Tolerant Tomato Research Articles

SlMYB99-mediated auxin and abscisic acid antagonistically regulate ascorbic acids biosynthesis in tomato.

Ascorbic acid (AsA) is a water-soluble antioxidant that plays important roles in plant development and human health. Understanding the regulatory mechanism underlying AsA biosynthesis is imperative to the development of high AsA plants. In this study, we reveal that the auxin response factor SlARF4 transcriptionally inhibits SlMYB99, which subsequently modulates AsA accumulation via transcriptional activation of AsA biosynthesis genes GPP, GLDH, and DHAR. The auxin-dependent transcriptional cascade of SlARF4-SlMYB99-GPP/GLDH/DHAR modulates AsA synthesis, while mitogen-activated protein kinase SlMAPK8 not only phosphorylates SlMYB99, but also activates its transcriptional activity. Both SlMYB99 and SlMYB11 proteins physically interact with each other, thereby synergistically regulating AsA biosynthesis by upregulating the expression of GPP, GLDH, and DHAR genes. Collectively, these results demonstrate that auxin and abscisic acid antagonistically regulate AsA biosynthesis during development and drought tolerance in tomato via the SlMAPK8-SlARF4-SlMYB99/11 module. These findings provide new insights into the mechanism underlying phytohormone regulation of AsA biosynthesis and provide a theoretical basis for the future development of high AsA plants via molecular breeding.

CRISPR-Cas9-based precise engineering of SlHyPRP1 protein towards multi-stress tolerance in tomato.

Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. tomato (Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. sp. lycopersici, which causes fusarium wilt. CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding.

Exploring tomato phenotypic variability under combined nitrogen and water deficit

BackgroundDespite drought and nitrogen (N) deficit being two of the most important crops’ growth limiting factors, only few studies have explored tomato phenotypic variability in response to both abiotic stresses.AimsIn this study, we aimed to perform a phenotypic evaluation and an analysis of the growth traits of 40 tomato genotypes (mostly focusing on old cultivars, but also including modern hybrids and wild tomato relatives’ accessions as anchors) grown in pots that were subjected to combined N and water deficit.MethodsEach genotype was divided into two groups: control (100% N, 100% field capacity) and combined deficit (20% N, 50% field capacity). A total of 14 morpho-physiological traits were evaluated and further analyzed using multivariate statistical methods.ResultsThe Principal Component Analyses revealed considerable phenotypical diversity among tomato genotypes, with four principal components explaining 82% of the variability. Data integration on a cluster analysis separated the studied genotypes into three distinct clusters based on their ability to handle the combined deficit. Tolerance was associated with traits such as lower specific leaf area, lower leaf area ratio and higher water use efficiency, comparing to the sensitive genotypes.ConclusionsThis study shows that tomato tolerance to combined N and water deficit largely varies between genotypes and that old cultivars represent a valuable gene pool towards more sustainable food production systems.

Ethylene inhibition with silver nitrate (AgNO3) and pyrazinamide (PZA) ameliorates in vitro salt tolerance of tomato (Lycopersicon esculentum L) plantlets

Ethylene inhibition with silver nitrate (AgNO3) and pyrazinamide (PZA) ameliorates in vitro salt tolerance of tomato (Lycopersicon esculentum L) plantlets

Anthocyanin synthesis is critical for melatonin-induced chromium stress tolerance in tomato

Chromium (Cr) is a toxic heavy metal for both animals and plants. The multifunctional signaling molecule melatonin can confer plant tolerance to heavy metal stress, but the mechanisms remain largely unknown. Here, we unveiled the critical role of the secondary metabolite anthocyanin in melatonin-induced Cr stress tolerance. Excess Cr caused severe phytotoxicity, which was manifested by leaf yellowing, stunted growth, reduced Fv/Fm, and increased accumulation of reactive oxygen species and malondialdehyde in a dose-dependent manner. Interestingly, leaf anthocyanin content increased under Cr stress and was the highest under 100 µM Cr (7.67-fold), while exogenous melatonin further increased anthocyanin accumulation with the highest being with 100 µM melatonin (by 90.72 %). In addition, exogenous melatonin increased endogenous melatonin content and alleviated Cr stress; however, suppression of melatonin accumulation aggravated Cr phytotoxicity and inhibited anthocyanin accumulation by downregulating the transcript levels of key structural genes. Melatonin also reduced the Cr content in roots and leaves. Crucially, suppression of anthocyanin biosynthesis by silencing an anthocyanin biosynthetic gene ANTHOCYANIDIN SYNTHASE (ANS) significantly compromised melatonin-induced anthocyanin accumulation and alleviation of Cr phytotoxicity, suggesting that anthocyanin potentially acts downstream of melatonin and its accumulation is essential for melatonin-induced Cr stress tolerance in tomato plants.

S‐nitrosylation of ACO homolog 4 improves ethylene synthesis and salt tolerance in tomato

Crop loss due to soil salinization is a global threat to agriculture. Nitric oxide (NO) and ethylene involve in multiple plant tolerance. However, their interaction in salt resistance remains largely elusive. We tested the mutual induction between NO and ethylene, and then identified an 1-aminocyclopropane-1-carboxylate oxidase homolog 4 (ACOh4) that influences ethylene synthesis and salt tolerance through NO-mediated S-nitrosylation. Both NO and ethylene positively responded to salt stress. Furthermore, NO participated in salt-induced ethylene production. Salt tolerance evaluation showed that function of NO was abolished by inhibiting ethylene production. Conversely, function of ethylene was little influenced by blocking NO generation. ACO was identified as the target of NO to control ethylene synthesis. In vitro and invivo results suggested that ACOh4 was S-nitrosylated at Cys172, resulting in its enzymatic activation. Moreover, ACOh4 was induced by NO through transcriptional manner. Knockdown of ACOh4 abolished NO-induced ethylene production and salt tolerance. At physiological status, ACOh4 positively regulates the Na+ and H+ efflux, and keeps K+ /Na+ homeostasis by promoting salt-resistive genes' transcripts. Our findings validate a role of NO-ethylene module in salt tolerance and uncover a novel mechanism of how NO promoting ethylene synthesis against adversity.

"Transcriptomic Analysis of AREB1 and AREB2 Genes Playing Important Roles in Drought Stress Tolerance in Tomato under in vitro Drought Stress"

"Transcriptomic Analysis of AREB1 and AREB2 Genes Playing Important Roles in Drought Stress Tolerance in Tomato under in vitro Drought Stress"

Regulation of miR319b-Targeted SlTCP10 during the Tomato Response to Low-Potassium Stress.

Potassium deficiency confines root growth and decreases root-to-shoot ratio, thereby limiting root K+ acquisition. This study aimed to identify the regulation network of microRNA319 involved in low-K+ stress tolerance in tomato (Solanum lycopersicum). SlmiR319b-OE roots demonstrated a smaller root system, a lower number of root hairs and lower K+ content under low-K+ stress. We identified SlTCP10 as the target of miR319b using a modified RLM-RACE procedure from some SlTCPs' predictive complementarity to miR319b. Then, SlTCP10-regulated SlJA2 (an NAC transcription factor) influenced the response to low-K+ stress. CR-SlJA2 (CRISPR-Cas9-SlJA2) lines showed the same root phenotype to SlmiR319-OE compared with WT lines. OE-SlJA2(Overexpression-SlJA2) lines showed higher root biomass, root hair number and K+ concentration in the roots under low-K+ conditions. Furthermore, SlJA2 has been reported to promote abscisic acid (ABA) biosynthesis. Therefore, SlJA2 increases low-K+ tolerance via ABA. In conclusion, enlarging root growth and K+ absorption by the expression of SlmiR319b-regulated SlTCP10, mediating SlJA2 in roots, could provide a new regulation mechanism for increasing K+ acquisition efficiency under low-K+ stress.

Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato.

Salinity stress is a major problem affecting plant growth and crop productivity. While plant biostimulants have been reported to be an effective solution to tackle salinity stress in different crops, the key genes and metabolic pathways involved in these tolerance processes remain unclear. This study focused on integrating phenotypic, physiological, biochemical and transcriptome data obtained from different tissues of Solanum lycopersicum L. plants (cv. Micro-Tom) subjected to a saline irrigation water program for 61 days (EC: 5.8 dS/m) and treated with a combination of protein hydrolysate and Ascophyllum nodosum-derived biostimulant, namely PSI-475. The biostimulant application was associated with the maintenance of higher K+/Na+ ratios in both young leaf and root tissue and the overexpression of transporter genes related to ion homeostasis (e.g., NHX4, HKT1;2). A more efficient osmotic adjustment was characterized by a significant increase in relative water content (RWC), which most likely was associated with osmolyte accumulation and upregulation of genes related to aquaporins (e.g., PIP2.1, TIP2.1). A higher content of photosynthetic pigments (+19.8% to +27.5%), increased expression of genes involved in photosynthetic efficiency and chlorophyll biosynthesis (e.g., LHC, PORC) and enhanced primary carbon and nitrogen metabolic mechanisms were observed, leading to a higher fruit yield and fruit number (47.5% and 32.5%, respectively). Overall, it can be concluded that the precision engineered PSI-475 biostimulant can provide long-term protective effects on salinity stressed tomato plants through a well-defined mode of action in different plant tissues.

Supplementary Material of Research Article "Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato"

Supplementary Material of Research Article "Transcriptome, Biochemical and Phenotypic Analysis of the Effects of a Precision Engineered Biostimulant for Inducing Salinity Stress Tolerance in Tomato"

Plant Genotype Shapes the Soil Nematode Community in the Rhizosphere of Tomatoes with Different Resistance to Meloidognye incognita

Soil nematodes are considered indicators of soil quality due to their immediate responses to changes in the soil environment and plants. However, little is known about the effects of plant genotypes on the soil nematode community. To elucidate this, high-throughput sequencing and gas chromatography/mass spectrometry analysis was conducted to analyze the soil nematode community and the structure of root exudates in the rhizosphere of tomatoes with different resistance to Meloidognye incognita. The dominant soil nematode group in the soil of resistant tomatoes was Acrobeloides, while the soil nematode group in the rhizosphere of the susceptible and tolerant tomatoes was Meloidognye. Hierarchical clustering analysis and non-metric multidimensional scaling showed that the three soil nematode communities were clustered into three groups according to the resistance level of the tomato cultivars. The soil nematode community of the resistant tomatoes had a higher maturity index and a low plant-parasite index, Wasilewska index and disease index compared to the values of the susceptible and tolerant tomatoes. Redundancy analysis revealed that the disease index and root exudates were strongly related to the soil nematode community of three tomato cultivars. Taken together, the resistance of the tomato cultivars and root exudates jointly shapes the soil nematode community. This study provided a valuable contribution to understanding the mechanism of plant genotypes shaping the soil nematode community.

Transcriptomic and proteomic analysis reveals (E)-2-hexenal modulates tomato resistance against Botrytis cinerea by regulating plant defense mechanism.

In a previous study, we observed that (E)-2-hexenal stimulated systemic resistance against B. cinerea in tomato plants. However, the molecular mechanisms underlying (E)-2-hexenal-mediated regulation of systemic immunity against B. cinerea remained unclear. In the current study, the global mechanism underlying (E)-2-hexenal-meidated regulation of biotic stress tolerance in tomato was investigated using RNA-seq- and LC-MS/MS- integrated transcriptomic and proteomic analyses. Compared to control plants, (E)-2-hexenal-treated plants exhibited reduced susceptibility to B. cinerea, with a 50.51% decrease in lesion diameters. Meanwhile, (E)-2-hexenal vapor fumigation significantly increased total phenolic content and activities of various antioxidant enzymes peroxidase (POD), phenylalanine ammonia lyase (PAL), and lipoxygenase (LOX). A total of 233 differentially expressed genes (DEGs) and 400 differentially expressed proteins (DEPs), respectively, were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that (E)-2-hexenal treatment markedly affected the expression of genes involved in multiple metabolic pathways, especially glutathione metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, and MAPK signaling pathway. Notably, proteomic analysis revealed modulation of the activities of several defense response proteins, such as pathogenesis-related (PR) proteins (Solyc02g031950.3.1, Solyc02g031920.4.1, and Solyc04g064870.3.1), peroxidases (Solyc06g050440.3.1, Solyc01g105070.3.1, Solyc01g015080.3.1, Solyc03g025380.3.1 and Solyc06g076630.3.1). Our results provide a comprehensive analysis of the effects of (E)-2-hexenal treatment on the transcriptome and proteome of tomato plants, which might be used as a reference in further studies on plant defense responses against pathogens.

Membrane lipid metabolism and scavenging of reactive oxygen species are essential for cold resistance of Solanum habrochaites (LA1777): Lipidomics and transcriptomes analysis

Low temperature significantly limits the production of tomato (Solanum lycopersicum) in greenhouse. Solanum habrochaites is a wild species of tomato closely related to Solanum lycopersicum and is more tolerant to cold than S. lycopersicum. However, the mechanism by which S. habrochaites resists low temperature is unknown. This study aimed to analyze the physiology, transcriptomics, and lipidomics of LA1777 (S. habrochaites variety) after exposure to cold stress for 3 days to assess its specific mechanism to cold tolerance. A cold-sensitive cultivated tomato variety AC was used as controls. Physiological analysis showed that the two tomato varieties were significantly affected by cold stress. However, LA1777 had more stable physiological characteristics and maintained relatively good photosynthetic capacity than the AC variety. Transcriptome analysis suggested that the stability of cell membrane structure and function might promote cold tolerance of LA1777. Furthermore, 243 differentially expressed genes (DEGs) involved in lipid metabolism were identified. Particularly, the upregulation of temperature-induced lipocalin genes (TILs) and fatty acid desaturase genes (FADs) promoted cold tolerance by inhibiting the excessive accumulation of reactive oxygen species (ROS) and increasing membrane lipid unsaturation, respectively. Meanwhile, lipidomics data indicated that monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) levels were significantly reduced in both plants under cold stress. MGDG levels decreased by 28% and 54% in LA1777 and AC, respectively, while DGDG decreased by 7% and 29%, respectively. Further results showed that high phosphoinositide (PI) content may increase cold tolerance in LA1777 by maintaining phosphatidylinositol signaling system. Also, results showed that adenosine triphosphate (ATP) produced via β-oxidation of fatty acids in mitochondria can maintain a higher metabolic level of wild tomato under cold stress. A metabolic model of the mechanism of cold tolerance in tomato was also constructed. This study provides a basis for further analysis of the molecular mechanism of cold resistance in tomato, which is crucial for genetic improvement.

Triacylglycerol lipase a10 (SlTGLa10) gene negatively regulates cold tolerance, vegetative and reproductive growth in tomato

Triacylglycerol (TAG) Lipases (TGLs) are the main enzymes involved in triacylglycerol catabolism. TGLs hydrolyze long-chain fatty acid triglycerides, which are involved in plant development and abiotic stress responses. Cold stress severely affects the yield of tomato plants; however, the mechanisms are not clear. Here, we show that Triacylglycerol Lipase a10 (SlTGLa10) negatively regulates cold tolerance and growth in tomato. Compared to wild-type (WT) plants, SlTGLa10 overexpression (OE) plants showed increased sensitivity to cold stress associated with higher levels of malondialdehyde, ion leakage and lipid peroxidation, lower activities of antioxidant enzymes, and reduced expression of several cold-responsive genes (SlCBF1, SlDRCi7, etc.) while the SlTGLa10 knockdown (KD) plants were moderately more cold-tolerant than WT. Expression of lipid transport and metabolism-related genes, and the TAG contents and composition were differentially affected in the OE and KD plants. The OE plants exhibited lighter seed weight, inhibited seed germination, shorter shoots and smaller fruits than WT plants, whereas KD plants showed the opposite phenotypes (heavier seeds, improved seed germination, longer shoots and larger fruits compared to the WT plants). Although our data indicated that SlMYB16 might not bind to conventional MYB-recognition sites in the promoter region of SlTGLa10, the transcript levels of SlTGLa10 were reduced in tomato plants overexpressing SlMYB16. The results suggest that SlMYB16 may function as a negative factor for the transcription of SlTGLa10. Overall, our work emphasizes the importance of SlTGLa10 for cold tolerance and growth and development in tomato and can provide a straightforward breeding strategy to develop crops that are cold-resistant and high-yielding.

MicroRNA162 regulates stomatal conductance in response to low night temperature stress via abscisic acid signaling pathway in tomato

MicroRNAs (miRNAs) mediate the degradation of target mRNA and inhibit mRNA translation to regulate gene expression at the transcriptional and post-transcriptional levels in response to environmental stress in plants. We characterized the post-transcriptional mechanism by deep sequencing small RNA (sRNA) to examine how miRNAs were involved in low night temperature (LNT) stress in tomato and whether the molecular mechanism depended on the abscisic acid (ABA) signaling pathway. We annotated conserved miRNAs and novel miRNAs with four sRNA libraries composed of wild-type (WT) tomato plants and ABA-deficient mutant (sit) plants under normal growth and LNT stress conditions. Reverse genetics analysis suggested that miR162 participated in LNT resistance and the ABA-dependent signaling pathway in tomato. miR162-overexpressing (pRI-miR162) and miR162-silenced (pRNAi-miR162) transgenic tomato plants were generated to evaluate miR162 functions in response to LNT stress. miR162 deficiency exhibited high photosynthetic capacity and regulated stomatal opening, suggesting negative regulation of miR162 in the ABA-dependent signaling pathway in response to LNT stress. As feedback regulation, miR162 positively regulated ABA to maintain homeostasis of tomato under diverse abiotic stresses. The mRNA of DICER-LIKE1 (DCL1) was targeted by miR162, and miR162 inhibited DCL1 cleavage in LNT response, including the regulation of miRNA160/164/171a and their targets. The DCL1-deficient mutants (dcl1) with CRISPR/Cas9 prevented stomatal opening to influence photosynthesis in the ABA signaling pathway under LNT stress. Finally, we established the regulatory mechanism of ABA-miR162-DCL1, which systematically mediated cold tolerance in tomato. This study suggests that post-transcriptional modulators acted as systemic signal responders via the stress hormone signaling pathway, and the model at the post-transcriptional level presents a new direction for research in plant abiotic stress resistance.

Desert endophytic fungi improve reproductive, morphological, biochemical, yield and fruit quality characteristics of tomato under drought stress

PurposeCrops are increasingly affected by drought; hence, the current study explored the potential role of three desert endophytic fungi, Aspergillus fumigatus, Aspergillus terreus and Talaromyces variabilis, in conferring drought tolerance in tomato plants.Design/methodology/approachPreserved endophytic fungi from a Rhazya stricta desert plant were adopted to obtain the required fungal treatment; tomatoes received fungal treatments directly in plastic trays and subsequently in pots. Drought was applied using 15% of PEG-6000 at two stages: flowering and fruiting. The following parameters were measured: pollen sterility, growth characteristics, morphological analysis and biochemical analysis, including proline, gibberellic acid (GA3) and chlorophyll measurements; thus, the data were analyzed statistically using SPSS software.FindingsAll applied endophytes significantly promoted pollen viability and tomato yield under stressed and nonstressed conditions. Interestingly, these endophytes significantly enhanced the number of trichomes under drought stress and promoted tomato fruit quality. The colonized tomato plants accumulated a high proline level under drought stress but lower than un-inoculated stressed plants. Also, a significant rise in growth characteristics was observed by A. fumigatus and A. terreus under normal conditions. Moreover, both raised GA3 levels under drought-stressed and nonstressed conditions. Also these two endophytes enhanced chlorophyll and carotenoid contents under drought stress. Fruit characteristics were enhanced by nonstressed T. variabilis and stressed A. fumigatus.Originality/valueThe present endophytic fungi provide impressive benefits to their host in normal and drought-stressed conditions. Consequently, they represent valuable sources as sustainable and environmentally friendly alternatives to mitigate drought stress.

Supplementary File "From root to leaf, a precision engineered biostimulant's mech-anism for inducing salinity stress tolerance in tomato"

Supplementary File "From root to leaf, a precision engineered biostimulant's mech-anism for inducing salinity stress tolerance in tomato"

Characterization of volatile organic compounds in grafted tomato plants upon potyvirus necrotic infection

A headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME/GC-MS) method was used to study the volatile organic compounds (VOCs) associated with the differential immune response of tomato plants infected with the recombinant strain of potato virus Y (PVYC-to), necrogenic to tomato. Analysis was carried out in UC82 (UC), a virus susceptible tomato variety, comparing the same UC plants grafted or not onto a virus tolerant tomato ecotype, Manduria (Ma); the three types of samples used for the GC-MS analysis were mock-inoculated UC/Ma plants, UC/Ma+PVYC-to and UC+PVYC-to plants; the VOCs obtained were 111. Results from symptomatic PVYC-to-infected UC plants showed a VOCs composition enriched in alcohols, fatty acid derivates, benzenoids, and salicylic acid derivatives, while in mock-inoculated UC/Ma plants VOCs were mainly characterized by methyl ester compounds. The VOC profile was in line with RNAseq data analyses, denoting that PVYC-to viral RNA accumulation and disease symptoms induce the specific transcriptional activation of genes involved in VOCs biosynthesis. Furthermore, principal component analysis highlighted that VOCs of PVYC-to-infected and mock-inoculated grafted plants were much closer each other than that of symptomatic PVYC-to-infected non-grafted UC plants. These results suggest that VOCs profiles of tomato plants are related to the viral RNA accumulation, disease intensity and graft-derived tolerance to PVYC-to infection.

Exogenous Application of Calcium Ameliorates Salinity Stress Tolerance of Tomato (Solanum lycopersicum L.) and Enhances Fruit Quality.

Tomato is affected by various biotic and abiotic stresses, especially salinity, which drastically hinders the growth and yield of tomato. Calcium (Ca) is a vital macronutrient which plays physiological and biochemical roles in plants. Hence, we studied the protective roles of Ca against salinity stress in tomato. There were eight treatments comprising control (nutrient solution), 5 mM Ca, 10 mM Ca, 15 mM Ca, 12 dS m-1 NaCl, 12 dS m-1 NaCl + 5 mM Ca, 12 dS m-1 NaCl + 10 mM Ca and 12 dS m-1 NaCl + 15 mM Ca, and two tomato varieties: BARI tomato-2 and Binatomato-5. Salinity significantly decreased the plant-growth and yield attributes, relative water content (RWC), photosynthetic pigments (SPAD value) and the uptake of K, Ca and Mg in leaves and roots. Salinity-induced oxidative stress was present in the form of increased Na+ ion concentration, hydrogen peroxide (H2O2) content and lipid peroxidation (MDA). Ca application reduced oxidative stress through the boosting of antioxidant enzymatic activity. Exogenous Ca application enhanced proline and glycine betaine content and reduced Na+ uptake, which resulted in the inhibition of ionic toxicity and osmotic stress, respectively. Hence, Ca application significantly increased the growth and yield attributes, RWC, SPAD value, and uptake of K, Ca and Mg. Calcium application also had a significant effect on the fruit quality of tomato and the highest total soluble solid, total sugar, reducing sugar, β-carotene, vitamin C and juice pH were found for the combined application of NaCl and Ca. Therefore, application of Ca reversed the salt-induced changes through increasing osmoprotectants, activation of antioxidants enzymes, and by optimizing mineral nutrient status.

CRISPR/Cas9-Mediated Mutation in XSP10 and SlSAMT Genes Impart Genetic Tolerance to Fusarium Wilt Disease of Tomato (Solanum lycopersicum L.)

Fusarium wilt is a major devastating fungal disease of tomato (Solanum lycopersicum L.) caused by Fusarium oxysporum f. sp. lycopersici (Fol) which reduces the yield and production. Xylem sap protein 10 (XSP10) and Salicylic acid methyl transferase (SlSAMT) are two putative negative regulatory genes associated with Fusarium wilt of tomato. Fusarium wilt tolerance in tomato can be developed by targeting these susceptible (S) genes. Due to its efficiency, high target specificity, and versatility, CRISPR/Cas9 has emerged as one of the most promising techniques for knocking out disease susceptibility genes in a variety of model and agricultural plants to increase tolerance/resistance to various plant diseases in recent years. Though alternative methods, like RNAi, have been attempted to knock down these two S genes in order to confer resistance in tomato against Fusarium wilt, there has been no report of employing the CRISPR/Cas9 system for this specific intent. In this study, we provide a comprehensive downstream analysis of the two S genes via CRISPR/Cas9-mediated editing of single (XSP10 and SlSAMT individually) and dual-gene (XSP10 and SlSAMT simultaneously). Prior to directly advancing on to the generation of stable lines, the editing efficacy of the sgRNA-Cas9 complex was first validated using single cell (protoplast) transformation. In the transient leaf disc assay, the dual-gene editing showed strong phenotypic tolerance to Fusarium wilt disease with INDEL mutations than single-gene editing. In stable genetic transformation of tomato at the GE1 generation, dual-gene CRISPR transformants of XSP10 and SlSAMT primarily exhibited INDEL mutations than single-gene-edited lines. The dual-gene CRISPR-edited lines (CRELs) of XSP10 and SlSAMT at GE1 generation conferred a strong phenotypic tolerance to Fusarium wilt disease compared to single-gene-edited lines. Taken together, the reverse genetic studies in transient and stable lines of tomato revealed that, XSP10 and SlSAMT function together as negative regulators in conferring genetic tolerance to Fusarium wilt disease.