Reduced γ-glutamyl hydrolase activity likely contributes to high folate levels in Periyakulam-1 tomato.

Tomato cultivars show wide variation in nutraceutical folate in ripe fruits, yet the loci regulating folate levels in fruits remain unexplored. To decipher regulatory points, we compared two contrasting tomato cultivars: Periyakulam-1 (PKM-1) with high folate and Arka Vikas (AV) with low folate. The progression of ripening in PKM-1 was nearly similar to AV but had substantially lower ethylene emission. In parallel, the levels of phytohormones salicylic acid, ABA, and jasmonic acid were substantially lower than AV. The fruits of PKM-1 were metabolically distinct from AV, with upregulation of several amino acids. Consistent with higher °Brix, the red ripe fruits also showed upregulation of sugars and sugar-derived metabolites. In parallel with higher folate, PKM-1 fruits also had higher carotenoid levels, especially lycopene and ß-carotene. The proteome analysis showed upregulation of carotenoid sequestration and folate metabolism-related proteins in PKM-1. The deglutamylation pathway mediated by γ-glutamyl hydrolase (GGH) was substantially reduced in PKM-1 at the red-ripe stage. The red-ripe fruits had reduced transcript levels of GGHs and lower GGH activity than AV. Conversely, the percent polyglutamylation of folate was much higher in PKM-1. Our analysis indicates the regulation of GGH activity as a potential target to elevate folate levels in tomato fruits.

A Field Collection of Indigenous Grapevines as a Valuable Repository for Applied Research.

The grapevine is an economically important plant, with a historical connection to the development of human culture. Currently, over 6000 accessions are known as individual grapevine varieties, some of which are important to national heritage, valuable for current viticultural practices, and as genetic resources to maintain plasticity under changing climatic conditions, environmental sustainability, and market demands. Recently, the diversity of cultivated grapevines has declined significantly, due to the increased focus of global wine industries on a few major cultivars. Moreover, due to biotic and abiotic stresses, the wild V. vinifera germplasm's genetic diversity has declined, with some varieties on the verge of extinction. Vitis germplasm conservation can be achieved via either in situ (e.g., protected areas) or Ex situ (e.g., field collections, seed banks, and tissue culture collections) methods. This study aims to highlight the importance of Vitis field bank collections. We demonstrate the research done in the Israeli indigenous Vitis vinifera collection. The multi-layer analysis of the varieties enabled the identification of drought stress-resistant varieties, and suggested a mechanism for this resistance through noting the dramatic phenological differences in foliage development between resistant and sensitive varieties. In addition, we show a general characterization of the varieties via major grape characteristics, including bunch and berry shape, as well as their possible utilization based on their aromatic and phenolic profiles.

Seeing the unseen: a trifoliate (MYB117) mutant allele fortifies folate and carotenoids in tomato fruits.

In tomato (Solanum lycopersicum), mutations in the gene encoding the R2R3-MYB117 transcription factor elicit trifoliate leaves and initiate the formation of axillary meristems; however, their effects on fruit ripening remain unexplored. The fruits of a new trifoliate (tf) mutant (tf-5) were firmer and had higher °Brix values and higher folate and carotenoid contents. The transcriptome, proteome, and metabolome profiling of tf-5 reflected a broad-spectrum change in cellular homeostasis. The tf-5 allele enhanced the fruit firmness by suppressing cell wall softening-related proteins. tf-5 fruit displayed a substantial increase in amino acids, particularly γ-aminobutyric acid, with a parallel reduction in aminoacyl-tRNA synthases. The increased lipoxygenase protein and transcript levels seemingly elevated jasmonic acid levels. In addition, increased abscisic acid hydrolase transcript levels coupled with reduced precursor supply lowered abscisic acid levels. The upregulation of carotenoids was mediated by modulation of methylerythreitol and plastoquinone pathways and increased the levels of carotenoid isomerization proteins. The upregulation of folate in tf-5 was connoted by the increase in the precursor p-aminobenzoic acid and transcript levels of several folate biosynthesis genes. The reduction in pterin-6-carboxylate levels and γ-glutamyl hydrolase activity indicated that reduced folate degradation in tf-5 increased folate levels. Our study delineates that in addition to leaf development, MYB117 also influences fruit metabolism. The tf-5 allele can be used to increase γ-aminobutyric acid, carotenoid, and folate levels in tomato.

Extraction and Analysis of Phenolic Compounds from Grape Berries.

Phenolics are ubiquitous compounds that represent the most abundant and diverse class of plant metabolites. From an analytical point of view, phenolics can be divided into soluble phenolics such as phenolic acids, phenylpropanoids, flavonoids and quinones, and nonsoluble compounds such as proanthocyanidins, lignins, and cell wall-bound hydroxycinnamic acids. Extraction of phenolics from the sample material is the first step toward their analysis. Biochemical methods for determination of total phenolics content were widely used in the past but modern chromatographic and mass spectrometric methods for identification and quantification of individual compounds are available in recent years. In this chapter, we describe methods for phenolic compounds extraction used in our laboratories from berries of Vitis vinifera and analytical methods including HPLC coupled to DAD detector and Q-TOF LC/MS for their analysis.

Cytokinin but not gibberellin application had major impact on the phenylpropanoid pathway in grape.

Cytokinin and gibberellic acid (GA) are growth regulators used to increase berry size in seedless grapes and it is of interest to understand their effects on the phenylpropanoid pathway and on ripening processes. GA3 and synthetic cytokinin forchlorfenuron (N-(2-chloro-4-pyridyl)-N'-phenylurea, CPPU) and their combination were applied to 6 mm diameter fruitlets of 'Sable Seedless', and berries were sampled 51 and 70 days (d) following application. All treatments increased berry size and delayed sugar accumulation and acid degradation with a stronger effect of CPPU. CPPU, but not GA, reduced berry color and the levels of anthocyanins. While CPPU reduced the levels of anthocyanins by more than 50%, the combined treatment of GA+CPPU reduced the levels by about 25% at 51 d. CPPU treatment had minor effects on flavonols content but increased the levels of monomeric flavan-3-ols by more than two-fold. Phloroglucinol analysis using HPLC showed that proanthocyanidin content was significantly increased by CPPU, whereas mean degree of polymerization was reduced from 26 to 19. Volatile analysis by GC-MS showed changes in composition with CPPU or GA treatment with potential impact on flavor. RNA-seq analysis showed that GA had a minor overall effect on the transcriptome whereas CPPU had pronounced effects on gene expression at both 51 and 70 d. Comparing the control and CPPU at similar Brix of ca. 19.7°, a reduced expression of stilbene synthases (STSs) including their regulators MYB14 and MYB15, and other phenylpropanoid-related genes was observed in CPPU-treated grapes. Overall, our study shows that CPPU had a major influence on the phenylpropanoid pathway and affected multiple ripening-related processes.

The new kid on the block: a dominant-negative mutation of phototropin1 enhances carotenoid content in tomato fruits.

Phototropins, the UVA-blue light photoreceptors, endow plants to detect the direction of light and optimize photosynthesis by regulating positioning of chloroplasts and stomatal gas exchange. Little is known about their functions in other developmental responses. A tomato Non-phototropic seedling1 (Nps1) mutant, bearing an Arg495His substitution in the vicinity of LOV2 domain in phototropin1, dominant-negatively blocks phototropin1 responses. The fruits of Nps1 mutant were enriched in carotenoids, particularly lycopene, compared with its parent, Ailsa Craig. On the contrary, CRISPR/CAS9-edited loss of function phototropin1 mutants displayed subdued carotenoids compared with the parent. The enrichment of carotenoids in Nps1 fruits is genetically linked with the mutation and exerted in a dominant-negative fashion. Nps1 also altered volatile profiles with high levels of lycopene-derived 6-methyl 5-hepten2-one. The transcript levels of several MEP and carotenogenesis pathway genes were upregulated in Nps1. Nps1 fruits showed altered hormonal profiles with subdued ethylene emission and reduced respiration. Proteome profiles showed a causal link between higher carotenogenesis and increased levels of protein protection machinery, which may stabilize proteins contributing to MEP and carotenogenesis pathways. The enhancement of carotenoid content by Nps1 in a dominant-negative fashion offers a potential tool for high lycopene-bearing hybrid tomatoes.

Girdling of table grapes at fruit set can divert the phenylpropanoid pathway towards accumulation of proanthocyanidins and change the volatile composition.

Girdling is an important horticultural practice that allows increased yields or modulated ripening but not much is known how it affects metabolic processes. Trunk girdling was performed at fruit set using a single-blade knife on two table grape cultivar SUPERIOR SEEDLESS® and SABLE SEEDLESS®. Sampling of berries was carried out 1 or 9 weeks after girdling in 2017 from both cultivars and 7 and 9 weeks after girdling of 'Sable' in 2018. As expected, girdling resulted in consistent increase in berry size but total soluble content of mature 'Superior' berries was not affected and in 'Sable' it was slightly reduced in one of the two seasons examined. One week after girdling, abscisic acid and gibberellin content was higher in fruitlets from girdled vines and genes of the phenylpropanoid pathway were induced in both cultivars. Berry color development of 'Sable' measured both by auto-fluorescence and concentration of anthocyanins was reduced upon girdling. In contrast, flavan-3-ol and flavonol content, and total proanthcyanidins (PA) content increased 1.8-fold while the mean degree polymerization of the PA decreased from 26 to 21 upon girdling. Girdling reduced the levels of fatty acid derived volatiles in berries of 'Superior' and 'Sable'. In 'Sable', the total terpene level and the level of volatiles released after acid hydrolysis, decreased upon girdling. Overall, our study indicates that girdling can divert metabolic pathways in a manner that may have significant effect on the taste and flavor of grapes.

Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population.

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next-generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty-five genes belonging to carotenoids and folate metabolism were PCR-amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600-bp amplicons were directly sequenced in a non-overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128-fold pooling. An evaluation of six different software programs (camba, crisp, gatk unified genotyper, lofreq, snver and vipr) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.

Natural variation in folate levels among tomato (Solanum lycopersicum) accessions.

Folate content was estimated in tomato (Solanum lycopersicum) accessions using microbiological assay (MA) and by LC-MS. The MA revealed that in red-ripe fruits folate levels ranged from 4 to 60µg/100g fresh weight. The LC-MS estimation of red-ripe fruits detected three folate forms, 5-CH3-THF, 5-CHO-THF, 5,10-CH(+)THF and folate levels ranged from 14 to 46µg/100g fresh weight. In mature green and red ripe fruit, 5-CH3-THF was the most abundant folate form. Comparison of LC-MS with MA revealed that MA inaccurately estimates folate levels. The accumulation of folate forms and their distribution varied among accessions. The single nucleotide polymorphism was examined in the key genes of the folate pathway to understand its linkage with folate levels. Despite the significant variation in folate levels among tomato accessions, little polymorphism was found in folate biosynthesis genes. Our results indicate that variation in folate level is governed by a more complex regulation at cellular homeostasis level.

Tomato Fruits Show Wide Phenomic Diversity but Fruit Developmental Genes Show Low Genomic Diversity.

Domestication of tomato has resulted in large diversity in fruit phenotypes. An intensive phenotyping of 127 tomato accessions from 20 countries revealed extensive morphological diversity in fruit traits. The diversity in fruit traits clustered the accessions into nine classes and identified certain promising lines having desirable traits pertaining to total soluble salts (TSS), carotenoids, ripening index, weight and shape. Factor analysis of the morphometric data from Tomato Analyzer showed that the fruit shape is a complex trait shared by several factors. The 100% variance between round and flat fruit shapes was explained by one discriminant function having a canonical correlation of 0.874 by stepwise discriminant analysis. A set of 10 genes (ACS2, COP1, CYC-B, RIN, MSH2, NAC-NOR, PHOT1, PHYA, PHYB and PSY1) involved in various plant developmental processes were screened for SNP polymorphism by EcoTILLING. The genetic diversity in these genes revealed a total of 36 non-synonymous and 18 synonymous changes leading to the identification of 28 haplotypes. The average frequency of polymorphism across the genes was 0.038/Kb. Significant negative Tajima'D statistic in two of the genes, ACS2 and PHOT1 indicated the presence of rare alleles in low frequency. Our study indicates that while there is low polymorphic diversity in the genes regulating plant development, the population shows wider phenotype diversity. Nonetheless, morphological and genetic diversity of the present collection can be further exploited as potential resources in future.

High performance liquid chromatography coupled to mass spectrometry for profiling and quantitative analysis of folate monoglutamates in tomato.

Folates are essential micronutrients for animals as they play a major role in one carbon metabolism. Animals are unable to synthesize folates and obtain them from plant derived food. In the present study, a high performance liquid chromatography coupled to mass spectrometric (HPLC-MS/MS) method was developed for the high throughput screening and quantitative analysis of folate monoglutamates in tomato fruits. For folate extraction, several parameters were optimized including extraction conditions, pH range, amount of tri-enzyme and boiling time. After processing the extract was purified using ultra-filtration with 10 kDa membrane filter. The ultra-filtered extract was chromatographed on a RP Luna C18 column using gradient elution program. The method was validated by determining linearity, sensitivity and recovery. This method was successfully applied to folate estimation in spinach, capsicum, and garden pea and demonstrated that this method offers a versatile approach for accurate and fast determination of different folate monoglutamates in vegetables.