Fig 1.
Mutating crel suppresses the e simple leaf phenotype and produces very compound leaves.
(A-H) Mature 5th leaves of the indicated genotypes. A-C suppression of e by crel-1; D-H: leaves of 5 different crel alleles. Scale bars: 2cm. (I) Early leaf development in the fifth leaf of wild type and crel-2. P4- P7 designate the developmental stages, where P4 is the forth youngest leaf primordium. Scale bars: 0.1 mm (P4), 0.5 mm (P6), 2 mm (P7), 2 cm (expanded leaf). (J) Quantification of the number of leaflets in a mature 5th leaf of the indicated crel alleles, compared to the wild type. 1st, 2nd and 3rd represent primary, secondary and tertiary leaflets, respectivley, where primary leaflets arise from the rachis, secondary leaflets arise from primary leaflets etc. (K) Leaflet production over time by the fifth leaf) L5) of the indicated genotypes.
Fig 2.
CREL acts relatively late in leaf development to promote differentiation and blade growth.
(A-F, I, J) Mature 5th leaves. (K,L) Mature 9th leaves. (G, H) whole plants. Scale bars: 2cm. FIL>>gene refers to genotypes generated by the LhG4-OP transactivation system, where the gene is expressed in the FIL expression domain. The FIL promoter is expressed in leaf primordia [52]; EdII-GUS is a stabilized form of E fused to the GUS reporter; ARF10m is a mutant form of ARF10 that is mutated in the miR160 binding site. A-D—epistasis of La-2/+, in which differentiation is accelerated, over crel. E, F—enhancment of crel by clau, in which differentiation is delayed. G-J–enhancment of genotypes with narrow leaves due to reduced auxin response by crel. K, L–suppression of the ectopic blade growth of FIL>>miR160 by crel, similar to the effect on e.
Fig 3.
CREL encodes a VRN5/VIL1 homolog expressed at late stages of leaf development.
(A) A diagram of the CREL (Solyc05g018390) gene. The boxes indicate exons and the combining lines introns. The location of the mutation in 4 crel alleles is indicated. (B) A phylogenetic tree of the tomato, Arabidopsis, rice and pepper VIL proteins, constructed using MEGA X [54,55] using a Maximum Likelihood method. Branch lenghts represent the expected number of substitutions per site. The blue arrow points to CREL. (C) qRT-PCR analysis of CREL mRNA expression at successive developmental stages of the 5th leaf. m+2, 3, or 4 represents the meristem and the 2, 3, or 4 youngest leaf primordia, respectively. P4-P9 represent isolated leaf primordia at the respective developmental stage (see Fig 1). Error bars represent the SE of at least three biological replicates. (D-G) Confocal images of leaf primordia of the indicated stages, expressing pCREL>>YFP, using the transactivation system, as in Fig 2. P4-P7 represent the 4th-7th youngest leaf primordia, respectively. In G, a leaflet from a P7 primordium is shown. Scale bars: 0.1 mm.
Fig 4.
CREL promotes stem vasculature maturation and differentiation.
(A, B) Whole plants of the indicated genotypes and ages. Scale bars: 10 cm. (C-H) stem cross sections, dissected from the first internode of the plant (between the hypocotyl and first leaves) at the indicated times after germination and stained with Toluidine blue. Scale bars: 500 mm. (I-N) Confocal images of stem cross sections, taken from the first internode of the plant at the indicated times after germination. Yellow arrowheads point to differentiated (WT) or undifferentiated (crel-2) xylem/ vasculature. Scale bars: 100 mm (I,J); 200 mm (L); 500 mm (K,M,N).
Fig 5.
CREL promotes root development and differentiation.
(A-F) Confocal images of root cross sections of the indicated genotypes dissected from the upper part of the primary root. Scale bars: 200μm (A, B, G); 500μm (C-F). (G) Root system volume at different times after germination, calculated with WinRhizo software. Shown are averages and SE of 3 plants (n = 3). Asterisks indicate statistically significant differences between crel-2 and WT, by Student’s t test, *p < 0.05. (H) Root system length at different times after germination (DAG), calculated withWinRhizo software. Shown are averages and SE of 3 plants (n = 3). Asterisks indicate statistically significant differences between crel-2 and WT, by Student’s t test, *p < 0.05.
Fig 6.
CREL promotes flowering, floral organ growth, and differentiation.
(A) Flowering time, measured by number of leaves produced before flowering, of the indicated genotypes. Error bars represent the SE; p-values indicate differences from WT, as determined by Dunnett`s test. n = 12 (wt, crel-1) and 4 (crel-2). (B, C) Stereoscope images of mature flowers. Scale bars: 1mm. (D-G) Scanning electron microscope (SEM) micrographs of the indicated genotypes at 2 early developmental stages. D, F—0.5 mm long stage 6 flowers; E, G—1 mm long stage 11 flowers (according to [57]. Yellow arrowheads point to normal (WT, D) and distorted (crel-2, F) young petals, and to normal (WT, E) and distorted (crel-2, F) stamens and stigma. Scale bars: 1mm (E, G); 0.5mm (D, F).
Fig 7.
CREL mediates H3K27me3 modifications at a subset of polycomb-target sites.
(A) Normalized H3K27me3 signal displayed across all protein-coding genes in WT and crel-2 mutant shoot apices. (B) Heatmaps of H3K27me3 signals at 7,469 sites identified as reproducibly enriched in WT but lost in crel-2 (crel-lost sites). Signals are centered in the middle of each enriched region and extend +/- 1 kb in each direction. (C) Heatmaps of H3K27me3 signals at 1,295 sites identified as reprodicbly enriched in crel-2 plants but not in WT (crel-gained sites), displayed as in (B). (D) Average plots of WT and crel-2 H3K27me3 at the 7,469 crel-lost sites shown in panel B. (E) Average plots of WT and crel-2 H3K27me3 at the 1,295 crel-gained sites shown in panel C.
Fig 8.
Changes in H3K27me3 deposition in crel mutant plants correlate to gene expression changes as measured by RNA-Seq.
(A) Differential gene expression analysis of WT and crel shoot apices by RNA-seq. These data are illustrated in a volcano plot, with purple dots representing upregulated transcripts (adjusted p value < Bonferroni corrected significance threshold of 1.4e-6 and log2 fold change > 0.6) and yellow dots representing downregulated transcripts (adjusted p value < 1.4e-6 and log2 fold change < -0.6). Vertical dashed lines delineate log2 fold change thresholds of -0.6 and +0.6. Four hundred and thirty one (431) transcripts were significantly upregulated and 265 were significantly downregulated in crel plants compared to WT. (B) Venn diagram comparing the overlap of upregulated transcripts (purple) and downregulated transcripts (yellow) to genes associated with H3K27me3 peaks unique to WT (crel-lost sites; green) and unique to crel-2 (crel-gained sites; green). The black box highlights overlap between upregulated transcripts and genes associated with WT unique peaks (crel-lost sites).