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.

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.

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.

Engineering Quantitative Trait Variation for Crop Improvement by Genome Editing.

Major advances in crop yields are needed in the coming decades. However, plant breeding is currently limited by incremental improvements in quantitative traits that often rely on laborious selection of rare naturally occurring mutations in gene-regulatory regions. Here, we demonstrate that CRISPR/Cas9 genome editing of promoters generates diverse cis-regulatory alleles that provide beneficial quantitative variation for breeding. We devised a simple genetic scheme, which exploits trans-generational heritability of Cas9 activity in heterozygous loss-of-function mutant backgrounds, to rapidly evaluate the phenotypic impact of numerous promoter variants for genes regulating three major productivity traits in tomato: fruit size, inflorescence branching, and plant architecture. Our approach allows immediate selection and fixation of novel alleles in transgene-free plants and fine manipulation of yield components. Beyond a platform to enhance variation for diverse agricultural traits, our findings provide a foundation for dissecting complex relationships between gene-regulatory changes and control of quantitative traits.

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.

Strigolactones promote flowering by inducing the miR319-LA-SFT module in tomato.

Strigolactones are a class of phytohormones with various functions in plant development, stress responses, and in the interaction with (micro)organisms in the rhizosphere. While their effects on vegetative development are well studied, little is known about their role in reproduction. We investigated the effects of genetic and chemical modification of strigolactone levels on the timing and intensity of flowering in tomato (Solanum lycopersicum L.) and the molecular mechanisms underlying such effects. Results showed that strigolactone levels in the shoot, whether endogenous or exogenous, correlate inversely with the time of anthesis and directly with the number of flowers and the transcript levels of the florigen-encoding gene SINGLE FLOWER TRUSS (SFT) in the leaves. Transcript quantifications coupled with metabolite analyses demonstrated that strigolactones promote flowering in tomato by inducing the activation of the microRNA319-LANCEOLATE module in leaves. This, in turn, decreases gibberellin content and increases the transcription of SFT. Several other floral markers and morpho-anatomical features of developmental progression are induced in the apical meristems upon treatment with strigolactones, affecting floral transition and, more markedly, flower development. Thus, strigolactones promote meristem maturation and flower development via the induction of SFT both before and after floral transition, and their effects are blocked in plants expressing a miR319-resistant version of LANCEOLATE. Our study positions strigolactones in the context of the flowering regulation network in a model crop species.

Immunity priming uncouples the growth-defense trade-off in tomato.

Plants have developed an array of mechanisms to protect themselves against pathogen invasion. The deployment of defense mechanisms is imperative for plant survival, but can come at the expense of plant growth, leading to the 'growth-defense trade-off' phenomenon. Following pathogen exposure, plants can develop resistance to further attack. This is known as induced resistance, or priming. Here, we investigated the growth-defense trade-off, examining how defense priming via systemic acquired resistance (SAR), or induced systemic resistance (ISR), affects tomato development and growth. We found that defense priming can promote, rather than inhibit, plant development, and that defense priming and growth trade-offs can be uncoupled. Cytokinin response was activated during induced resistance, and found to be required for the observed growth and disease resistance resulting from ISR activation. ISR was found to have a stronger effect than SAR on plant development. Our results suggest that growth promotion and induced resistance can be co-dependent, and that, in certain cases, defense priming can drive developmental processes and promote plant yield.

A novel DCL2-dependent miRNA pathway in tomato affects susceptibility to RNA viruses.

Tomato Dicer-like2 (slDCL2) is a key component of resistance pathways against potato virus X (PVX) and tobacco mosaic virus (TMV). It is also required for production of endogenous small RNAs, including miR6026 and other noncanonical microRNAs (miRNAs). The slDCL2 mRNAs are targets of these slDCL2-dependent RNAs in a feedback loop that was disrupted by target mimic RNAs of miR6026. In lines expressing these RNAs, there was correspondingly enhanced resistance against PVX and TMV. These findings illustrate a novel miRNA pathway in plants and a crop protection strategy in which miRNA target mimicry elevates expression of defense-related mRNAs.

Maximizing saffron apocarotenoid production in varied tomato fruit carotenoid contexts.

Saffron spice owes its commercial appreciation to its specific apocarotenoids: crocins, picrocrocin, and safranal. In Crocus sativus, these compounds are biosynthesized from zeaxanthin through oxidative cleavage by the carotenoid cleavage dioxygenase 2 (CCD2). Transgenic tomato plants expressing CsCCD2 in the fruit, named Tomaffron, accumulate high levels of saffron apocarotenoids despite the low substrate availability for CsCCD2. In the present study, CsCCD2 has been introduced into Xantomato; this tomato variety accumulates high levels of zeaxanthin and ß-carotene in ripe fruit due to a combination of four mutant alleles. Xantomato and Tomaffron genotypes have been combined to optimize apocarotenoid production. The best transgenic lines accumulated 15 and 14 times more crocins and picrocrocin than Tomaffron, alongside a fourfold increase in ß-carotene compared to Xantomato, albeit at a cost in fruit yield. Segregation of the four mutations has been carried out to find the best combination for obtaining high levels of saffron apocarotenoids without adverse effects on fruit yield. Plants harboring the high-pigmented 3 (hp3) and BETA (BSh) mutations accumulated 6 and 15 times more crocins and picrocrocin than Tomaffron, without observable pleiotropic effects. Additionally, those high levels of saffron apocarotenoids were obtained in fruit accumulating high levels of both lycopene and ß-carotene independently or in combination, suggesting a regulatory role for the apocarotenoids produced and indicating that it is possible to increase the levels of both types of healthy promoting molecules simultaneously.

SlWRKY55 coordinately acts with SlVQ11 to enhance tomato thermotolerance by activating SlHsfA2.

High temperature (HT) severely restricts plant growth, development, and productivity. Plants have evolved a set of mechanisms to cope with HT, including the regulation of heat stress transcription factors (Hsfs) and heat shock proteins (Hsps). However, it is not clear how the transcriptional and translational levels of Hsfs and Hsps are controlled in tomato. Here, we reported that the HT-induced transcription factor SlWRKY55 recruited SlVQ11 to coordinately regulate defense against HT. SlWRKY55 directly bound to the promoter of SlHsfA2 and promoted its expression, which was increased by SlVQ11. Moreover, both SlWRKY55 and SlVQ11 physically interacted with SlHsfA2 to enhance the transcriptional activity of SlHsfA2. Thus, our results revealed a molecular mechanism that the SlWRKY55/SlVQ11-SlHsfA2 cascade enhanced thermotolerance and provided potential target genes for improving the adaptability of crops to HT.

Ploidy-specific transcriptomes shed light on the heterogeneous identity and metabolism of developing tomato pericarp cells.

Endoreduplication, during which cells increase their DNA content through successive rounds of full genome replication without cell division, is the major source of endopolyploidy in higher plants. Endoreduplication plays pivotal roles in plant growth and development and is associated with the activation of specific transcriptional programmes that are characteristic of each cell type, thereby defining their identity. In plants, endoreduplication is found in numerous organs and cell types, especially in agronomically valuable ones, such as the fleshy fruit (pericarp) of tomato presenting high ploidy levels. We used the tomato pericarp tissue as a model system to explore the transcriptomes associated with endoreduplication progression during fruit growth. We confirmed that expression globally scales with ploidy level and identified sets of differentially expressed genes presenting only developmental-specific, only ploidy-specific expression patterns or profiles resulting from an additive effect of ploidy and development. When comparing ploidy levels at a specific developmental stage, we found that non-endoreduplicated cells are defined by cell division state and cuticle synthesis while endoreduplicated cells are mainly defined by their metabolic activity changing rapidly over time. By combining this dataset with publicly available spatiotemporal pericarp expression data, we proposed a map describing the distribution of ploidy levels within the pericarp. These transcriptome-based predictions were validated by quantifying ploidy levels within the pericarp tissue. This in situ ploidy quantification revealed the dynamic progression of endoreduplication and its cell layer specificity during early fruit development. In summary, the study sheds light on the complex relationship between endoreduplication, cell differentiation and gene expression patterns in the tomato pericarp.

Synergistic regulation at physiological, transcriptional and metabolic levels in tomato plants subjected to a combination of salt and heat stress.

With global warming and climate change, abiotic stresses often simultaneously occur. Combined salt and heat stress was a common phenomenon that was severe, particularly in arid/semi-arid lands. We aimed to reveal the systematic responsive mechanisms of tomato genotypes with different salt/heat susceptibilities to combined salt and heat stress. Morphological and physiological responses of salt-tolerant/sensitive and heat-tolerant/sensitive tomatoes at control, heat, salt and combined stress were investigated. Based on leaf Fv /Fm and H2 O2 content, samples from tolerant genotype at the four treatments for 36 h were taken for transcriptomics and metabolomics. We found that plant height, dry weight and net photosynthetic rate decreased while leaf Na+ concentration increased in all four genotypes under salt and combined stress than control. Changes in physiological indicators such as photosynthetic parameters and defence enzyme activities in tomato under combined stress were regulated by the expression of relevant genes and the accumulation of key metabolites. We screened five key pathways in tomato responding to a combination of salt and heat stress, such as oxidative phosphorylation (map00190). Synergistic regulation at morphological, physiological, transcriptional and metabolic levels in tomato plants was induced by combined stress. Heat stress was considered as a dominant stressor for tomato plants under the current combined stress. The oxidative phosphorylation pathway played a key role in tomato in response to combined stress, where tapped key genes (e.g. alternative oxidase, Aox1a) need further functional analysis. Our study will provide a valuable resource important for studying stress combination and improving tomato tolerance.

Detection of trait donors and QTL boundaries for early blight resistance using local ancestry inference in a library of genomic sequences for tomato.

By conducting hierarchical clustering along a sliding window, we generated haplotypes across hundreds of re-sequenced genomes in a few hours. We leveraged our method to define cryptic introgressions underlying disease resistance in tomato (Solanum lycopersicum L.) and to discover resistant germplasm in the tomato seed bank. The genomes of 9 accessions with early blight (Alternaria linariae) disease resistance were newly sequenced and analyzed together with published sequences for 770 tomato and wild species accessions, most of which are available in germplasm collections. Identification of common ancestral haplotypes among resistant germplasm enabled rapid fine mapping of recently discovered quantitative trait loci (QTL) conferring resistance and the identification of possible causal variants. The source of the early blight QTL EB-9 was traced to a vintage tomato named 'Devon Surprise'. Another QTL, EB-5, as well as resistance to bacterial spot disease (Xanthomonas spp.), was traced to Hawaii 7998. A genomic survey of all accessions forecasted EB-9-derived resistance in several heirloom tomatoes, accessions of S. lycopersicum var. cerasiforme, and S. pimpinellifolium PI 37009. Our haplotype-based predictions were validated by screening the accessions against the causal pathogen. There was little evidence of EB-5 prevalence in surveyed contemporary germplasm, presenting an opportunity to bolster tomato disease resistance by adding this rare locus. Our work demonstrates practical insights that can be derived from the efficient processing of large genome-scale datasets, including rapid functional prediction of disease resistance QTL in diverse genetic backgrounds. Finally, our work finds more efficient ways to leverage public genetic resources for crop improvement.

Helper NLRs Nrc2 and Nrc3 act codependently with Prf/Pto and activate MAPK signaling to induce immunity in tomato.

Plant intracellular immune receptors, primarily nucleotide-binding, leucine-rich repeat proteins (NLRs), detect pathogen effector proteins and activate NLR-triggered immunity (NTI). Recently, 'sensor' NLRs have been reported to function with 'helper' NLRs to activate immunity. We investigated the role of two helper NLRs, Nrc2 and Nrc3, on immunity in tomato to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) mediated by the sensor NLR Prf and the Pto kinase. An nrc2/nrc3 mutant no longer activated Prf/Pto-mediated NTI to Pst containing the effectors AvrPto and AvrPtoB. An nrc3 mutant showed intermediate susceptibility between wild-type plants and a Prf mutant, while an nrc2 mutant developed only mild disease. These observations indicate that Nrc2 and Nrc3 act additively in Prf-/Pto-mediated immunity. We examined at what point Nrc2 and Nrc3 act in the Prf/Pto-mediated immune response. In the nrc2/3 mutant, programmed cell death (PCD) normally induced by constitutively active variants of AvrPtoB, Pto, or Prf was abolished, but that induced by M3Kα or Mkk2 was not. PCD induced by a constitutively active Nrc3 was also abolished in a Nicotiana benthamiana line with reduced expression of Prf. MAPK activation triggered by expression of AvrPto in the wild-type tomato plants was completely abolished in the nrc2/3 mutant. These results indicate that Nrc2 and Nrc3 act with Prf/Pto and upstream of MAPK signaling. Nrc2 and Nrc3 were not required for PCD triggered by Ptr1, another sensor NLR-mediating Pst resistance, although these helper NLRs do appear to be involved in resistance to certain Pst race 1 strains.

Specific ABA-independent tomato transcriptome reprogramming under abiotic stress combination.

Crops often have to face several abiotic stresses simultaneously, and under these conditions, the plant's response significantly differs from that observed under a single stress. However, up to the present, most of the molecular markers identified for increasing plant stress tolerance have been characterized under single abiotic stresses, which explains the unexpected results found when plants are tested under real field conditions. One important regulator of the plant's responses to abiotic stresses is abscisic acid (ABA). The ABA signaling system engages many stress-responsive genes, but many others do not respond to ABA treatments. Thus, the ABA-independent pathway, which is still largely unknown, involves multiple signaling pathways and important molecular components necessary for the plant's adaptation to climate change. In the present study, ABA-deficient tomato mutants (flacca, flc) were subjected to salinity, heat, or their combination. An in-depth RNA-seq analysis revealed that the combination of salinity and heat led to a strong reprogramming of the tomato transcriptome. Thus, of the 685 genes that were specifically regulated under this combination in our flc mutants, 463 genes were regulated by ABA-independent systems. Among these genes, we identified six transcription factors (TFs) that were significantly regulated, belonging to the R2R3-MYB family. A protein-protein interaction network showed that the TFs SlMYB50 and SlMYB86 were directly involved in the upregulation of the flavonol biosynthetic pathway-related genes. One of the most novel findings of the study is the identification of the involvement of some important ABA-independent TFs in the specific plant response to abiotic stress combination. Considering that ABA levels dramatically change in response to environmental factors, the study of ABA-independent genes that are specifically regulated under stress combination may provide a remarkable tool for increasing plant resilience to climate change.

Deciphering salt stress responses in Solanum pimpinellifolium through high-throughput phenotyping.

Soil salinity is a major environmental stressor affecting agricultural productivity worldwide. Understanding plant responses to salt stress is crucial for developing resilient crop varieties. Wild relatives of cultivated crops, such as wild tomato, Solanum pimpinellifolium, can serve as a useful resource to further expand the resilience potential of the cultivated germplasm, S. lycopersicum. In this study, we employed high-throughput phenotyping in the greenhouse and field conditions to explore salt stress responses of a S. pimpinellifolium diversity panel. Our study revealed extensive phenotypic variations in response to salt stress, with traits such as transpiration rate, shoot mass, and ion accumulation showing significant correlations with plant performance. We found that while transpiration was a key determinant of plant performance in the greenhouse, shoot mass strongly correlated with yield under field conditions. Conversely, ion accumulation was the least influential factor under greenhouse conditions. Through a Genome Wide Association Study, we identified candidate genes not previously associated with salt stress, highlighting the power of high-throughput phenotyping in uncovering novel aspects of plant stress responses. This study contributes to our understanding of salt stress tolerance in S. pimpinellifolium and lays the groundwork for further investigations into the genetic basis of these traits, ultimately informing breeding efforts for salinity tolerance in tomato and other crops.

PP2C phosphatase Pic14 negatively regulates tomato Pto/Prf-triggered immunity by inhibiting MAPK activation.

Type 2C protein phosphatases (PP2Cs) are emerging as important regulators of plant immune responses, although little is known about how they might impact nucleotide-binding, leucine-rich repeat (NLR)-triggered immunity (NTI). We discovered that expression of the PP2C immunity-associated candidate 14 gene (Pic14) is induced upon activation of the Pto/Prf-mediated NTI response in tomato. Pto/Prf recognizes the effector AvrPto translocated into plant cells by the pathogen Pseudomonas syringae pv. tomato (Pst) and activate a MAPK cascade and other responses which together confer resistance to bacterial speck disease. Pic14 encodes a PP2C with an N-terminal kinase-interacting motif (KIM) and a C-terminal phosphatase domain. Upon inoculation with Pst-AvrPto, Pto/Prf-expressing tomato plants with loss-of-function mutations in Pic14 developed less speck disease, specifically in older leaves, compared to wild-type plants. Transient expression of Pic14 in leaves of Nicotiana benthamiana and tomato inhibited cell death typically induced by Pto/Prf and the MAPK cascade members M3Kα and Mkk2. The cell death-suppressing activity of Pic14 was dependent on the KIM and the catalytic phosphatase domain. Pic14 inhibited M3Kα- and Mkk2-mediated activation of immunity-associated MAPKs and Pic14 was shown to be an active phosphatase that physically interacts with and dephosphorylates Mkk2 in a KIM-dependent manner. Together, our results reveal Pic14 as an important negative regulator of Pto/Prf-triggered immunity by interacting with and dephosphorylating Mkk2.