Functional characterization of plant specific Indeterminate Domain (IDD) transcription factors in tomato (Solanum lycopersicum L.).

Plant-specific transcription factors (TFs) are responsible for regulating the genes involved in the development of plant-specific organs and response systems for adaptation to terrestrial environments. This includes the development of efficient water transport systems, efficient reproductive organs, and the ability to withstand the effects of terrestrial factors, such as UV radiation, temperature fluctuations, and soil-related stress factors, and evolutionary advantages over land predators. In rice and Arabidopsis, INDETERMINATE DOMAIN (IDD) TFs are plant-specific TFs with crucial functions, such as development, reproduction, and stress response. However, in tomatoes, IDD TFs remain uncharacterized. Here, we examined the presence, distribution, structure, characteristics, and expression patterns of SlIDDs. Database searches, multiple alignments, and motif alignments suggested that 24 TFs were related to Arabidopsis IDDs. 18 IDDs had two characteristic C2H2 domains and two C2HC domains in their coding regions. Expression analyses suggest that some IDDs exhibit multi-stress responsive properties and can respond to specific stress conditions, while others can respond to multiple stress conditions in shoots and roots, either in a tissue-specific or universal manner. Moreover, co-expression database analyses suggested potential interaction partners within IDD family and other proteins. This study functionally characterized SlIDDs, which can be studied using molecular and bioinformatics methods for crop improvement.

Plant growth-promoting rhizobacteria Pseudomonas aeruginosa HG28-5 improves salt tolerance by regulating Na+/K+ homeostasis and ABA signaling pathway in tomato.

Salinity stress badly restricts the growth, yield and quality of vegetable crops. Plant growth-promoting rhizobacteria (PGPR) is a friendly and effective mean to enhance plant growth and salt tolerance. However, information on the regulatory mechanism of PGPR on vegetable crops in response to salt stress is still incomplete. Here, we screened a novel salt-tolerant PGPR strain Pseudomonas aeruginosa HG28-5 by evaluating the tomatoes growth performance, chlorophyll fluorescence index, and relative electrolyte leakage (REL) under normal and salinity conditions. Results showed that HG28-5 colonization improved seedling growth parameters by increasing the plant height (23.7%), stem diameter (14.6%), fresh and dry weight in the shoot (60.3%, 91.1%) and root (70.1%, 92.5%), compared to salt-stressed plants without colonization. Likewise, HG28-5 increased levels of maximum photochemical efficiency of PSII (Fv/Fm) (99.3%), the antioxidant enzyme activities as superoxide dismutase (SOD, 85.5%), peroxidase (POD, 35.2%), catalase (CAT, 20.6%), and reduced the REL (48.2%), MDA content (41.3%) and ROS accumulation in leaves of WT tomatoes under salt stress in comparison with the plants treated with NaCl alone. Importantly, Na+ content of HG28-5 colonized salt-stressed WT plants were decreased by15.5% in the leaves and 26.6% in the roots in the corresponding non-colonized salt-stressed plants, which may be attributed to the higher K+ concentration and SOS1, SOS2, HKT1;2, NHX1 transcript levels in leaves of colonized plants under saline condition. Interestingly, increased abscisic acid (ABA) content and upregulation of ABA pathway genes (ABA synthesis-related genes NCED1, NCED2, NCED4, NECD6 and signal genes ABF4, ABI5, and AREB) were observed in HG28-5 inoculated salt-stressed WT plants. ABA-deficient mutant (not) with NCED1 deficiency abolishes the effect of HG28-5 on alleviating salt stress in tomato, as exhibited by the substantial rise of REL and ROS accumulation and sharp drop of Fv/Fm in the leaves of not mutant plants. Notably, HG28-5 colonization enhances tomatoes fruit yield by 54.9% and 52.4% under normal and saline water irrigation, respectively. Overall, our study shows that HG28-5 colonization can significantly enhance salt tolerance and improved fruit yield by a variety of plant protection mechanism, including reducing oxidative stress, regulating plant growth, Na+/K+ homeostasis and ABA signaling pathways in tomato. The findings not only deepen our understanding of PGPR regulation plant growth and salt tolerance but also allow us to apply HG28-5 as a microbial fertilizer for agricultural production in high-salinity areas.

Treatment of tomato paste wastewater by electrochemical and membrane processes: process optimization and cost calculation.

This study investigated the treatment of wastewater from tomato paste (TP) production using electrocoagulation (EC) and electrooxidation (EO). The effectiveness of water recovery from the pretreated water was then investigated using the membrane process. For this purpose, the effects of independent control variables, including electrode type (aluminum, iron, graphite, and stainless steel), current density (25-75 A/m2), and electrolysis time (15-120 min) on chemical oxygen demand (COD) and color removal were investigated. The results showed that 81.0% of COD and 100% of the color removal were achieved by EC at a current density of 75 A/m2, a pH of 6.84 and a reaction time of 120 min aluminum electrodes. In comparison, EO with graphite electrodes achieved 55.6% of COD and 100% of the color removal under similar conditions. The operating cost was calculated to be in the range of $0.56-30.62/m3. Overall, the results indicate that EO with graphite electrodes is a promising pretreatment process for the removal of various organics. In the membrane process, NP030, NP010, and NF90 membranes were used at a volume of 250 mL and 5 bar. A significant COD removal rate of 94% was achieved with the membrane. The combination of EC and the membrane process demonstrated the feasibility of water recovery from TP wastewater.

SlCathB2 as a negative regulator mediates a novel regulatory pathway upon high-temperature stress response in tomato.

High-temperature stress (HS) is a major abiotic stress that affects the yield and quality of plants. Cathepsin B-like protease 2 (CathB2) has been reported to play a role in developmental processes and stress response, but its involvement in HS response has not been identified. Here, overexpression, virus-induced gene silencing (VIGS)and RNA-sequencing analysis were performed to uncover the functional characteristics of SlCathB2-1 and SlCathB2-2 genes for HS response in tomato. The results showed that overexpression of SlCathB2-1 and SlCathB2-2 resulted in reduced heat tolerance of tomato to HS while silencing the genes resulted in enhanced heat tolerance. RNA-sequencing analysis revealed that the heat shock proteins (HSPs) exhibited higher expression in WT than in SlCathB2-1 and SlCathB2-2 overexpression lines. Furthermore, the possible molecular regulation mechanism underlying SlCathB2-1 and SlCathB2-2-mediated response to HS was investigated. We found that SlCathB2-1 and SlCathB2-2 negatively regulated antioxidant capacity by regulating a set of genes involved in antioxidant defence and reactive oxygen species (ROS) signal transduction. We also demonstrated that SlCathB2-1 and SlCathB2-2 positively regulated ER-stress-induced PCD (ERSID) by regulating unfolded protein response (UPR) gene expression. Furthermore, SlCathB2-1 and SlCathB2-2 interacting with proteasome subunit beta type-4 (PBA4) was identified in the ERSID pathway using yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) screening. Overall, the study identified both SlCathB2-1 and SlCathB2-2 as new negative regulators to HS and presented a new HS response pathway. This provided the foundation for the construction of heat-tolerant molecular mechanisms and breeding strategies aiming to improve the thermotolerance of tomato plants.

A distal enhancer guides the negative selection of toxic glycoalkaloids during tomato domestication.

Steroidal glycoalkaloids (SGAs) are major plant defense metabolites against pests, while they are considered poisonous in food. The genetic basis that guides negative selection of SGAs production during tomato domestication remains poorly understood. Here, we identify a distal enhancer, GAME Enhancer 1 (GE1), as the key regulator of SGAs metabolism in tomato. GE1 recruits MYC2-GAME9 transcriptional complex to regulate the expression of GAME cluster genes via the formation of chromatin loops located in the neighboring DNA region. A naturally occurring GE176 allelic variant is found to be more active in stimulating GAME expression. We show that the weaker GE1 allele has been the main driver for selecting reduced SGAs levels during tomato domestication. Unravelling the "TFs-Enhancer-Promoter" regulatory mechanism operating in SGAs metabolism opens unprecedented prospects for SGAs manipulation in Solanaceae via precision breeding strategies.

Curcumin: An innovative approach for postharvest control of Alternaria alternata induced black rot in cherry tomatoes.

Curcumin, a natural bioactive compound derived from Curcuma longa, has been widely recognized for its antifungal properties. In this study, we investigated the effects of curcumin on the phytopathogenic fungus Alternaria alternata and its pathogenicity in cherry tomato fruit. The results demonstrated that curcumin treatment significantly inhibited mycelial growth and spore germination of A. alternata in a dose-dependent manner. Scanning electron microscopy revealed alterations in the morphology of A. alternata mycelia treated with curcumin. Furthermore, curcumin treatment led to an increase in malondialdehyde and hydrogen peroxide contents, indicating cell membrane damage in A. alternata. Moreover, curcumin exhibited a remarkable inhibitory effect on the incidence and lesion diameters of black rot caused by A. alternata in cherry tomato fruit. Gene expression analysis revealed upregulation of defense-related genes (POD, SOD, and CAT) in tomato fruit treated with curcumin. Additionally, curcumin treatment resulted in decreased activity of exocellular pathogenic enzymes (polygalacturonase, pectin lyase, and endo-1,4-ß-d-glucanase) in A. alternata. Overall, our findings highlight the potential of curcumin as an effective antifungal agent against A. alternata, providing insights into its inhibitory mechanisms on mycelial growth, spore germination, and pathogenicity in cherry tomato fruit.

Bacteria from the skin of amphibians promote growth of Arabidopsis thaliana and Solanum lycopersicum by modifying hormone-related transcriptome response.

Plants and microorganisms establish beneficial associations that can improve their development and growth. Recently, it has been demonstrated that bacteria isolated from the skin of amphibians can contribute to plant growth and defense. However, the molecular mechanisms involved in the beneficial effect for the host are still unclear. In this work, we explored whether bacteria isolated from three tropical frogs species can contribute to plant growth. After a wide screening, we identified three bacterial strains with high biostimulant potential, capable of modifying the root structure of Arabidopsis thaliana plants. In addition, applying individual bacterial cultures to Solanum lycopersicum plants induced an increase in their growth. To understand the effect that these microorganisms have over the host plant, we analysed the transcriptomic profile of A. thaliana during the interaction with the C32I bacterium, demonstrating that the presence of the bacteria elicits a transcriptional response associated to plant hormone biosynthesis. Our results show that amphibian skin bacteria can function as biostimulants to improve agricultural crops growth and development by modifying the plant transcriptomic responses.

Enhancing Botrytis disease management in tomato plants: insights from a Pseudomonas putida strain with biocontrol activity.

AIMS: This study explores the biocontrol potential of Pseudomonas putida Z13 against Botrytis cinerea in tomato plants, addressing challenges posed by the pathogen's fungicide resistance. The aims of the study were to investigate the in vitro and in silico biocontrol traits of Z13, identify its plant-colonizing efficacy, evaluate the efficacy of different application strategies against B. cinerea in planta, and assess the capacity of Z13 to trigger induced systemic resistance (ISR) in plants. METHODS AND RESULTS: The in vitro experiments revealed that Z13 inhibits the growth of B. cinerea, produces siderophores, and exhibits swimming and swarming activity. Additionally, the Z13 genome harbors genes that encode compounds triggering ISR, such as pyoverdine and pyrroloquinoline quinone. The in planta experiments demonstrated Z13's efficacy in effectively colonizing the rhizosphere and leaves of tomato plants. Therefore, three application strategies of Z13 were evaluated against B. cinerea: root drenching, foliar spray, and the combination of root drenching and foliar spray. It was demonstrated that the most effective treatment of Z13 against B. cinerea was the combination of root drenching and foliar spray. Transcriptomic analysis showed that Z13 upregulates the expression of the plant defense-related genes PR1 and PIN2 upon B. cinerea inoculation. CONCLUSION: The results of the study demonstrated that Z13 possesses significant biocontrol traits, such as the production of siderophores, resulting in significant plant protection against B. cinerea when applied as a single treatment to the rhizosphere or in combination with leaf spraying. Additionally, it was shown that Z13 root colonization primes plant defenses against the pathogen.

Mutation mapping of a variegated EMS tomato reveals an FtsH-like protein precursor potentially causing patches of four phenotype classes in the leaves with distinctive internal morphology.

BACKGROUND: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F2 population to reveal candidate genes responsible for the variegation. RESULTS: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation. CONCLUSION: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation.

Bacterial biota associated with the invasive insect pest Tuta absoluta (Meyrick).

Tuta absoluta (the tomato pinworm) is an invasive insect pest with a highly damaging effect on tomatoes causing between 80 and 100% yield losses if left uncontrolled. Resistance to chemical pesticides have been reported in some T. absoluta populations. Insect microbiome plays an important role in the behavior, physiology, and survivability of their host. In a bid to explore and develop an alternative control method, the associated microbiome of this insect was studied. In this study, we unraveled the bacterial biota of T. absoluta larvae and adults by sequencing and analyzing the 16S rRNA V3-V4 gene regions using Illumina NovaSeq PE250. Out of 2,092,015 amplicon sequence variants (ASVs) recovered from 30 samples (15 larvae and 15 adults), 1,268,810 and 823,205 ASVs were obtained from the larvae and adults, respectively. A total of 433 bacterial genera were shared between the adults and larval samples while 264 and 139 genera were unique to the larvae and adults, respectively. Amplicon metagenomic analyses of the sequences showed the dominance of the phylum Proteobacteria in the adult samples while Firmicutes and Proteobacteria dominated in the larval samples. Linear discriminant analysis effect size (LEfSe) comparison revealed the genera Pseudomonas, Delftia and Ralstonia to be differentially enriched in the adult samples while Enterococcus, Enterobacter, Lactococcus, Klebsiella and Wiessella were differentially abundant in the larvae. The diversity indices showed that the bacterial communities were not different between the insect samples collected from different geographical regions. However, the bacterial communities significantly differed based on the sample type between larvae and adults. A co-occurrence network of significantly correlated taxa revealed a strong interaction between the microbial communities. The functional analysis of the microbiome using FAPROTAX showed that denitrification, arsenite oxidation, methylotrophy and methanotrophy as the active functional groups of the adult and larvae microbiomes. Our results have revealed the core taxonomic, functional, and interacting microbiota of T. absoluta and these indicate that the larvae and adults harbor a similar but transitory set of bacteria. The results provide a novel insight and a basis for exploring microbiome-based biocontrol strategy for this invasive insect pest as well as the ecological significance of some of the identified microbiota is discussed.

Tomato seed extract promotes health of the gut microbiota and demonstrates a potential new way to valorize tomato waste.

The current effort to valorize waste byproducts to increase sustainability and reduce agricultural loss has stimulated interest in potential utilization of waste components as health-promoting supplements. Tomato seeds are often discarded in tomato pomace, a byproduct of tomato processing, yet these seeds are known to contain an array of compounds with biological activity and prebiotic potential. Here, extract from tomato seeds (TSE), acquired from pomace, was evaluated for their ability to effect changes on the gut microbiota using an ex vivo strategy. The results found that TSE significantly increased levels of the beneficial taxa Bifidobacteriaceae in a donor-independent manner, from a range of 18.6-24.0% to 27.0-51.6% relative abundance following treatment, yet the specific strain of Bifidobacteriaceae enhanced was inter-individually variable. These structural changes corresponded with a significant increase in total short-chain fatty acids, specifically acetate and propionate, from an average of 13.3 to 22.8 mmol/L and 4.6 to 7.4 mmol/L, respectively. Together, these results demonstrated that TSE has prebiotic potential by shaping the gut microbiota in a donor-independent manner that may be beneficial to human health. These findings provide a novel application for TSE harvested from tomato pomace and demonstrate the potential to further valorize tomato waste products.

Transcriptome Analysis of Tomato Leaves Reveals Candidate Genes Responsive to Tomato Brown Rugose Fruit Virus Infection.

Tomato brown rugose fruit virus (ToBRFV) is a newly-emerging tobamovirus which was first reported on tomatoes in Israel and Jordan, and which has now spread rapidly in Asia, Europe, North America, and Africa. ToBRFV can overcome the resistance to other tobamoviruses conferred by tomato Tm-1, Tm-2, and Tm-22 genes, and it has seriously affected global crop production. The rapid and comprehensive transcription reprogramming of host plant cells is the key to resisting virus attack, but there have been no studies of the transcriptome changes induced by ToBRFV in tomatoes. Here, we made a comparative transcriptome analysis between tomato leaves infected with ToBRFV for 21 days and those mock-inoculated as controls. A total of 522 differentially expressed genes were identified after ToBRFV infection, of which 270 were up-regulated and 252 were down-regulated. Functional analysis showed that DEGs were involved in biological processes such as response to wounding, response to stress, protein folding, and defense response. Ten DEGs were selected and verified by qRT-PCR, confirming the reliability of the high-throughput sequencing data. These results provide candidate genes or signal pathways for the response of tomato leaves to ToBRFV infection.

Identification and virus-induced gene silencing (VIGS) analysis of methyltransferase affecting tomato (Solanum lycopersicum) fruit ripening.

MAIN CONCLUSION: Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases. Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.

Editing of SlWRKY29 by CRISPR-activation promotes somatic embryogenesis in Solanum lycopersicum cv. Micro-Tom.

At present, the development of plants with improved traits like superior quality, high yield, or stress resistance, are highly desirable in agriculture. Accelerated crop improvement, however, must capitalize on revolutionary new plant breeding technologies, like genetically modified and gene-edited crops, to heighten food crop traits. Genome editing still faces ineffective methods for the transformation and regeneration of different plant species and must surpass the genotype dependency of the transformation process. Tomato is considered an alternative plant model system to rice and Arabidopsis, and a model organism for fleshy-fruited plants. Furthermore, tomato cultivars like Micro-Tom are excellent models for tomato research due to its short life cycle, small size, and capacity to grow at high density. Therefore, we developed an indirect somatic embryo protocol from cotyledonary tomato explants and used this to generate epigenetically edited tomato plants for the SlWRKY29 gene via CRISPR-activation (CRISPRa). We found that epigenetic reprogramming for SlWRKY29 establishes a transcriptionally permissive chromatin state, as determined by an enrichment of the H3K4me3 mark. A whole transcriptome analysis of CRISPRa-edited pro-embryogenic masses and mature somatic embryos allowed us to characterize the mechanism driving somatic embryo induction in the edited tomato cv. Micro-Tom. Furthermore, we show that enhanced embryo induction and maturation are influenced by the transcriptional effector employed during CRISPRa, as well as by the medium composition and in vitro environmental conditions such as osmotic components, plant growth regulators, and light intensity.

Alleviation potential of green-synthesized selenium nanoparticles for cadmium stress in Solanum lycopersicum L: modulation of secondary metabolites and physiochemical attributes.

KEY MESSAGE: Selenium nanoparticles reduce cadmium absorption in tomato roots, mitigating heavy metal effects. SeNPs can efficiently help to enhance growth, yield, and biomolecule markers in cadmium-stressed tomato plants. In the present study, the effects of selenium nanoparticles (SeNPs) were investigated on the tomato plants grown in cadmium-contaminated soil. Nanoparticles were synthesized using water extract of Nigella sativa and were characterized for their size and shape. Two application methods (foliar spray and soil drench) with nanoparticle concentrations of 0, 100, and 300 mg/L were used to observe their effects on cadmium-stressed plants. Growth, yield, biochemical, and stress parameters were studied. Results showed that SeNPs positively affected plant growth, mitigating the negative effects of cadmium stress. Shoot length (SL), root length (RL), number of branches (NB), number of leaves per plant (NL), and leaf area (LA) were significantly reduced by cadmium stress but enhanced by 45, 51, 506, 208, and 82%, respectively, by soil drench treatment of SeNPs. Similarly, SeNPs increased the fruit yield (> 100%) and fruit weight (> 100%), and decreased the days to fruit initiation in tomato plants. Pigments were also positively affected by the SeNPs, particularly in foliar treatment. Lycopene content was also enhanced by the addition of NPs (75%). Furthermore, the addition of SeNPs improved the ascorbic acid, protein, phenolic, flavonoid, and proline contents of the tomato plants under cadmium stress, whereas stress enzymes also showed enhanced activities under cadmium stress. It is concluded from the present study that the addition of selenium nanoparticles enhanced the growth and yield of Cd-stressed plants by reducing the absorption of cadmium and increasing the stress management of plants.

HD-Zip proteins modify floral structures for self-pollination in tomato.

Cleistogamy is a type of self-pollination that relies on the formation of a stigma-enclosing floral structure. We identify three homeodomain-leucine zipper IV (HD-Zip IV) genes that coordinately promote the formation of interlocking trichomes at the anther margin to unite neighboring anthers, generating a closed anther cone and cleistogamy (flower morphology necessitating strict self-pollination). These HD-Zip IV genes also control style length by regulating the transition from cell division to endoreduplication. The expression of these HD-Zip IV genes and their downstream gene, Style 2.1, was sequentially modified to shape the cleistogamy morphology during tomato evolution and domestication. Our results provide insights into the molecular basis of cleistogamy in modern tomato and suggest targets for improving fruit set and preventing pollen contamination in genetically modified crops.

Tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of the PpPGF to regulate peach softening during fruit ripening.

Peach fruit rapidly soften after harvest, a significant challenge for producers and marketers as it results in rotting fruit and significantly reduces shelf life. In this study, we identified two tandem genes, PpNAC1 and PpNAC5, within the sr (slow ripening) locus. Phylogenetic analysis showed that NAC1 and NAC5 are highly conserved in dicots and that PpNAC1 is the orthologous gene of Non-ripening (NOR) in tomato. PpNAC1 and PpNAC5 were highly expressed in peach fruit, with their transcript levels up-regulated at the onset of ripening. Yeast two-hybrid and bimolecular fluorescence complementation assays showed PpNAC1 interacting with PpNAC5 and this interaction occurs with the tomato and apple orthologues. Transient gene silencing experiments showed that PpNAC1 and PpNAC5 positively regulate peach fruit softening. Yeast one-hybrid and dual luciferase assays and LUC bioluminescence imaging proved that PpNAC1 and PpNAC5 directly bind to the PpPGF promoter and activate its transcription. Co-expression of PpNAC1 and PpNAC5 showed higher levels of PpPGF activation than expression of PpNAC1 or PpNAC5 alone. In summary, our findings demonstrate that the tandem transcription factors PpNAC1 and PpNAC5 synergistically activate the transcription of PpPGF to regulate fruit softening during peach fruit ripening.

Identification of Volatiles in Tomato Fruit Using Headspace Solid-Phase-Micro-Extraction (HS-SPME) Coupled with Gas Chromatography-Mass Spectrometry (GC-MS).

Plant volatile organic compounds (VOCs) are organic chemicals that plants release as part of their natural biological processes. Various plant tissues produce VOCs, including leaves, stems, flowers, and roots. VOCs are essential in plant communication, defense against pests and pathogens, aroma and flavor, and attracting pollinators. The study of plant volatiles has become an increasingly important area of research in recent years, as scientists have recognized these compounds' important roles in plant physiology. As a result, there has been a growing interest in developing methods for collecting and analyzing plant VOCs. HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) is commonly used for plant volatile analysis due to its high sensitivity and selectivity. This chapter describes an efficient method for extracting and identifying volatile compounds by HS-SPME coupled with GC-MS in tomato fruits.

A Comprehensive Protocol for Assembly of Multiple gRNAs into a Direct Vector for Genome Editing in Tomato.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas 9 (CRISPR-associated protein 9) is a robust DNA-encoded, RNA-mediated sequence-specific nuclease system widely used for genome editing of various plants. Although there are many reports on the assembly of gRNAs and plant transformation, there is no single resource for the complete gene editing methodology in tomato. This chapter provides a comprehensive protocol for designing gRNAs, their assembly into the vector, plant transformation, and final mutant analysis in tomato.

Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster.

Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE (SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1, and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.