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.

Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour-related volatiles in genome-edited tomato RIN mutants.

The tomato (Solanum lycopersicum) ripening inhibitor (rin) mutation is known to completely repress fruit ripening. The heterozygous (RIN/rin) fruits have extended shelf life, ripen normally, but have inferior taste/flavour. To address this, we used genome editing to generate newer alleles of RIN (rinCR ) by targeting the K-domain. Unlike previously reported CRISPR alleles, the rinCR alleles displayed delayed onset of ripening, suggesting that the mutated K-domain represses the onset of ripening. The rinCR fruits had extended shelf life and accumulated carotenoids at an intermediate level between rin and progenitor line. Besides, the metabolites and hormonal levels in rinCR fruits were more akin to rin. To overcome the negative attributes of rin, we crossed the rinCR alleles with Nps1, a dominant-negative phototropin1 mutant, which enhances carotenoid levels in tomato fruits. The resulting Nps1/rinCR hybrids had extended shelf life and 4.4-7.1-fold higher carotenoid levels than the wild-type parent. The metabolome of Nps1/rinCR fruits revealed higher sucrose, malate, and volatiles associated with tomato taste and flavour. Notably, the boosted volatiles in Nps1/rinCR were only observed in fruits bearing the homozygous Nps1 mutation. The Nps1 introgression into tomato provides a promising strategy for developing cultivars with extended shelf life, improved taste, and flavour.

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.

Augmenting tomato functional genomics with a genome-wide induced genetic variation resource.

Induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but have an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. Genome-wide induced mutation resources can improve the targeted breeding of tomatoes and facilitate functional genomics. In this study, we sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected approximately 41 million novel mutations and 5.5 million short InDels not present in the parental cultivar. Approximately 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than one-third of genes in the mutagenized population had one or more deleterious mutations predicted by Sorting Intolerant From Tolerant (SIFT). Nearly one-fourth of deleterious genes mapped on tomato metabolic pathways modulate multiple pathway steps. In addition to the reported GC>AT transition bias for EMS, our population also had a substantial number of AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is the least mutagenic toward EMS. The validation of a potato leaf-like mutation, reduction in carotenoids in ζ-carotene isomerase mutant fruits, and chloroplast relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our database makes a large repertoire of mutations accessible to functional genomics studies and breeding of tomatoes.

ACTH-Modulated Membrane Guanylate Cyclase Signaling System: Origin and Creation.

The membrane guanylate cyclase (MGC) cellular signaling pathway consists of seven signaling pathways and is critical for the survival of prokaryotes eukaryotes, and highly complex vertebrate organisms. A sequel to the author's earlier comprehensive reviews, covering the field of MGC from its origin to its establishment to the year 2014, this article exclusively deals with the history of its development from the year 1963 to 1987. It narrates the efforts involved in building on small projects, brick by brick, and its emergence from the chasm of disbelief, through steady, continuous work. To make the presentation simple and chronologically continuous, the subject matters of the earlier reviews and publication of these authors have been freely borrowed with appropriate citations.

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.

Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation.

In ripening tomato fruits, the leaf-specific carotenoids biosynthesis mediated by phytoene synthase 2 (PSY2) is replaced by a fruit-specific pathway by the expression of two chromoplast-specific genes: phytoene synthase 1 (PSY1) and lycopene-ß-cyclase (CYCB). Though both PSY1 and PSY2 genes express in tomato fruits, the functional role of PSY2 is not known. To decipher whether PSY2-mediated carotenogenesis operates in ripening fruits, we blocked the in vivo activity of lycopene-ß-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-ß-cyclases. The CPTA-treatment induced accumulation of lycopene in leaves, immature-green and ripening fruits. Even in psy1 mutants V7 and r that are deficient in fruit-specific carotenoid biosynthesis, CPTA triggered lycopene accumulation but lowered the abscisic acid level. Differing from fruit-specific carotenogenesis, CPTA-treated V7 and r mutant fruits accumulated lycopene but not phytoene and phytofluene. The lack of phytoene and phytofluene accumulation was reminiscent of PSY2-mediated leaf-like carotenogenesis, where phytoene and phytofluene accumulation is never seen. The lycopene accumulation was associated with the partial transformation of chloroplasts to chromoplasts bearing thread-like structures. Our study uncovers the operation of a parallel carotenogenesis pathway mediated by PSY2 that provides precursors for abscisic acid biosynthesis in ripening tomato fruits.

Multilimbed membrane guanylate cyclase signaling system, evolutionary ladder.

One monumental discovery in the field of cell biology is the establishment of the membrane guanylate cyclase signal transduction system. Decoding its fundamental, molecular, biochemical, and genetic features revolutionized the processes of developing therapies for diseases of endocrinology, cardio-vasculature, and sensory neurons; lastly, it has started to leave its imprints with the atmospheric carbon dioxide. The membrane guanylate cyclase does so via its multi-limbed structure. The inter-netted limbs throughout the central, sympathetic, and parasympathetic systems perform these functions. They generate their common second messenger, cyclic GMP to affect the physiology. This review describes an historical account of their sequential evolutionary development, their structural components and their mechanisms of interaction. The foundational principles were laid down by the discovery of its first limb, the ACTH modulated signaling pathway (the companion monograph). It challenged two general existing dogmas at the time. First, there was the question of the existence of a membrane guanylate cyclase independent from a soluble form that was heme-regulated. Second, the sole known cyclic AMP three-component-transduction system was modulated by GTP-binding proteins, so there was the question of whether a one-component transduction system could exclusively modulate cyclic GMP in response to the polypeptide hormone, ACTH. The present review moves past the first question and narrates the evolution and complexity of the cyclic GMP signaling pathway. Besides ACTH, there are at least five additional limbs. Each embodies a unique modular design to perform a specific physiological function; exemplified by ATP binding and phosphorylation, Ca2+-sensor proteins that either increase or decrease cyclic GMP synthesis, co-expression of antithetical Ca2+ sensors, GCAP1 and S100B, and modulation by atmospheric carbon dioxide and temperature. The complexity provided by these various manners of operation enables membrane guanylate cyclase to conduct diverse functions, exemplified by the control over cardiovasculature, sensory neurons and, endocrine systems.

Evaluation of high salinity tolerance in Pongamia pinnata (L.) Pierre by a systematic analysis of hormone-metabolic network.

Salinity stress results in significant losses in plant productivity and loss of cultivable lands. Although Pongamia pinnata is reported to be a salt-tolerant semiarid biofuel tree, the adaptive mechanisms to saline environments are elusive. Despite a reduction in carbon exchange rate (CER), the unchanged relative water content provides no visible salinity induced symptoms in leaves of hydroponic cultivated Pongamia seedlings for 8 days. Our Na+ -specific fluorescence results demonstrated that there was an effective apoplastic sodium sequestration in the roots. Salinity stress significantly increased zeatin (~5.5-fold), and jasmonic acid (~3.8-fold) levels in leaves while zeatin (~2.5-fold) content increased in leaves as well as in roots of salt-treated plants. Metabolite analysis suggested that osmolytes such as myo-inositol and mannitol were enhanced by ~12-fold in leaves and roots of salt-treated plants. Additionally, leaves of Pongamia showed a significant enhancement in carbohydrate content, while fatty acids were accumulated in roots under salt stress condition. At the molecular level, salt stress enhanced the expression of genes related to transporters, including the Salt Overly Sensitive 2 gene (SOS2), SOS3, vacuolar-cation/proton exchanger, and vacuolar-proton/ATPase exclusively in leaves, whereas the Sodium Proton Exchanger1 (NHX1), Cation Calcium Exchanger (CCX), and Cyclic Nucleotide Gated Channel 5 (CNGC5) were up-regulated in roots. Antioxidant gene expression analysis clearly demonstrated that peroxidase levels were significantly enhanced by ~10-fold in leaves, while Catalase and Fe-superoxide Dismutase (Fe-SOD) genes were increased in roots under salt stress. The correlation interaction studies between phytohormones and metabolites revealed new insights into the molecular and metabolic adaptations that confer salinity tolerance to Pongamia.

iTRAQ-based proteome profiling revealed the role of Phytochrome A in regulating primary metabolism in tomato seedling.

In plants, during growth and development, photoreceptors monitor fluctuations in their environment and adjust their metabolism as a strategy of surveillance. Phytochromes (Phys) play an essential role in plant growth and development, from germination to fruit development. FR-light (FR) insensitive mutant (fri) carries a recessive mutation in Phytochrome A and is characterized by the failure to de-etiolate in continuous FR. Here we used iTRAQ-based quantitative proteomics along with metabolomics to unravel the role of Phytochrome A in regulating central metabolism in tomato seedlings grown under FR. Our results indicate that Phytochrome A has a predominant role in FR-mediated establishment of the mature seedling proteome. Further, we observed temporal regulation in the expression of several of the late response proteins associated with central metabolism. The proteomics investigations identified a decreased abundance of enzymes involved in photosynthesis and carbon fixation in the mutant. Profound accumulation of storage proteins in the mutant ascertained the possible conversion of sugars into storage material instead of being used or the retention of an earlier profile associated with the mature embryo. The enhanced accumulation of organic sugars in the seedlings indicates the absence of photomorphogenesis in the mutant.

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.

Mutations in tomato 1-aminocyclopropane carboxylic acid synthase2 uncover its role in development beside fruit ripening.

The role of ethylene in plant development is mostly inferred from its exogenous application. The usage of mutants affecting ethylene biosynthesis proffers a better alternative to decipher its role. In tomato (Solanum lycopersicum), 1-aminocyclopropane carboxylic acid synthase2 (ACS2) is a key enzyme regulating ripening-specific ethylene biosynthesis. We characterised two contrasting acs2 mutants; acs2-1 overproduces ethylene, has higher ACS activity, and has increased protein levels, while acs2-2 is an ethylene underproducer, displays lower ACS activity, and has lower protein levels than wild type. Consistent with high/low ethylene emission, the mutants show opposite phenotypes, physiological responses, and metabolomic profiles compared with the wild type. The acs2-1 mutant shows early seed germination, faster leaf senescence, and accelerated fruit ripening. Conversely, acs2-2 has delayed seed germination, slower leaf senescence, and prolonged fruit ripening. The phytohormone profiles of mutants were mostly opposite in the leaves and fruits. The faster/slower senescence of acs2-1/acs2-2 leaves correlated with the endogenous ethylene/zeatin ratio. The genetic analysis showed that the metabolite profiles of respective mutants co-segregated with the homozygous mutant progeny. Our results uncover that besides ripening, ACS2 participates in the vegetative and reproductive development of tomato. The distinct influence of ethylene on phytohormone profiles indicates the intertwining of ethylene action with other phytohormones in regulating plant development.

Reanalysis of genome sequences of tomato accessions and its wild relatives: development of Tomato Genomic Variation (TGV) database integrating SNPs and INDELs polymorphisms.

MOTIVATION: Facilitated by technological advances and expeditious decrease in the sequencing costs, whole-genome sequencing is increasingly implemented to uncover variations in cultivars/accessions of many crop plants. In tomato (Solanum lycopersicum), the availability of the genome sequence, followed by the resequencing of tomato cultivars and its wild relatives, has provided a prodigious resource for the improvement of traits. A high-quality genome resequencing of 84 tomato accessions and wild relatives generated a dataset that can be used as a resource to identify agronomically important alleles across the genome. Converting this dataset into a searchable database, including information about the influence of single-nucleotide polymorphisms (SNPs) on protein function, provides valuable information about the genetic variations. The database will assist in searching for functional variants of a gene for introgression into tomato cultivars. RESULTS: A recent release of better-quality tomato genome reference assembly SL3.0, and new annotation ITAG3.2 of SL3.0, dropped 3857 genes, added 4900 novel genes and updated 20 766 genes. Using the above version, we remapped the data from the tomato lines resequenced under the '100 tomato genome resequencing project' on new tomato genome assembly SL3.0 and made an online searchable Tomato Genomic Variations (TGVs) database. The TGV contains information about SNPs and insertion/deletion events and expands it by functional annotation of variants with new ITAG3.2 using SIFT4G software. This database with search function assists in inferring the influence of SNPs on the function of a target gene. This database can be used for selecting SNPs, which can be potentially deployed for improving tomato traits. AVAILABILITY AND IMPLEMENTATION: TGV is freely available at http://psd.uohyd.ac.in/tgv.

Root-specific expression of chickpea cytokinin oxidase/dehydrogenase 6 leads to enhanced root growth, drought tolerance and yield without compromising nodulation.

Cytokinin group of phytohormones regulate root elongation and branching during post-embryonic development. Cytokinin-degrading enzymes cytokinin oxidases/dehydrogenases (CKXs) have been deployed to investigate biological activities of cytokinin and to engineer root growth. We expressed chickpea cytokinin oxidase 6 (CaCKX6) under the control of a chickpea root-specific promoter of CaWRKY31 in Arabidopsis thaliana and chickpea having determinate and indeterminate growth patterns, respectively, to study the effect of cytokinin depletion on root growth and drought tolerance. Root-specific expression of CaCKX6 led to a significant increase in lateral root number and root biomass in Arabidopsis and chickpea without any penalty to vegetative and reproductive growth of shoot. Transgenic chickpea lines showed increased CKX activity in root. Soil-grown advanced chickpea transgenic lines exhibited higher root-to-shoot biomass ratio and enhanced long-term drought tolerance. These chickpea lines were not compromised in root nodulation and nitrogen fixation. The seed yield in some lines was up to 25% higher with no penalty in protein content. Transgenic chickpea seeds possessed higher levels of zinc, iron, potassium and copper. Our results demonstrated the potential of cytokinin level manipulation in increasing lateral root number and root biomass for agronomic trait improvement in an edible legume crop with indeterminate growth habit.

Is naphthylphthalamic acid a specific phytotropin? It elevates ethylene and alters metabolic homeostasis in tomato.

In higher plants, phytohormone indole-3-acetic acid is characteristically transported from the apex towards the base of the plant, termed as polar auxin transport (PAT). Among the inhibitors blocking PAT, N-1-naphthylphthalamic acid (NPA) that targets ABCB transporters is most commonly used. NPA-treated light-grown Arabidopsis seedlings show severe inhibition of hypocotyl and root elongation. In light-grown tomato seedlings, NPA inhibited root growth, but contrary to Arabidopsis stimulated hypocotyl elongation. The NPA-stimulation of hypocotyl elongation was milder in blue, red, and far-red light-grown seedlings. The NPA-treatment stimulated emission of ethylene from the seedlings. The scrubbing of ethylene by mercuric perchlorate reduced NPA-stimulated hypocotyl elongation. NPA action on hypocotyl elongation was antagonized by 1-methylcyclopropene, an inhibitor of ethylene action. NPA-treated seedlings had reduced levels of indole-3-butyric acid and higher levels of zeatin in the shoots. NPA did not alter indole-3-acetic levels in shoots. The analysis of metabolic networks indicated that NPA-treatment induced moderate shifts in the networks compared to exogenous ethylene that induced a drastic shift in metabolic networks. Our results indicate that in addition to ethylene, NPA-stimulated hypocotyl elongation in tomato may also involve zeatin and indole-3- butyric acid. Our results indicate that NPA-mediated physiological responses may vary in a species-specific fashion.

Leaf mass area determines water use efficiency through its influence on carbon gain in rice mutants.

Saving water and enhancing rice productivity are consensually the most important research goals globally. While increasing canopy cover would enhance growth rates by higher photosynthetic carbon gain, an accompanied increase in transpiration would have a negative impact on saving water as well as for sustainability under water-limited conditions. Increased water use efficiency (WUE) by virtue of higher carbon assimilatory capacity can significantly circumvent this trade-off. Here, we report leaf mass area (LMA) has an important canopy architecture trait which when combined with superior carboxylation efficiency (CE) would achieve higher water productivity in rice. A set of 130 ethyl methanesulfonate induced mutants of an upland cultivar Nagina-22 (N22), was screened for leaf morphological traits leading to the identification of mutants differing in LMA. The wild-type, N22, along with a selected low-LMA (380-4-3) and two high-LMA mutants (392-9-1 and 457-1-3), all with comparable total leaf area, were raised under well-watered (100% Field Capacity (FC)) and water-limited (60% FC) conditions. Low Δ13 C and a higher RuBisCO content in high-LMA mutants indicated higher carboxylation efficiency, leading to increased carbon gain. Single parent backcross populations developed by crossing high and the low-LMA mutants with N22, separately, were screened for LMA, Δ13 C and growth traits. Comparison of dry matter accumulation per unit leaf area among the progenies differing in LMA and Δ13 C reiterated the association of LMA with CE. Results illustrated that high-LMA when combined with higher CE (low Δ13 C) lead to increased WUE and growth rates.

A Low-Cost High-Throughput Method for Plant Genomic DNA Isolation.

Many of the functional genomics methods require isolation of genomic DNA from large population of plants. The selection of DNA isolation protocols depends on several factors such as choice of starting material, ease of handling, time and labor required for isolation, the final quantity as well as the quality of genomic DNA. We outline here a high-throughput method of DNA extraction from different plant species including cereal crops. The protocol can be used for extraction of DNA in single tubes as well as for large formats in 96-well plates. The protocol includes steps for eliminating interfering secondary products such as phenolics. This protocol can be applied for high-throughput isolation of DNA for varied applications such as TILLING, mapping, fingerprinting, etc. as a cost-effective protocol compared to commercial kits.