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The VIL gene CRAWLING ELEPHANT controls maturation and differentiation in tomato via polycomb silencing

['Ido Shwartz', 'Institute Of Plant Sciences', 'Genetics In Agriculture', 'The Robert H. Smith Faculty Of Agriculture', 'Food', 'Environment', 'The Hebrew University Of Jerusalem', 'Rehovot', 'Chen Yahav', 'Neta Kovetz']

Date: 2022-05

VERNALIZATION INSENSITIVE 3-LIKE (VIL) proteins are PHD-finger proteins that recruit the repressor complex Polycomb Repressive Complex 2 (PRC2) to the promoters of target genes. Most known VIL targets are flowering repressor genes. Here, we show that the tomato VIL gene CRAWLING ELEPHANT (CREL) promotes differentiation throughout plant development by facilitating the trimethylation of Histone H3 on lysine 27 (H3K27me3). We identified the crel mutant in a screen for suppressors of the simple-leaf phenotype of entire (e), a mutant in the AUX/IAA gene ENTIRE/SlIAA9, involved in compound-leaf development in tomato. crel mutants have increased leaf complexity, and suppress the ectopic blade growth of e mutants. In addition, crel mutants are late flowering, and have delayed and aberrant stem, root and flower development. Consistent with a role for CREL in recruiting PRC2, crel mutants show drastically reduced H3K27me3 enrichment at approximately half of the 14,789 sites enriched in wild-type plants, along with upregulation of many underlying genes. Interestingly, this reduction in H3K27me3 across the genome in crel is also associated with gains in H3K27me3 at a smaller number of sites that normally have modest levels of the mark in wild-type plants, suggesting that PRC2 activity is no longer limiting in the absence of CREL. Our results uncover a wide role for CREL in plant and organ differentiation in tomato and suggest that CREL is required for targeting PRC2 activity to, and thus silencing, a specific subset of polycomb targets.

Plants form organs continuously throughout their lives, and the number and shape of their organs is determined in a flexible manner according to the internal and external circumstances. Alongside this flexibility, plants maintain basic developmental programs to ensure proper functioning. Among the ways by which plants achieve flexible development is by tuning the pace of their maturation and differentiation, at both the plant and organ levels. One of the ways plants regulate the rate of maturation and differentiation is by changing gene expression. Here, we identified a gene that promotes plant and organ maturation and differentiation. This gene, CRAWLING ELEPHANT (CREL) acts by bringing a repressing complex to target genes. We show the importance of CREL in multiple developmental processes and in the expression of multiple genes throughout the tomato genome.

Funding: This work was supported by grants from the United States – Israel Binational Science Foundation (BSF, 2015093) to N.O and R.B.D., German-Israel Project Cooperation Foundation (OR309/1-1;FE552/12-1) to N.O. and J.M.J.-and from the U.S. – Israel Binational Agricultural Research and Development Fund (IS4531-12(c) and IS5103-18R) and the Israel Science Foundation (grants number 2407-18 and 248-19) to NO. AI thanks the Azrieli Foundation for the award of an Azrieli fellowship. E.G.K. was supported by NIH training grant 5T32GM008490. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Most relevant data are within the manuscript and its Supporting Information files. RNAseq reads used for mapping crel mutantsare available at https://www.ncbi.nlm.nih.gov/sra under project numbers PRJNA347502 (M82) and PRJNA723668 (crel-2 and crel-2). All shoot apex ChIP-seq and RNA-seq datasets have been deposited to the NCBI GEO database and are available under accession number GSE174416.

Copyright: © 2022 Shwartz et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, a screen for suppressors of the e simple-leaf phenotype identified the crawling elephant (crel) mutant, which substantially suppresses the ectopic blade growth of e. We found CREL to encode a tomato VIL gene, related to Arabidopsis VIL1/VRN5. crel mutants affect many aspects of tomato development, including plant and organ maturation. Comparison of H3K27me3 modifications between wild type and crel plants showed that CREL is required for H3K27me3 deposition at approximately half of the H3K27me3-enriched sites found in wild-type plants. Therefore, CREL promotes maturation throughout the plant life by promoting selective deposition of H3K27me3 and gene silencing at a subset of PRC2 targets.

Tomato plants have compound leaves, which are composed of multiple leaflets [ 27 ]. The elaboration of compound leaves depends on slow maturation of the developing leaf, which enables an extended organogenesis activity at the leaf margin, during which leaflets are formed [ 28 – 32 ]. Leaflets are formed by differential growth at the leaf margin, where regions of blade growth are separated by intercalary regions of growth inhibition [ 33 ]. Auxin has been shown to promote growth and its response is inhibited in the intercalary domains [ 34 – 40 ]. Mutations in the tomato gene SlIAA9/ENTIRE (E), encoding an auxin-response inhibitor from the Aux/IAA family that specifies the intercalary domain, result in simplified leaves due to ectopic blade growth in the intercalary domain [ 34 , 41 , 42 ].

VIL proteins have been identified from several plant species [ 16 – 25 ]. They have been shown to promote flowering in all tested species, including species that do not have an FLC ortholog and/or do not respond to vernalization. In rice, the OsLF and OsLFL1 genes encode transcription factors that inhibit flowering and have been identified as VIL targets [ 18 , 20 ]. A VIN3 ortholog has also been identified in tomato [ 24 ]. While the vast majority of research on VIL proteins concerned their involvement in flowering induction, several studies reported additional developmental effects. For example, Arabidopsis vrn5 mutants had increased leaf curling, increased numbers of petals, and distorted siliques [ 13 ]. In rice, leaf inclination2 (lc2) mutants had an altered leaf angle, curled leaves and severe sterility, and other rice VIL genes were found to affect spikelet development, branching and grain yield [ 16 , 17 , 19 , 26 ]. Silencing the Brachypodium distachyon BdVIL4, which is similar to VIN3, led to increased branching [ 21 ]. Pepper cavil1 mutants affect leaf development, apical dominance and branching [ 25 ]. However, the knowledge about the involvement of VIL proteins in these and other developmental processes is limited, and their role in compound-leaf development has not been explored. In addition, it is not clear whether VIL proteins recruit PRC2 mainly to targets involved in the induction to flowering or whether they have broader roles in plant development.

Polycomb Repressive Complex 2 (PRC2) is a conserved complex that represses gene expression by trimethylating lysine 27 of histone H3 proteins (H3K27me3)[ 1 – 3 ]. PRC2 activity counteracts, and is counteracted by, the transcription-promoting functions of trithorax-group proteins [ 4 ]. The core PRC2 is composed of 4 subunits. In plants, some of these subunits are encoded by small gene families, allowing the formation of multiple, distinct complexes. Different plant PRC2 complexes have been shown to regulate specific developmental processes such as endosperm development, flowering time and flower development [ 2 , 3 ]. As PRC2 complexes do not have DNA binding domains, they are recruited to target loci by interacting proteins [ 2 , 5 – 10 ]. One of the most characterized PRC2-regulated processes in Arabidopsis is the induction of flowering in response to prolonged cold, termed vernalization. In response to vernalization, PRC2 promotes flowering by silencing the flowering inhibitor FLC. The vernalization-specific VRN-PRC2 complex is recruited to FLC by complexing with PHD proteins from the VERNALIZATION INSENSITIVE 3-LIKE (VIL) family [ 7 , 11 – 13 ]. In Arabidopsis, the VIL family consists of 4 members, including VIN3 and VRN5. Vernalization induces VIN3 expression, while VRN5 is expressed constitutively. VIL proteins also repress additional members of the FLC family during vernalization, and VRN5 and VIL2 are also involved in other flowering pathways [ 7 , 11 , 14 , 15 ].

Results

CREL is a VRN5 homolog To identify the CREL gene, we genetically mapped the crel-1 mutation using an F2 mapping population from a cross between the crel-1 mutant, in the Solanum lycopersicum M82 background, and S. pimpinellifolium. crel-1 was mapped to chromosome 5. Further mapping was hampered by an introgression of S. pimpinellifolium sequences in the M82 line in this region [53]. We therefore used RNA-seq to identify possible causative mutations in crel-1 and crel-2, which led to the identification of mutations in the gene Solyc05g018390 in both crel-1 and crel-2. In crel-1, a G to A substitution at position 4264 from the transcription start site (TSS) led to a stop codon in exon III. The fast neutron allele crel-2 contains a 12,826-bp-long deletion, which results in the elimination of exon I and II and part of exon III (Fig 3A). Sequencing the Solyc05g018390 gene in two additional crel alleles identified a 1-bp deletion in the first exon at position 322 from the TSS in crel-3, and an A to T substitution in position 3630 leading to a stop codon in the third exon in crel-5 (Fig 3A). We therefore concluded that Solyc05g018390 is CREL. CREL is predicted to encode a plant homeodomain (PHD) finger protein (Fig 3A and 3B). It is most similar to the Arabidopsis VRN5 gene. PPT PowerPoint slide

PNG larger image

TIFF original image Download: 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. https://doi.org/10.1371/journal.pgen.1009633.g003

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