Application of genome editing techniques to regulate gene expression in crops.

BACKGROUND: Enhanced agricultural production is urgently required to meet the food demands of the increasing global population. Abundant genetic diversity is expected to accelerate crop development. In particular, the development of the CRISPR/Cas genome editing technology has greatly enhanced our ability to improve crop's genetic diversity through direct artificial gene modification. However, recent studies have shown that most crop improvement efforts using CRISPR/Cas techniques have mainly focused on the coding regions, and there is a relatively lack of studies on the regulatory regions of gene expression. RESULTS: This review briefly summarizes the development of CRISPR/Cas system in the beginning. Subsequently, the importance of gene regulatory regions in plants is discussed. The review focuses on recent developments and applications of mutations in regulatory regions via CRISPR/Cas techniques in crop breeding. CONCLUSION: Finally, an outline of perspectives for future crop breeding using genome editing technologies is provided. This review provides new research insights for crop improvement using genome editing techniques.

Optimization of ATAC-seq in wheat seedling roots using INTACT-isolated nuclei.

BACKGROUND: The genetic information contained in the genome of an organism is organized in genes and regulatory elements that control gene expression. The genomes of multiple plants species have already been sequenced and the gene repertory have been annotated, however, cis-regulatory elements remain less characterized, limiting our understanding of genome functionality. These elements act as open platforms for recruiting both positive- and negative-acting transcription factors, and as such, chromatin accessibility is an important signature for their identification. RESULTS: In this work we developed a transgenic INTACT [isolation of nuclei tagged in specific cell types] system in tetraploid wheat for nuclei purifications. Then, we combined the INTACT system together with the assay for transposase-accessible chromatin with sequencing [ATAC-seq] to identify open chromatin regions in wheat root tip samples. Our ATAC-seq results showed a large enrichment of open chromatin regions in intergenic and promoter regions, which is expected for regulatory elements and that is similar to ATAC-seq results obtained in other plant species. In addition, root ATAC-seq peaks showed a significant overlap with a previously published ATAC-seq data from wheat leaf protoplast, indicating a high reproducibility between the two experiments and a large overlap between open chromatin regions in root and leaf tissues. Importantly, we observed overlap between ATAC-seq peaks and cis-regulatory elements that have been functionally validated in wheat, and a good correlation between normalized accessibility and gene expression levels. CONCLUSIONS: We have developed and validated an INTACT system in tetraploid wheat that allows rapid and high-quality nuclei purification from root tips. Those nuclei were successfully used to performed ATAC-seq experiments that revealed open chromatin regions in the wheat genome that will be useful to identify cis-regulatory elements. The INTACT system presented here will facilitate the development of ATAC-seq datasets in other tissues, growth stages, and under different growing conditions to generate a more complete landscape of the accessible DNA regions in the wheat genome.

Shared distal regulatory regions may contribute to the coordinated expression of human ribosomal protein genes.

To identify the potential distal regulatory regions of human ribosomal protein genes (RPGs) and to understand their characteristics, we studied the chromatin interactions in seven cell lines and four primary cell types. We identified 22,797 putative regulatory regions that directly or indirectly interact with human RPG promoters. A large proportion of these regions are only present in one cell line or one cell type, implying that RPGs may be differentially regulated across experimental conditions. We also noticed that groups of RPGs, which are the same groups across cell lines and cell types, share common regulatory regions. These shared regulatory regions by RPGs may contribute to their coordinated regulation. By studying the overrepresented motifs in the identified regulatory regions, we showed that there are about two dozen motifs in these regions shared across cell lines and cell types. Our study shed new light on the coordinated transcriptional regulation of human RPGs.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Modern human changes in regulatory regions implicated in cortical development.

BACKGROUND: Recent paleogenomic studies have highlighted a very small set of proteins carrying modern human-specific missense changes in comparison to our closest extinct relatives. Despite being frequently alluded to as highly relevant, species-specific differences in regulatory regions remain understudied. Here, we integrate data from paleogenomics, chromatin modification and physical interaction, and single-cell gene expression of neural progenitor cells to identify derived regulatory changes in the modern human lineage in comparison to Neanderthals/Denisovans. We report a set of genes whose enhancers and/or promoters harbor modern human single nucleotide changes and are active at early stages of cortical development. RESULTS: We identified 212 genes controlled by regulatory regions harboring modern human changes where Neanderthals/Denisovans carry the ancestral allele. These regulatory regions significantly overlap with putative modern human positively-selected regions and schizophrenia-related genetic loci. Among the 212 genes, we identified a substantial proportion of genes related to transcriptional regulation and, specifically, an enrichment for the SETD1A histone methyltransferase complex, known to regulate WNT signaling for the generation and proliferation of intermediate progenitor cells. CONCLUSIONS: This study complements previous research focused on protein-coding changes distinguishing our species from Neanderthals/Denisovans and highlights chromatin regulation as a functional category so far overlooked in modern human evolution studies. We present a set of candidates that will help to illuminate the investigation of modern human-specific ontogenetic trajectories.

Cis-regulatory genetic variants in the CCR5 gene and natural HIV-1 control in black South Africans.

Studies have investigated CCR5 haplotypes (HHA, HHB, HHC, HHD, HHE, HHF*1, HHF*2, HHG*1, HHG*2), defined by seven 5'UTR single nucleotide polymorphisms (SNPs), CCR2-V64I and CCR5Δ32, in HIV-1 disease. CCR5 cis-regulatory regions were sequenced, CCR2-V64I and CCR5Δ32 genotyped, and compared in HIV-1-infected black South Africans: 71 HIV-1 controllers (23 elite controllers, 37 viraemic controllers (VCs), 11 high viral load long-term non-progressors) and 74 progressors. The HHE haplotype and 3'UTR +2919 T > G SNP heterozygosity were underrepresented in total controllers and VCs vs. progressors (p = .004; p = .007 and p = .002, pbonferroni = 0.032; p = .004, respectively). Possession of the +2919 T > G SNP (dominant mode) was associated with HIV-1 progression (controllers vs. progressors: p = .001, pbonferroni = 0.016). The +2919 T > G SNP is in linkage disequilibrium (LD; r2 = 0.73) with two 5'UTR SNPs (-2459G > A and -2135 T > C; r2 = 1: 5'UTR-2SNP-hap). The 5'UTR-2SNP-hap was lower in total controllers and VCs vs. progressors (p = .003, pbonferroni = 0.048; p = .01, respectively). Results suggest -2459G > A, -2135 T > C, and + 2919 T > G as key CCR5 variants in HIV-1 control.

A k-mer grammar analysis to uncover maize regulatory architecture.

BACKGROUND: Only a small percentage of the genome sequence is involved in regulation of gene expression, but to biochemically identify this portion is expensive and laborious. In species like maize, with diverse intergenic regions and lots of repetitive elements, this is an especially challenging problem that limits the use of the data from one line to the other. While regulatory regions are rare, they do have characteristic chromatin contexts and sequence organization (the grammar) with which they can be identified. RESULTS: We developed a computational framework to exploit this sequence arrangement. The models learn to classify regulatory regions based on sequence features - k-mers. To do this, we borrowed two approaches from the field of natural language processing: (1) "bag-of-words" which is commonly used for differentially weighting key words in tasks like sentiment analyses, and (2) a vector-space model using word2vec (vector-k-mers), that captures semantic and linguistic relationships between words. We built "bag-of-k-mers" and "vector-k-mers" models that distinguish between regulatory and non-regulatory regions with an average accuracy above 90%. Our "bag-of-k-mers" achieved higher overall accuracy, while the "vector-k-mers" models were more useful in highlighting key groups of sequences within the regulatory regions. CONCLUSIONS: These models now provide powerful tools to annotate regulatory regions in other maize lines beyond the reference, at low cost and with high accuracy.

Genome-Wide Analysis of Putative G-Quadruplex Sequences (PGQSs) in Onion Yellows Phytoplasma (Strain OY-M): An Emerging Plant Pathogenic Bacteria.

Phytoplasma, an emerging plant pathogen is an endocellular obligate parasite of plant phloem tissues with highly reduced genomes and low GC content. They contain a minimal set of genes essential for survival as an intracellular parasite. The role of G-Quadruplexes in pathogenicity has been reported in a variety of microbial pathogens. Detailed investigation on the genome wide occurrence and distribution of Putative G-Quadruplex forming Sequences (PGQSs) in the AT-rich genome of Onion yellows phytoplasma (strain OY-M) was carried out. Relative enrichment and depletion of these putative secondary structures in different genomic regions of OY-M was investigated with an aim to unravel their association with functionally important genomic locations. PGQSs density of 0.4407/Kbp was detected in the genome of OY-M phytoplasma, which is significantly higher than the average PGQSs density (0.136/Kbp) reported for other members of its phylum, namely Tenericutes. A non-random distribution of PGQSs across the length of the genome was observed. Putative promoter regions of OY-M were found to be particularly enriched in PGQSs followed by genic regions. The repeat rich regions were identified to have minimum PGQSs density. Presence of PGQSs in important genes such as those involved in secretory pathways of virulent factors, transport related functions, rRNA and tRNA was particularly intriguing. Our study reports for the first time a detailed investigation on the genome-wide locations of putative G-Quadruplexes in phytoplasma and highlights the need to further investigate their role in the metabolism and also in the mechanism of pathogenicity.

Patterns of variation in cis-regulatory regions: examining evidence of purifying selection.

BACKGROUND: With only 2 % of the human genome consisting of protein coding genes, functionality across the rest of the genome has been the subject of much debate. This has gained further impetus in recent years due to a rapidly growing catalogue of genomic elements, based primarily on biochemical signatures (e.g. the ENCODE project). While the assessment of functionality is a complex task, the presence of selection acting on a genomic region is a strong indicator of importance. In this study, we apply population genetic methods to investigate signals overlaying several classes of regulatory elements. RESULTS: We disentangle signals of purifying selection acting directly on regulatory elements from the confounding factors of demography and purifying selection linked to e.g. nearby protein coding regions. We confirm the importance of regulatory regions proximal to coding sequence, while also finding differential levels of selection at distal regions. We note differences in purifying selection among transcription factor families. Signals of constraint at some genomic classes were also strongly dependent on their physical location relative to coding sequence. In addition, levels of selection efficacy across genomic classes differed between African and non-African populations. CONCLUSIONS: In order to assign a valid signal of selection to a particular class of genomic sequence, we show that it is crucial to isolate the signal by accounting for the effects of demography and linked-purifying selection. Our study highlights the intricate interplay of factors affecting signals of selection on functional elements.

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

A Significant Regulatory Mutation Burden at a High-Affinity Position of the CTCF Motif in Gastrointestinal Cancers.

Somatic mutations drive cancer and there are established ways to study those in coding sequences. It has been shown that some regulatory mutations are over-represented in cancer. We develop a new strategy to find putative regulatory mutations based on experimentally established motifs for transcription factors (TFs). In total, we find 1,552 candidate regulatory mutations predicted to significantly reduce binding affinity of many TFs in hepatocellular carcinoma and affecting binding of CTCF also in esophagus, gastric, and pancreatic cancers. Near mutated motifs, there is a significant enrichment of (1) genes mutated in cancer, (2) tumor-suppressor genes, (3) genes in KEGG cancer pathways, and (4) sets of genes previously associated to cancer. Experimental and functional validations support the findings. The strategy can be applied to identify regulatory mutations in any cell type with established TF motifs and will aid identifications of genes contributing to cancer.

Genome-wide analysis of G-quadruplexes in herpesvirus genomes.

BACKGROUND: G-quadruplexes are increasingly recognized as regulatory elements in human, animal, bacterial and plant genomes. The presence and function of G-quadruplexes are not well studied among herpesviruses; in particular, there are no systematic genome-wide analysis of these important secondary structures in herpesvirus genomes. RESULTS: We performed genome-wide analysis of putative quadruplex sequences (PQS) in human herpesviruses. We found unusually high PQS densities among human herpesviruses. PQS are enriched in the repeat regions and regulatory regions of human herpesviruses. Interestingly, PQS densities are higher in regulatory regions of immediate early genes compared to early and late genes in most herpesviruses. In addition, the majority of genes functionally conserved across human herpesviruses contain one or more PQS within the regulatory regions. We also describe the existence of unique intramolecular PQS repeats or repetitive G-quadruplex motifs in herpesviruses. Functional studies confirm a role for G-quadruplexes in regulating the gene expression of human herpesviruses. CONCLUSION: The pervasiveness of PQS, their enrichment and conservation at specific genomic locations suggest that these structural entities may represent a novel class of functional elements in herpesviruses. Our findings provide the necessary framework for studies on the biological role of G-quadruplexes in herpesviruses.

Cancer somatic mutations cluster in a subset of regulatory sites predicted from the ENCODE data.

BACKGROUND: Transcriptional regulation of gene expression is essential for cellular differentiation and function, and defects in the process are associated with cancer. The ENCODE project has mapped potential regulatory sites across the complete genome in many cell types, and these regions have been shown to harbour many of the somatic mutations that occur in cancer cells, suggesting that their effects may drive cancer initiation and development. The ENCODE data suggests a very large number of regulatory sites, and methods are needed to identify those that are most relevant and to connect them to the genes that they control. METHODS: Predictive models of gene expression were developed by integrating the ENCODE data for regulation, including transcription factor binding and DNase1 hypersensitivity, with RNA-seq data for gene expression. A penalized regression method was used to identify the most predictive potential regulatory sites for each transcript. Known cancer somatic mutations from the COSMIC database were mapped to potential regulatory sites, and we examined differences in the mapping frequencies associated with sites chosen in regulatory models and other (rejected) sites. The effects of potential confounders, for example replication timing, were considered. RESULTS: Cancer somatic mutations preferentially occupy those regulatory regions chosen in our models as most predictive of gene expression. CONCLUSION: Our methods have identified a significantly reduced set of regulatory sites that are enriched in cancer somatic mutations and are more predictive of gene expression. This has significance for the mechanistic interpretation of cancer mutations, and the understanding of genetic regulation.

A medium-scale assay for enhancer validation in amniotes.

BACKGROUND: Enhancers are key elements to control gene expression in time and space and thus orchestrate gene function during development, homeostasis, and disease. Whole genome approaches and bioinformatic predictions have generated a tremendous pool of potential enhancers, however their spatiotemporal activity often remains to be validated in vivo. Despite recent progress in developing high throughput strategies for enhancer evaluation, these remain mainly restricted to invertebrates and in vitro cell culture. RESULTS: Here we design a medium-scale method to validate potential enhancers in an amniote embryo, the chick. Using a unique barcode for different reporter vectors allows us to detect the activity of nine separate enhancers in a single embryo by one-step RT-PCR. The assay is sufficiently sensitive to expand its capacity further by generating additional barcoded vectors. CONCLUSIONS: As a rapid, sensitive, and cost-effective way to assess enhancer activity in an amniote vertebrate, this method provides a major advance and a useful alternative to the generation of transgenic animals.

Xenopus tropicalis osteoblast-specific open chromatin regions reveal promoters and enhancers involved in human skeletal phenotypes and shed light on early vertebrate evolution.

While understanding the genetic underpinnings of osteogenesis has far-reaching implications for skeletal diseases and evolution, a comprehensive characterization of the osteoblastic regulatory landscape in non-mammalian vertebrates is still lacking. Here, we compared the ATAC-Seq profile of Xenopus tropicalis (Xt) osteoblasts to a variety of non mineralizing control tissues, and identified osteoblast-specific nucleosome free regions (NFRs) at 527 promoters and 6747 distal regions. Sequence analyses, Gene Ontology, RNA-Seq and ChIP-Seq against four key histone marks confirmed that the distal regions correspond to bona fide osteogenic transcriptional enhancers exhibiting a shared regulatory logic with mammals. We report 425 regulatory regions conserved with human and globally associated to skeletogenic genes. Of these, 35 regions have been shown to impact human skeletal phenotypes by GWAS, including one trps1 enhancer and the runx2 promoter, two genes which are respectively involved in trichorhinophalangeal syndrome type I and cleidocranial dysplasia. Intriguingly, 60 osteoblastic NFRs also align to the genome of the elephant shark, a species lacking osteoblasts and bone tissue. To tackle this paradox, we chose to focus on dlx5 because its conserved promoter, known to integrate regulatory inputs during mammalian osteogenesis, harbours an osteoblast-specific NFR in both frog and human. Hence, we show that dlx5 is expressed in Xt and elephant shark odontoblasts, supporting a common cellular and genetic origin of bone and dentine. Taken together, our work (i) unravels the Xt osteogenic regulatory landscape, (ii) illustrates how cross-species comparisons harvest data relevant to human biology and (iii) reveals that a set of genes including bnc2, dlx5, ebf3, mir199a, nfia, runx2 and zfhx4 drove the development of a primitive form of mineralized skeletal tissue deep in the vertebrate lineage.

Benchmarking tools for transcription factor prioritization.

Spatiotemporal regulation of gene expression is controlled by transcription factor (TF) binding to regulatory elements, resulting in a plethora of cell types and cell states from the same genetic information. Due to the importance of regulatory elements, various sequencing methods have been developed to localise them in genomes, for example using ChIP-seq profiling of the histone mark H3K27ac that marks active regulatory regions. Moreover, multiple tools have been developed to predict TF binding to these regulatory elements based on DNA sequence. As altered gene expression is a hallmark of disease phenotypes, identifying TFs driving such gene expression programs is critical for the identification of novel drug targets. In this study, we curated 84 chromatin profiling experiments (H3K27ac ChIP-seq) where TFs were perturbed through e.g., genetic knockout or overexpression. We ran nine published tools to prioritize TFs using these real-world datasets and evaluated the performance of the methods in identifying the perturbed TFs. This allowed the nomination of three frontrunner tools, namely RcisTarget, MEIRLOP and monaLisa. Our analyses revealed opportunities and commonalities of tools that will help to guide further improvements and developments in the field.

Patterns of enrichment and acceleration in evolutionary rates of promoters suggest a role of regulatory regions in cetacean gigantism.

BACKGROUND: Cetaceans (whales, porpoises, and dolphins) are a lineage of aquatic mammals from which some species became giants. Only recently, gigantism has been investigated from the molecular point of view. Studies focused mainly on coding regions, and no data on the influence of regulatory regions on gigantism in this group was available. Accordingly, we investigated the molecular evolution of non-coding regulatory regions of genes already described in the literature for association with size in mammals, focusing mainly on the promoter regions. For this, we used Ciiider and phyloP tools. Ciiider identifies significantly enriched transcription factor binding sites, and phyloP estimates the molecular evolution rate of the promoter. RESULTS: We found evidence of enrichment of transcription binding factors related to large body size, with distinct patterns between giant and non-giant cetaceans in the IGFBP7 and NCAPG promoters, in which repressive agents are present in small cetaceans and those that stimulate transcription, in giant cetaceans. In addition, we found evidence of acceleration in the IGF2, IGFBP2, IGFBP7, and ZFAT promoters. CONCLUSION: Our results indicate that regulatory regions may also influence cetaceans' body size, providing candidate genes for future research to understand the molecular basis of the largest living animals.

Systematic identification of disease-causing promoter and untranslated region variants in 8,040 undiagnosed individuals with rare disease.

Background: Both promoters and untranslated regions (UTRs) have critical regulatory roles, yet variants in these regions are largely excluded from clinical genetic testing due to difficulty in interpreting pathogenicity. The extent to which these regions may harbour diagnoses for individuals with rare disease is currently unknown. Methods: We present a framework for the identification and annotation of potentially deleterious proximal promoter and UTR variants in known dominant disease genes. We use this framework to annotate de novo variants (DNVs) in 8,040 undiagnosed individuals in the Genomics England 100,000 genomes project, which were subject to strict region-based filtering, clinical review, and validation studies where possible. In addition, we performed region and variant annotation-based burden testing in 7,862 unrelated probands against matched unaffected controls. Results: We prioritised eleven DNVs and identified an additional variant overlapping one of the eleven. Ten of these twelve variants (82%) are in genes that are a strong match to the individual's phenotype and six had not previously been identified. Through burden testing, we did not observe a significant enrichment of potentially deleterious promoter and/or UTR variants in individuals with rare disease collectively across any of our region or variant annotations. Conclusions: Overall, we demonstrate the value of screening promoters and UTRs to uncover additional diagnoses for previously undiagnosed individuals with rare disease and provide a framework for doing so without dramatically increasing interpretation burden.

In Embryo Gene Reporter Assays for Evaluation of Cis-Regulatory Regions.

Gene expression reporter assays measure the relevance of cis-regulatory elements and DNA-binding proteins in modulating transcriptional activity. Commonly, they are performed in cell lines. However, regulation of transcriptional activity during development is complex and dynamic, and not many cell lines reproduce the embryonic conditions. Thus, conclusions derived from cell line data provide limited information about embryonic development. On the other hand, one of the major hurdles for embryonic assays is delivering reporter plasmids in a tissue-specific manner. In this sense, the chick embryo is a good model system to perform these assays. Electroporation of chick embryos provides temporal and spatially controlled plasmid delivery. Further, it is a well-established, easy, and an economical procedure. Here, we describe in detail how to measure in the chick neural tube (1) enhancer activity with GFP, (2) enhancer activity with luciferase, and (3) 3'UTR activity with luciferase.

Targeting editing of tomato SPEECHLESS cis-regulatory regions generates plants with altered stomatal density in response to changing climate conditions.

Flexible developmental programs enable plants to customize their organ size and cellular composition. In leaves of eudicots, the stomatal lineage produces two essential cell types, stomata and pavement cells, but the total numbers and ratio of these cell types can vary. Central to this flexibility is the stomatal lineage initiating transcription factor, SPEECHLESS (SPCH). Here we show, by multiplex CRISPR/Cas9 editing of SlSPCH cis-regulatory sequences in tomato, that we can identify variants with altered stomatal development responses to light and temperature cues. Analysis of tomato leaf development across different conditions, aided by newly-created tools for live-cell imaging and translational reporters of SlSPCH and its paralogues SlMUTE and SlFAMA, revealed the series of cellular events that lead to the environmental change-driven responses in leaf form. Plants bearing the novel SlSPCH variants generated in this study are powerful resources for fundamental and applied studies of tomato resilience in response to climate change.