis of age-dependent gene expression in vegetative SAMs. Showing scaled expression of 195 genes that show consistent Spearman’s correlation with number of leaves produced in vegetative SAMs (ρ > 0.25 in dst sft apices, ρ > 0.25 in 190 WT, sft and dst SAM at the vegetative phase, Supplementary Table 7). 574 meristems of four genotypes are shown as columns, and ordered according to their resolved temporal lineages. Top panel is showing 155 of the age-dependent genes (79%) with low Spearman’s correlation to order of WT meristems (−0.35 < ρ < 0.35). Lower panel display scaled expression of 40 genes that maintain both age-dependent and transition-dependent dynamics. Dots below matrix indicate the number of leaves produced by each meristem, colored by genotype. f) Normalized expression (UMI per 100k UMIs) in 574 WT, sft, dst and ever vegetative dst sft meristems. Top panel showing two genes of the SPL clade displaying strong evidence to age-related expression dynamics. Lower panel shows expression of S-WOX9 and HAN which is specifically linked to the process of floral transformation and abolished in ever-vegetative meristems. Developmental phases in all genotypes are shown as background colors, and color of dots indicates the initial morphology-based score assigned to each meristem.

Extended Data Fig. 9 Quality controls for SMT analysis of uf apices.

a) Images of uf apices corresponding to seven morphology-based developmental scores, including elongated TM2 (TM2E) detailed in Fig. 5e,f. Dashed white lines demarcate dissection region. Mean and SE of days after germination (DAG) per stage computed from distributions shown in b. Scale bar = 100 μm. b) Distribution of seven morphology-based scores assigned for uf apices during random sampling of uf plants at four consecutive days (9-12 DAGs). Bars showing fractions of stages observed in each day and SE of binomial sampling. Horizontal dashed lines indicate the respective averaged DAG of each stage in wild-type plants. n9 = 30, n10 = 31, n11 = 28, n12 = 14. c) Fractions of morphology-based scores assigned to uf apices that were collected during unbiased sampling shown in b (light bars) and out of meristems processed by SMT (darker bars). Bars display fractions and SE is computed based on binomial sampling. nSMT = 76, nunbiased = 103. d) Fractions of SAMs classified as at flowering meristem (FM) stage or later. The four genotypes were sampled over time as marked by the x-axis (red line indicates no data was collected at this DAG for the respective genotype). Bars indicate means. SE is based on binomial sampling. e) Comparison of log2 total-UMIs obtained from randomly selected half of the replicates in each uf meristem (denoted as replicate #1, x-axis) to the other half (replicate #2, y-axis). Single meristems are shown as dots and colored by morphology-based score. X-signs indicate replicates of designated empty wells. f) Spearman’s correlations between normalized expression levels (UMI per 100 K UMIs) of selected genes in two groups of technical replicates. 75 uf meristems (with both replicates covered by at least 100 K UMIs) are shown as dots and colored by morphology-based score. g) Image of line overexpressing UF under the constitute driver 35 S, showing severe developmental defects. h) Differential expression between pooled uf-tr meristems and WT meristems at trI and trII phases. X-axis showing log2-ratio between down-sampled counts (positive ratio – higher expression in uf meristems, negative ratio – higher in WT). Y-axis show average of log2 total-UMI counts of each gene in the two compared pools. Genes with highest expression fold-change are highlighted and annotated. Note the upregulation of CLV3 and CLE9, and downregulation of S-WOX9 and PUCHI in uf. i) Normalized expression (UMI per 100k UMIs) of CLE9 and CLV3 in 650 WT, sft, dst, uf and ever vegetative dst sft meristems. Developmental phases are shown as background colors. Meristems are shown as dots and colored by initial morphology-based score assigned to them.

Extended Data Fig. 10 Rescue of ever-vegetative plants and genetic dissection of floral transition programs.

a) Flowering time (counted by leaf number) in ever-vegetative dst sft plants expressing S-WOX9 or UF by the TCS promoter, or FUL2 by the constitutive 35 S promoter. N.A. indicates flowering is not observed even at >50 leaves. Dots indicate individual plants and bars showing mean and SE. b) Ectopic expression of the florigen-target MADS-box gene FUL2 is insufficient to rescue flowering in ever-vegetative dst sft plants. Wild-type (left), dst sft (middle) and p35S::FUL2 dst sft (right). Arrows mark inflorescences of the main (red) or sympodial (cyan) shoots. c) Summary of genetic hierarchies identified for components analysed in this study. Upper panel: Gradual buildup of signals via the DST florigen-independent pathway is largely mediated by FA. Middle panel: S-WOX9 acts downstream of UF. Lower panel: activation of local transition programs by S-WOX9 or UF bypasses the need for accumulation of signals acting downstream of floral transition boosters.

Supplementary information

Supplementary Information

Supplementary Figs. 1–7.

Reporting Summary

Supplementary Tables

Supplementary Table 1 List of gene modules maintaining stage-independent expression variance between individual SAMs. Supplementary Table 2 List of 288 classified stage-anchor genes and other annotated transition markers. Supplementary Table 3 Differential gene expression in matched pools of WT and sft SAMs. Supplementary Table 4 List of genetic markers used for whole-genome single-nucleotide polymorphism genotyping of dst-1 (e137) and its background genetic modifiers that restore fertility. Supplementary Table 5 Differential gene expression in matched pools of WT and dst SAMs. Supplementary Table 6 Differential gene expression in matched pools of WT and dst sft (ever-vegetative) SAMs. Supplementary Table 7 Correlations of gene expression with the number of leaves produced by SAMs. Supplementary Table 8 Screen for early-activated genes during SAM floral transition of WT plants (Fig. 5a,b and Supplementary Fig. 6a,b). Supplementary Table 9 Screen for early-activated genes during SAM floral transition of sft plants (Supplementary Fig. 6c–f). Supplementary Table 10 Screen for early-activated genes during SAM floral transition of dst plants (Supplementary Fig. 6g–j). Supplementary Table 11 List of primers used for cloning of cDNA, promoters and guide RNAs for CRISPR–Cas9-targeted mutagenesis of early-acting transition genes. Supplementary Table 12 CRISPR–Cas9-derived alleles of early-activated genes during transition. Supplementary Table 13 Differential gene expression in pools of WT and uf SAMs at early transition phases.

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Meir, Z., Aviezer, I., Chongloi, G.L. et al. Dissection of floral transition by single-meristem transcriptomes at high temporal resolution. Nat. Plants 7, 800–813 (2021). https://doi.org/10.1038/s41477-021-00936-8

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