A tomato ethylene-resistant mutant displays altered growth and higher β-carotene levels in fruit
Gupta S.K., Santisree P., Gupta P., Kilambi H.V., Sreelakshmi Y., Sharma R.
Article, Plant Physiology and Biochemistry, 2025, DOI Link
View abstract ⏷
The mutants resistant to ethylene are helpful in deciphering the role of ethylene in plant development. We isolated an ethylene-resistant tomato (Solanum lycopersicum) mutant by screening for acetylene-resistant (atr-1) seedlings. The atr-1 mutant displayed resistance to kinetin, suggesting attenuation of the ethylene sensing response. atr-1 also exhibited resistance to ABA- and glucose-mediated inhibition of seed germination. Unlike the Never-ripe (Nr) mutant seedlings that were hypersensitive to glucose, atr-1 seedlings were resistant to glucose, indicating ethylene sensing in atr-1 is compromised in a manner distinct from Nr. Metabolically, atr-1 seedlings had lower levels of amino acids but higher levels of several phytohormones, including ABA. atr-1 plants grew faster and produced more flowers, leading to a higher fruit set. However, the atr-1 fruits took a longer duration to reach the red-ripe (RR) stage. The ripened atr-1 fruits retained high β-carotene and lycopene levels post-RR stage and had longer on-vine longevity. The metabolome profiles of post-RR stage atr-1 fruits revealed increased levels of sugars. The atr-1 had a P279L mutation in the GAF domain of the ETR4, a key ethylene receptor regulating tomato ripening. The atr-1 exhibits phenotypic traits distinct from the Sletr4-1 (G154S) mutant, thus represents a new ETR4 allele named Sletr4-2. Our study highlights that novel alleles in ethylene receptors may aid in enhancing the nutritional quality of tomato.
Plant High-throughput Metabolomics: Recent Achievements, Applications and Future Perspectives
Darapu L.D., Tiwari S., Gupta P.
Book chapter, High-Throughput Plant Metabolomics, 2025, DOI Link
View abstract ⏷
Plants produce a vast array of metabolites, far more than those produced by most other organisms. Large-scale metabolite profiling assays now provide unmatched access to global data sets of metabolites and their correspond ing pathways, greatly enhancing our understanding of plant biology. High-throughput metabolome analysis platforms have accelerated the discovery of diverse biochemical metabolites and new pathways while enhancing our understanding of existing ones. Despite many metabolites remaining unidentified, metabolomics has signifi cantly advanced our comprehension of plant physiology and biology through the study of these small molecules. As well as the latest advancements in analytical techniques, the integration of metabolomics with other omics will greatly enhance our understanding of biological systems. In this chapter, we briefly describe the latest developments in the field of analytical techniques and their application in plant metabolomics.
Genome-Wide Identification and In Silico Analysis of Annexins in Chickpea (Cicer arietinum L.)
Swain B., Gupta P., Yadav D.
Article, Biochemical Genetics, 2025, DOI Link
View abstract ⏷
Annexins are a ubiquitous, evolutionarily conserved group of Ca2+-dependent phospholipid-binding proteins. They are a family of less numerous and more varied proteins that form a unique monophyletic group. They play an important role in various abiotic and biotic stress responses through Ca2+-mediated signaling. Chickpea (Cicer arietinum L.) is one of the most widely grown legume crops in the world. In recent years, intensive research has been carried out to identify and elucidate genes and molecular pathways that control stress responses in plants. The availability of the chickpea genome has hastened the functional genomics of chickpea. In the current study, we attempted Genome-wide identification and in silico analysis of Annexins in chickpea. Thirteen annexin sequences have been identified in the chickpea genome. Four conserved annexin domains were found in ten annexin members, while three annexins CaAnn5, CaAnn12, and CaAnn13, showed three, two, and one conserved domain, respectively. The gene structure analysis showed the presence of multiple exons in all thirteen annexins. Most Annexin genes are composed of 3–5 introns. Their chromosomal locations showed that out of thirteen genes, ten could be mapped on four chromosomes. Three genes were placed on the scaffold regions. The promoter sequence analysis of all thirteen annexins showed the presence of various elements related to growth and development and response to different phytohormones and abiotic stress. The gene expression data of different annexins in various tissues like leaf, shoot, root, flower bud, and young pod showed their differential expression. Analysis of expression data of roots in drought stress showed their differential expression with the different stages of plant growth. Overall, the current findings show the possible role of CaAnns in different stages of plant growth and development in normal and stressful conditions. Moreover, these findings will be helpful in the further characterization of CaAnn genes and their promoters.
Carotenoid Extraction from Plant Tissues
Priscilla K., Sharma V., Gautam A., Gupta P., Dagar R., Kishore V., Kumar R.
Book chapter, Methods in Molecular Biology, 2024, DOI Link
View abstract ⏷
Carotenoids are the natural pigments available in nature and exhibit different colors such as yellow, red, and orange. These are a class of phytonutrients that have anti-cancer, anti-inflammatory, anti-oxidant, immune-modulatory, and anti-aging properties. These were used in food, pharmaceutical, nutraceutical, and cosmetic industries. They are divided into two classes: carotenes and xanthophylls. The carotenes are non-oxygenated derivatives and xanthophylls are oxygenated derivatives. The major source of carotenoids are vegetables, fruits, and tissues. Carotenoids also perform the roles of photoprotection and photosynthesis. In addition to the roles mentioned above, they are also involved and act as precursor molecules for the biosynthesis of phytohormones such as strigolactone and abscisic acid. This chapter briefly introduces carotenoids and their extraction method from plant tissue. Proposed protocol describes the extraction of carotenoid using solvents chloroform and dichloromethane. Reverse-phase HPLC can be performed with C30 columns using gradient elution. The column C30 is preferred to the C18 column because the C30 column has salient features, which include selective nature in the separation of structural isomers and hydrophobic, long-chain compounds, and shows the best compatibility with highly aqueous mobile phases. A complete pipeline for the extraction of carotenoids from plant tissue is given in the present protocol.
Sample Preparation from Plant Tissue for Gas Chromatography–Mass Spectrometry (GC-MS)we
Dagar R., Gautam A., Priscilla K., Sharma V., Gupta P., Kumar R.
Book chapter, Methods in Molecular Biology, 2024, DOI Link
View abstract ⏷
Metabolites are intermediate products formed during metabolism. Metabolites play different roles, including providing energy, supporting structure, transmitting signals, catalyzing reactions, enhancing defense, and interacting with other species. Plant metabolomics research aims to detect precisely all metabolites found within tissues of plants through GC-MS. This chapter primarily focuses on extracting metabolites using chemicals such as methanol, chloroform, ribitol, MSTFA, and TMCS. The metabolic analysis method is frequently used according to the specific kind of sample or matrix being investigated and the analysis objective. Chromatography (LC, GC, and CE) with mass spectrometry and NMR spectroscopy is used in modern metabolomics to analyze metabolites from plant samples. The most frequently used method for metabolites analysis is the GC-MS. It is a powerful technique that combines gas chromatography’s separation capabilities with mass spectrometry, offering detailed information, including structural identification of each metabolite. This chapter contains an easy-to-follow guide to extract plant-based metabolites. The current protocol provides all the information needed for extracting metabolites from a plant, precautions, and troubleshooting.
Introgression of a dominant phototropin1 mutant enhances carotenoids and boosts flavour-related volatiles in genome-edited tomato RIN mutants
Nizampatnam N.R., Sharma K., Gupta P., Pamei I., Sarma S., Sreelakshmi Y., Sharma R.
Article, New Phytologist, 2024, DOI Link
View abstract ⏷
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.
Identification of Volatiles in Tomato Fruit Using Headspace Solid-Phase-Micro-Extraction (HS-SPME) Coupled with Gas Chromatography-Mass Spectrometry (GC-MS)
Gupta P., Dhanya A.J., Sharma R., Sreelakshmi Y.
Book chapter, Methods in Molecular Biology, 2024, DOI Link
View abstract ⏷
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.
Augmenting tomato functional genomics with a genome-wide induced genetic variation resource
Gupta P., Dholaniya P.S., Princy K., Madhavan A.S., Sreelakshmi Y., Sharma R.
Article, Frontiers in Plant Science, 2023, DOI Link
View abstract ⏷
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.
Reduced γ-glutamyl hydrolase activity likely contributes to high folate levels in Periyakulam-1 tomato
Tyagi K., Sunkum A., Gupta P., Kilambi H.V., Sreelakshmi Y., Sharma R.
Article, Horticulture Research, 2023, DOI Link
View abstract ⏷
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 β-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.
Two roads diverge in the yellow pigments metabolic pathway for ABA synthesis
Gupta P., Mann V., Hirschberg J.
Article, Molecular plant, 2022, DOI Link
The Genetic Components of a Natural Color Palette: A Comprehensive List of Carotenoid Pathway Mutations in Plants
Review, Frontiers in Plant Science, 2022, DOI Link
View abstract ⏷
Carotenoids comprise the most widely distributed natural pigments. In plants, they play indispensable roles in photosynthesis, furnish colors to flowers and fruit and serve as precursor molecules for the synthesis of apocarotenoids, including aroma and scent, phytohormones and other signaling molecules. Dietary carotenoids are vital to human health as a source of provitamin A and antioxidants. Hence, the enormous interest in carotenoids of crop plants. Over the past three decades, the carotenoid biosynthesis pathway has been mainly deciphered due to the characterization of natural and induced mutations that impair this process. Over the year, numerous mutations have been studied in dozens of plant species. Their phenotypes have significantly expanded our understanding of the biochemical and molecular processes underlying carotenoid accumulation in crops. Several of them were employed in the breeding of crops with higher nutritional value. This compendium of all known random and targeted mutants available in the carotenoid metabolic pathway in plants provides a valuable resource for future research on carotenoid biosynthesis in plant species.
Phytoene synthase 2 in tomato fruits remains functional and contributes to abscisic acid formation
Gupta P., Rodriguez-Franco M., Bodanapu R., Sreelakshmi Y., Sharma R.
Article, Plant Science, 2022, DOI Link
View abstract ⏷
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-β-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-β-cyclases in fruits of several carotenoids and ripening mutants by CPTA (2-(4-Chlorophenylthio)triethylamine hydrochloride), an inhibitor of lycopene-β-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.
Metabolomics intervention towards better understanding of plant traits
Sharma V., Gupta P., Priscilla K., Sharankumar, Hangargi B., Veershetty A., Ramrao D.P., Suresh S., Narasanna R., Naik G.R., Kumar A., Guo B., Zhuang W., Varshney R.K., Pandey M.K., Kumar R.
Review, Cells, 2021, DOI Link
View abstract ⏷
The majority of the most economically important plant and crop species are enriched with the availability of high-quality reference genome sequences forming the basis of gene discovery which control the important biochemical pathways. The transcriptomics and proteomics resources have also been made available for many of these plant species that intensify the understanding at expression levels. However, still we lack integrated studies spanning genomics–transcriptomics– proteomics, connected to metabolomics, the most complicated phase in phenotype expression. Nevertheless, for the past few decades, emphasis has been more on metabolome which plays a crucial role in defining the phenotype (trait) during crop improvement. The emergence of modern high throughput metabolome analyzing platforms have accelerated the discovery of a wide variety of biochemical types of metabolites and new pathways, also helped in improving the understanding of known existing pathways. Pinpointing the causal gene(s) and elucidation of metabolic pathways are very important for development of improved lines with high precision in crop breeding. Along with other-omics sciences, metabolomics studies have helped in characterization and annotation of a new gene(s) function. Hereby, we summarize several areas in the field of crop development where metabolomics studies have made its remarkable impact. We also assess the recent research on metabolomics, together with other omics, contributing toward genetic engineering to target traits and key pathway(s).
Reanalysis of genome sequences of tomato accessions and its wild relatives: Development of Tomato Genomic Variation (TGV) database integrating SNPs and INDELs polymorphisms
Gupta P., Dholaniya P.S., Devulapalli S., Tawari N.R., Sreelakshmi Y., Sharma R.
Article, Bioinformatics, 2020, DOI Link
View abstract ⏷
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.
A low-cost high-throughput method for plant genomic DNA isolation
Gupta P., Salava H., Sreelakshmi Y., Sharma R.
Book chapter, Methods in Molecular Biology, 2020, DOI Link
View abstract ⏷
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.
Next-generation sequencing (NGS)-based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population
Gupta P., Reddaiah B., Salava H., Upadhyaya P., Tyagi K., Sarma S., Datta S., Malhotra B., Thomas S., Sunkum A., Devulapalli S., Till B.J., Sreelakshmi Y., Sharma R.
Article, Plant Journal, 2017, DOI Link
View abstract ⏷
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.
Tomato fruits show wide phenomic diversity but fruit developmental genes show low genomic diversity
Mohan V., Gupta S., Thomas S., Mickey H., Charakana C., Chauhan V.S., Sharma K., Kumar R., Tyagi K., Sarma S., Gupta S.K., Kilambi H.V., Nongmaithem S., Kumari A., Gupta P., Sreelakshmi Y., Sharma R.
Article, PLoS ONE, 2016, DOI Link
View abstract ⏷
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 nonsynonymous 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.
A rapid and sensitive method for determination of carotenoids in plant tissues by high performance liquid chromatography
Gupta P., Sreelakshmi Y., Sharma R.
Article, Plant Methods, 2015, DOI Link
View abstract ⏷
Background: The dietary carotenoids serve as precursor for vitamin A and prevent several chronic-degenerative diseases. The carotenoid profiling is necessary to understand their importance on human health. However, the available high-performance liquid chromatography (HPLC) methods to resolve the major carotenoids require longer analysis times and do not adequately resolve the violaxanthin and neoxanthin. Results: A fast and sensitive HPLC method was developed using a C30 column at 20°C with a gradient consisting of methanol, methyl-tert-butyl ether and water. A total of 15 major carotenoids, including 14 all-trans forms and one cis form were resolved within 20 min. The method also distinctly resolved violaxanthin and neoxanthin present in green tissues. Additionally this method also resolved geometrical isomers of the carotenoids. Conclusion: The HPLC coupled with C30 column efficiently resolved fifteen carotenoids and their isomers in shorter runtime of 20 min. Application of this method to diverse matrices such as tomato fruits and leaves, Arabidopsis leaves and green pepper fruits showed the versatility and robustness of the method. The method would be useful for high throughput analysis of large number of samples.
