Peptide REF1 is a local wound signal promoting plant regeneration.

Plants frequently encounter wounding and have evolved an extraordinary regenerative capacity to heal the wounds. However, the wound signal that triggers regenerative responses has not been identified. Here, through characterization of a tomato mutant defective in both wound-induced defense and regeneration, we demonstrate that in tomato, a plant elicitor peptide (Pep), REGENERATION FACTOR1 (REF1), acts as a systemin-independent local wound signal that primarily regulates local defense responses and regenerative responses in response to wounding. We further identified PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) as the receptor perceiving REF1 signal for plant regeneration. REF1-PORK1-mediated signaling promotes regeneration via activating WOUND-INDUCED DEDIFFERENTIATION 1 (WIND1), a master regulator of wound-induced cellular reprogramming in plants. Thus, REF1-PORK1 signaling represents a conserved phytocytokine pathway to initiate, amplify, and stabilize a signaling cascade that orchestrates wound-triggered organ regeneration. Application of REF1 provides a simple method to boost the regeneration and transformation efficiency of recalcitrant crops.

Plant bioacoustics: The sound expression of stress.

Plants are not exactly known to be great conversationalists. In this issue of Cell, a new study highlights that when stressed by desiccation or cutting injury, tomato and tobacco plants can produce airborne ultrasonic emissions. These sounds are loud enough to be heard by insects and can be analytically categorized using trained neural networks, pointing to their potential informative value.

Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

Whitefly hijacks a plant detoxification gene that neutralizes plant toxins.

Plants protect themselves with a vast array of toxic secondary metabolites, yet most plants serve as food for insects. The evolutionary processes that allow herbivorous insects to resist plant defenses remain largely unknown. The whitefly Bemisia tabaci is a cosmopolitan, highly polyphagous agricultural pest that vectors several serious plant pathogenic viruses and is an excellent model to probe the molecular mechanisms involved in overcoming plant defenses. Here, we show that, through an exceptional horizontal gene transfer event, the whitefly has acquired the plant-derived phenolic glucoside malonyltransferase gene BtPMaT1. This gene enables whiteflies to neutralize phenolic glucosides. This was confirmed by genetically transforming tomato plants to produce small interfering RNAs that silence BtPMaT1, thus impairing the whiteflies' detoxification ability. These findings reveal an evolutionary scenario whereby herbivores harness the genetic toolkit of their host plants to develop resistance to plant defenses and how this can be exploited for crop protection.

Conserved pleiotropy of an ancient plant homeobox gene uncovered by cis-regulatory dissection.

Divergence of gene function is a hallmark of evolution, but assessing functional divergence over deep time is not trivial. The few alleles available for cross-species studies often fail to expose the entire functional spectrum of genes, potentially obscuring deeply conserved pleiotropic roles. Here, we explore the functional divergence of WUSCHEL HOMEOBOX9 (WOX9), suggested to have species-specific roles in embryo and inflorescence development. Using a cis-regulatory editing drive system, we generate a comprehensive allelic series in tomato, which revealed hidden pleiotropic roles for WOX9. Analysis of accessible chromatin and conserved cis-regulatory sequences identifies the regions responsible for this pleiotropic activity, the functions of which are conserved in groundcherry, a tomato relative. Mimicking these alleles in Arabidopsis, distantly related to tomato and groundcherry, reveals new inflorescence phenotypes, exposing a deeply conserved pleiotropy. We suggest that targeted cis-regulatory mutations can uncover conserved gene functions and reduce undesirable effects in crop improvement.

Major Impacts of Widespread Structural Variation on Gene Expression and Crop Improvement in Tomato.

Structural variants (SVs) underlie important crop improvement and domestication traits. However, resolving the extent, diversity, and quantitative impact of SVs has been challenging. We used long-read nanopore sequencing to capture 238,490 SVs in 100 diverse tomato lines. This panSV genome, along with 14 new reference assemblies, revealed large-scale intermixing of diverse genotypes, as well as thousands of SVs intersecting genes and cis-regulatory regions. Hundreds of SV-gene pairs exhibit subtle and significant expression changes, which could broadly influence quantitative trait variation. By combining quantitative genetics with genome editing, we show how multiple SVs that changed gene dosage and expression levels modified fruit flavor, size, and production. In the last example, higher order epistasis among four SVs affecting three related transcription factors allowed introduction of an important harvesting trait in modern tomato. Our findings highlight the underexplored role of SVs in genotype-to-phenotype relationships and their widespread importance and utility in crop improvement.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

Per-Nucleus Crossover Covariation and Implications for Evolution.

Crossing over is a nearly universal feature of sexual reproduction. Here, analysis of crossover numbers on a per-chromosome and per-nucleus basis reveals a fundamental, evolutionarily conserved feature of meiosis: within individual nuclei, crossover frequencies covary across different chromosomes. This effect results from per-nucleus covariation of chromosome axis lengths. Crossovers can promote evolutionary adaptation. However, the benefit of creating favorable new allelic combinations must outweigh the cost of disrupting existing favorable combinations. Covariation concomitantly increases the frequencies of gametes with especially high, or especially low, numbers of crossovers, and thus might concomitantly enhance the benefits of crossing over while reducing its costs. A four-locus population genetic model suggests that such an effect can pertain in situations where the environment fluctuates: hyper-crossover gametes are advantageous when the environment changes while hypo-crossover gametes are advantageous in periods of environmental stasis. These findings reveal a new feature of the basic meiotic program and suggest a possible adaptive advantage.

Tomato Multiomics Reveals Consequences of Crop Domestication and Improvement.

Genome-scale analyses of variation, gene expression, and metabolite accumulation in ancestral, early domesticates, and modern tomatoes by Zhu et al. identify genes underlying fruit chemistry and demonstrate that alleles affecting metabolic quality have been bred into modern varieties as a result of linkage drag. Similar metabolic hitchhikers are likely ubiquitous in other domesticated species.

Rewiring of the Fruit Metabolome in Tomato Breeding.

Humans heavily rely on dozens of domesticated plant species that have been further improved through intensive breeding. To evaluate how breeding changed the tomato fruit metabolome, we have generated and analyzed a dataset encompassing genomes, transcriptomes, and metabolomes from hundreds of tomato genotypes. The combined results illustrate how breeding globally altered fruit metabolite content. Selection for alleles of genes associated with larger fruits altered metabolite profiles as a consequence of linkage with nearby genes. Selection of five major loci reduced the accumulation of anti-nutritional steroidal glycoalkaloids in ripened fruits, rendering the fruit more edible. Breeding for pink tomatoes modified the content of over 100 metabolites. The introgression of resistance genes from wild relatives in cultivars also resulted in major and unexpected metabolic changes. The study reveals a multi-omics view of the metabolic breeding history of tomato, as well as provides insights into metabolome-assisted breeding and plant biology.

Bypassing Negative Epistasis on Yield in Tomato Imposed by a Domestication Gene.

Selection for inflorescence architecture with improved flower production and yield is common to many domesticated crops. However, tomato inflorescences resemble wild ancestors, and breeders avoided excessive branching because of low fertility. We found branched variants carry mutations in two related transcription factors that were selected independently. One founder mutation enlarged the leaf-like organs on fruits and was selected as fruit size increased during domestication. The other mutation eliminated the flower abscission zone, providing "jointless" fruit stems that reduced fruit dropping and facilitated mechanical harvesting. Stacking both beneficial traits caused undesirable branching and sterility due to epistasis, which breeders overcame with suppressors. However, this suppression restricted the opportunity for productivity gains from weak branching. Exploiting natural and engineered alleles for multiple family members, we achieved a continuum of inflorescence complexity that allowed breeding of higher-yielding hybrids. Characterizing and neutralizing similar cases of negative epistasis could improve productivity in many agricultural organisms. VIDEO ABSTRACT.

Oligomerization-mediated autoinhibition and cofactor binding of a plant NLR.

Nucleotide-binding leucine-rich repeat (NLR) proteins play a pivotal role in plant immunity by recognizing pathogen effectors1,2. Maintaining a balanced immune response is crucial, as excessive NLR expression can lead to unintended autoimmunity3,4. Unlike most NLRs, the plant NLR required for cell death 2 (NRC2) belongs to a small NLR group characterized by constitutively high expression without self-activation5. The mechanisms underlying NRC2 autoinhibition and activation are not yet understood. Here we show that Solanum lycopersicum (tomato) NRC2 (SlNRC2) forms dimers and tetramers and higher-order oligomers at elevated concentrations. Cryo-electron microscopy shows an inactive conformation of SlNRC2 in these oligomers. Dimerization and oligomerization not only stabilize the inactive state but also sequester SlNRC2 from assembling into an active form. Mutations at the dimeric or interdimeric interfaces enhance pathogen-induced cell death and immunity in Nicotiana benthamiana. The cryo-electron microscopy structures unexpectedly show inositol hexakisphosphate (IP6) or pentakisphosphate (IP5) bound to the inner surface of the C-terminal leucine-rich repeat domain of SlNRC2, as confirmed by mass spectrometry. Mutations at the inositol phosphate-binding site impair inositol phosphate binding of SlNRC2 and pathogen-induced SlNRC2-mediated cell death in N. benthamiana. Our study indicates a negative regulatory mechanism of NLR activation and suggests inositol phosphates as cofactors of NRCs.

Graph pangenome captures missing heritability and empowers tomato breeding.

Missing heritability in genome-wide association studies defines a major problem in genetic analyses of complex biological traits1,2. The solution to this problem is to identify all causal genetic variants and to measure their individual contributions3,4. Here we report a graph pangenome of tomato constructed by precisely cataloguing more than 19 million variants from 838 genomes, including 32 new reference-level genome assemblies. This graph pangenome was used for genome-wide association study analyses and heritability estimation of 20,323 gene-expression and metabolite traits. The average estimated trait heritability is 0.41 compared with 0.33 when using the single linear reference genome. This 24% increase in estimated heritability is largely due to resolving incomplete linkage disequilibrium through the inclusion of additional causal structural variants identified using the graph pangenome. Moreover, by resolving allelic and locus heterogeneity, structural variants improve the power to identify genetic factors underlying agronomically important traits leading to, for example, the identification of two new genes potentially contributing to soluble solid content. The newly identified structural variants will facilitate genetic improvement of tomato through both marker-assisted selection and genomic selection. Our study advances the understanding of the heritability of complex traits and demonstrates the power of the graph pangenome in crop breeding.

Unraveling the diversity and cultural heritage of fruit crops through paleogenomics.

Abundant and plentiful fruit crops are threatened by the loss of diverse legacy cultivars which are being replaced by a limited set of high-yielding ones. This article delves into the potential of paleogenomics that utilizes ancient DNA analysis to revive lost diversity. By focusing on grapevines, date palms, and tomatoes, recent studies showcase the effectiveness of paleogenomic techniques in identifying and understanding genetic traits crucial for crop resilience, disease resistance, and nutritional value. The approach not only tracks landrace dispersal and introgression but also sheds light on domestication events. In the face of major future environmental challenges, integrating paleogenomics with modern breeding strategies emerges as a promising avenue to significantly bolster fruit crop sustainability.

Phytosulfokine peptide optimizes plant growth and defense via glutamine synthetase GS2 phosphorylation in tomato.

Phytosulfokine (PSK) is a plant pentapeptide hormone that fulfills a wide range of functions. Although PSK has frequently been reported to function in the inverse regulation of growth and defense in response to (hemi)biotrophic pathogens, the mechanisms involved remain largely unknown. Using the tomato (Solanum lycopersicum) and Pseudomonas syringae pv. tomato (Pst) DC3000 pathogen system, we present compelling evidence that the PSK receptor PSKR1 interacts with the calcium-dependent protein kinase CPK28, which in turn phosphorylates the key enzyme of nitrogen assimilation glutamine synthetase GS2 at two sites (Serine-334 and Serine-360). GS2 phosphorylation at S334 specifically regulates plant defense, whereas S360 regulates growth, uncoupling the PSK-induced effects on defense responses and growth regulation. The discovery of these sites will inform breeding strategies designed to optimize the growth-defense balance in a compatible manner.

A gradient of the HD-Zip regulator Woolly regulates multicellular trichome morphogenesis in tomato.

Homeodomain (HD) proteins regulate embryogenesis in animals such as the fruit fly (Drosophila melanogaster), often in a concentration-dependent manner. HD-leucine zipper (Zip) IV family genes are unique to plants and often function in the L1 epidermal cell layer. However, our understanding of the roles of HD-Zip IV family genes in plant morphogenesis is limited. In this study, we investigated the morphogenesis of tomato (Solanum lycopersicum) multicellular trichomes, a type of micro-organ in plants. We found that a gradient of the HD-Zip IV regulator Woolly (Wo) coordinates spatially polarized cell division and cell expansion in multicellular trichomes. Moreover, we identified a TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATING CELL NUCLEAR ANTIGEN BINDING FACTOR (TCP) transcription factor-encoding gene, SlBRANCHED2a (SlBRC2a), as a key downstream target of Wo that regulates the transition from cell division to cell expansion. High levels of Wo promote cell division in apical trichome cells, whereas in basal trichome cells, Wo mediates a negative feedback loop with SlBRC2a that forces basal cells to enter endoreduplication. The restricted high and low activities of Wo pattern the morphogenesis of tomato multicellular trichomes. These findings provide insights into the functions of HD-Zip IV genes during plant morphogenesis.

Cell-cell communication and initial population composition shape the structure of potato spindle tuber viroid quasispecies.

RNA viruses and viroids replicate with high mutation rates, forming quasispecies, population of variants centered around dominant sequences. The mechanisms governing quasispecies remain unclear. Plasmodesmata regulate viroid movement and were hypothesized to impact viroid quasispecies. Here, we sequenced the progeny of potato spindle tuber viroid intermediate (PSTVd-I) strain from mature guard cells lacking plasmodesmal connections and from in vitro-cultivated mesophyll cell protoplasts from systemic leaves of early-infected tomato (Solanum lycopersicum) plants. Remarkably, more variants accumulated in guard cells compared to whole leaves. Similarly, after extended cell culture, we observed more variants in cultivated mesophyll protoplasts. Coinfection and single-cell sequencing experiments demonstrated that the same plant cell can be infected multiple times by the same or different PSTVd sequences. To study the impact of initial population composition on PSTVd-I quasispecies, we conducted coinfections with PSTVd-I and variants. Two inoculum ratios (10:1 or 1:10) established quasispecies with or without PSTVd-I as the master sequence. In the absence of the master sequence, the percentage of novel variants initially increased. Moreover, a 1:1 PSTVd-I/variant RNA ratio resulted in PSTVd-I dominating (>50%), while the variants reached 20%. After PSTVd-I-only infection, the variants reached around 10%, while after variant-only infection, the variants were significantly more than 10%. These results emphasize the role of cell-to-cell communication and initial population composition in shaping PSTVd quasispecies.

Expansin SlExp1 and endoglucanase SlCel2 synergistically promote fruit softening and cell wall disassembly in tomato.

Fruit softening, an irreversible process that occurs during fruit ripening, can lead to losses and waste during postharvest transportation and storage. Cell wall disassembly is the main factor leading to loss of fruit firmness, and several ripening-associated cell wall genes have been targeted for genetic modification, particularly pectin modifiers. However, individual knockdown of most cell wall-related genes has had minimal influence on cell wall integrity and fruit firmness, with the notable exception of pectate lyase. Compared to pectin disassembly, studies of the cell wall matrix, the xyloglucan-cellulose framework, and underlying mechanisms during fruit softening are limited. Here, a tomato (Solanum lycopersicum) fruit ripening-associated α-expansin (SlExpansin1/SlExp1) and an endoglucanase (SlCellulase2/SlCel2), which function in the cell wall matrix, were knocked out individually and together using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing. Simultaneous knockout of SlExp1 and SlCel2 enhanced fruit firmness, reduced depolymerization of homogalacturonan-type pectin and xyloglucan, and increased cell adhesion. In contrast, single knockouts of either SlExp1 or SlCel2 did not substantially change fruit firmness, while simultaneous overexpression of SlExp1 and SlCel2 promoted early fruit softening. Collectively, our results demonstrate that SlExp1 and SlCel2 synergistically regulate cell wall disassembly and fruit softening in tomato.

Null mutants of a tomato Rho of plants exhibit enhanced water use efficiency without a penalty to yield.

Improving water use efficiency in crops is a significant challenge as it involves balancing water transpiration and CO2 uptake through stomatal pores. This study investigates the role of SlROP9, a tomato Rho of Plants protein, in guard cells and its impact on plant transpiration. The results reveal that SlROP9 null mutants exhibit reduced stomatal conductance while photosynthetic CO2 assimilation remains largely unaffected. Notably, there is a notable decrease in whole-plant transpiration in the rop9 mutants compared to the wild type, especially during noon hours when the water pressure deficit is high. The elevated stomatal closure observed in rop9 mutants is linked to an increase in reactive oxygen species formation. This is very likely dependent on the respiratory burst oxidase homolog (RBOH) NADPH oxidase and is not influenced by abscisic acid (ABA). Consistently, activated ROP9 can interact with RBOHB in both yeast and plants. In diverse tomato accessions, drought stress represses ROP9 expression, and in Arabidopsis stomatal guard cells, ABA suppresses ROP signaling. Therefore, the phenotype of the rop9 mutants may arise from a disruption in ROP9-regulated RBOH activity. Remarkably, large-scale field experiments demonstrate that the rop9 mutants display improved water use efficiency without compromising fruit yield. These findings provide insights into the role of ROPs in guard cells and their potential as targets for enhancing water use efficiency in crops.

A catalogue of recombination coldspots in interspecific tomato hybrids.

Increasing natural resistance and resilience in plants is key for ensuring food security within a changing climate. Breeders improve these traits by crossing cultivars with their wild relatives and introgressing specific alleles through meiotic recombination. However, some genomic regions are devoid of recombination especially in crosses between divergent genomes, limiting the combinations of desirable alleles. Here, we used pooled-pollen sequencing to build a map of recombinant and non-recombinant regions between tomato and five wild relatives commonly used for introgressive tomato breeding. We detected hybrid-specific recombination coldspots that underscore the role of structural variations in modifying recombination patterns and maintaining genetic linkage in interspecific crosses. Crossover regions and coldspots show strong association with specific TE superfamilies exhibiting differentially accessible chromatin between somatic and meiotic cells. About two-thirds of the genome are conserved coldspots, located mostly in the pericentromeres and enriched with retrotransposons. The coldspots also harbor genes associated with agronomic traits and stress resistance, revealing undesired consequences of linkage drag and possible barriers to breeding. We presented examples of linkage drag that can potentially be resolved by pairing tomato with other wild species. Overall, this catalogue will help breeders better understand crossover localization and make informed decisions on generating new tomato varieties.