Buckwheat OMICS: present status and future prospects.

Buckwheat (Fagopyrum spp.) is an underutilized resilient crop of North Western Himalayas belonging to the family Polygonaceae and is a source of essential nutrients and therapeutics. Common Buckwheat and Tatary Buckwheat are the two main cultivated species used as food. It is the only grain crop possessing rutin, an important metabolite with high nutraceutical potential. Due to its inherent tolerance to various biotic and abiotic stresses and a short life cycle, Buckwheat has been proposed as a model crop plant. Nutritional security is one of the major concerns, breeding for a nutrient-dense crop such as Buckwheat will provide a sustainable solution. Efforts toward improving Buckwheat for nutrition and yield are limited due to the lack of available: genetic resources, genomics, transcriptomics and metabolomics. In order to harness the agricultural importance of Buckwheat, an integrated breeding and OMICS platforms needs to be established that can pave the way for a better understanding of crop biology and developing commercial varieties. This, coupled with the availability of the genome sequences of both Buckwheat species in the public domain, should facilitate the identification of alleles/QTLs and candidate genes. There is a need to further our understanding of the molecular basis of the genetic regulation that controls various economically important traits. The present review focuses on: the food and nutritional importance of Buckwheat, its various omics resources, utilization of omics approaches in understanding Buckwheat biology and, finally, how an integrated platform of breeding and omics will help in developing commercially high yielding nutrient rich cultivars in Buckwheat.

Identification of novel SNPs in Pun1 locus for pungency in Capsicum species.

BACKGROUND: Capsaicin and its analogues known as capsaicinoids are the principal sources of pungency in Capsicum spp. In this study, characterization of North-West Himalayan chilli germplasm and commercial landraces of different Indian states known for different pungency-color combinations was done based on capsaicin concentration. Moreover, molecular variation in pungency among high, medium and mild/not pungent Capsicum spp., especially those adapted to North-West Himalayas were elucidated. METHODS AND RESULTS: Forty-nine genotypes of chilli comprising breeding lines of Kashmiri origin, commercial landraces of Southern Indian origin and one of the world's hottest chilli Bhut Jolokia from Nagaland state of India were used as an experimental material. Wide variation in capsaicin content was observed among the genotypes, wherein, Bhut Jolokia (Capsicum chinense) expressed the highest capsaicin content (10,500.75 µg/g). Further, molecular analysis of PunI gene was done for discovering SNPs responsible for variations in pungency. In the non-pungent Nishat-1 (Capsicum annuum var. grossum), the 650 bp DNA fragment was not amplified due to 2.5 kb deletion spanning the putative promoter and first exon of AT3. The amplified DNA product for high and medium pungent was sequencing. Sequence alignment among revealed SNPs which were further observed responsible for variations in amino acid sequence and protein structure. CONCLUSION: The observed variation in protein structure might be responsible for high capsaicin production in one genotype as compared to the other and hence the protein conformation determines its interaction with the substrate.

Identification of Lipoxygenase gene repertoire of Cannabis sativa and functional characterization of CsLOX13 gene.

Lipoxygenase (LOX) enzymes play a pivotal role in the biosynthesis of oxylipins. The phyto-oxilipins have been implicated in diverse aspects of plant biology, from regulating plant growth and development to providing tolerance against biotic and abiotic stresses. C. sativa is renowned for its bioactive secondary metabolites, namely cannabinoids. LOX route is assumed to be involved in the biosynthesis of hexanoic acid, which is one of the precursors of cannabinoids of C. sativa. For obvious reasons, the LOX gene family deserves thorough investigation in the C. sativa. Genome-wide analysis revealed the presence of 21 LOX genes in C. sativa, which can be further grouped into 13-LOX and 9-LOX depending upon their phylogeny as well as the enzyme activity. The promoter regions of the CsLOX genes were predicted to contain cis-acting elements involved in phytohormones responsiveness and stress response. The qRT-PCR-based expression analysis of 21 LOX genes revealed their differential expression in different plant parts (root, stem, young leaf, mature leaf, sugar leaf, and female flower). The majority of CsLOX genes displayed preferential expression in the female flower, which is the primary site for the biosynthesis of cannabinoids. The highest LOX activity and expression level of a jasmonate marker gene were reported in the female flowers among all the plant parts. Several CsLOX genes were found to be upregulated by MeJA treatment. Based on the transient expression in Nicotiana benthamiana and the development of stable Nicotiana tabacum transgenic lines, we demonstrate that CsLOX13 encodes functional lipoxygenase and play an important role in the biosynthesis of oxylipins.

Two homeologous MATE transporter genes, NtMATE21 and NtMATE22, are involved in the modulation of plant growth and flavonol transport in Nicotiana tabacum.

The multidrug and toxic compound extrusion (MATE) protein family has been implicated in the transport of a diverse range of molecules, including specialized metabolites. In tobacco (Nicotiana tabacum), only a limited number of MATE transporters have been functionally characterized, and no MATE transporter has been studied in the context of flavonoid transport in this plant species so far. In the present study, we characterize two homeologous tobacco MATE genes, NtMATE21 and NtMATE22, and demonstrate their role in flavonol transport and in plant growth and development. The expression of these two genes was reported to be up-regulated in trichomes as compared with the trichome-free leaf. The transcript levels of NtMATE21 and NtMATE22 were found to be higher in flavonol overproducing tobacco transgenic lines as compared with wild type tobacco. The two transporters were demonstrated to be localized to the plasma membrane. Genetic manipulation of NtMATE21 and NtMATE22 led to altered growth phenotypes and modulated flavonol contents in N. tabacum. The ß-glucuronidase and green fluorescent protein fusion transgenic lines of promoter regions suggested that NtMATE21 and NtMATE22 are exclusively expressed in the trichome heads in the leaf tissue and petals. Moreover, in a transient transactivation assay, NtMYB12, a flavonol-specific MYB transcription factor, was found to transactivate the expression of NtMATE21 and NtMATE22 genes. Together, our results strongly suggest the involvement of NtMATE21 and NtMATE22 in flavonol transport as well as in the regulation of plant growth and development.

Comprehensive genome-wide identification, characterization, and expression profiling of MATE gene family in Nicotiana tabacum.

The transporters belonging to the MATE family are involved in the transportation of diverse ligands, including metal ions and small organic molecules, and, therefore, play an important role in plant biology. Our genome-wide analysis led to the identification of 138 MATE genes in N. tabacum, which were grouped into four major phylogenetic clades. The expression of several NtMATE genes was reported to be differential in different tissues, namely young leaf, mature leaf, stem, root, and mature flower. The upstream regions of the NtMATE genes were predicted to contain several cis-acting elements associated with hormonal, developmental, and stress responses. Some of the genes were found to display induced expression following methyl jasmonate treatment. The co-expression analysis revealed 126 candidate transcription factor genes that might be involved in the transcriptional regulation of 21 NtMATE genes. Certain MATE genes (NtMATE81, NtMATE82, NtMATE88, and NtMATE89) were predicted to be targeted by micro RNAs (nta-miR167a, nta-miR167b, nta-miR167c, nta-miR167d and nta-miR167e). The computational analysis of MATE transporters provided insights into the key amino acid residues involved in the binding of the alkaloids. Further, the putative function of some of the NtMATE transporters was also revealed. The present study develops a solid foundation for the functional characterization of MATE transporter genes in N. tabacum.

Membrane transporters: the key drivers of transport of secondary metabolites in plants.

KEY MESSAGE: This review summarizes the recent updates in the area of transporters of plant secondary metabolites, including their applied aspects in metabolic engineering of economically important secondary metabolites. Plants have evolved biosynthetic pathways to produce structurally diverse secondary metabolites, which serve distinct functions, including defense against pathogens and herbivory, thereby playing a pivotal role in plant ecological interactions. These compounds often display interesting bioactivities and, therefore, have been used as repositories of natural drugs and phytoceuticals for humans. At an elevated level, plant secondary metabolites could be cytotoxic to the plant cell itself; therefore, plants have developed sophisticated mechanisms to sequester these compounds to prevent cytotoxicity. Many of these valuable natural compounds and their precursors are biosynthesized and accumulated at diverse subcellular locations, and few are even transported to sink organs via long-distance transport, implying the involvement of compartmentalization via intra- and intercellular transport mechanisms. The transporter proteins belonging to different families of transporters, especially ATP binding cassette (ABC) and multidrug and toxic compound extrusion (MATE) have been implicated in membrane-mediated transport of certain plant secondary metabolites. Despite increasing reports on the characterization of transporter proteins and their genes, our knowledge about the transporters of several medicinally and economically important plant secondary metabolites is still enigmatic. A comprehensive understanding of the molecular mechanisms underlying the whole route of secondary metabolite transportome, in addition to the biosynthetic pathways, will aid in systematic and targeted metabolic engineering of high-value secondary metabolites. The present review embodies a comprehensive update on the progress made in the elucidation of transporters of secondary metabolites in view of basic and applied aspects of their transport mechanism.

Comprehensive transcriptome analysis provides insights into metabolic and gene regulatory networks in trichomes of Nicotiana tabacum.

KEY MESSAGE: Comprehensive transcriptome analysis suggested that the primary metabolism is modulated to augment the supply of substrates towards secondary metabolism operating in the glandular trichomes of Nicotiana tabacum. The comparative gene expression and co-expression network analysis revealed that certain members of transcription factor genes belonging to the MYB, HD-ZIP, ERF, TCP, SRS, WRKY and DOF families may be involved in the regulation of metabolism and/other aspects in the glandular trichomes of N. tabacum The glandular trichomes of Nicotiana tabacum are highly productive in terms of secondary metabolites and therefore have been projected to be used as a prognostic platform for metabolic engineering of valuable natural products. For obvious reasons, detailed studies pertaining to the metabolic and gene regulatory networks operating in the glandular trichomes of N. tabacum are of pivotal significance to be undertaken. We have carried out next-generation sequencing of glandular trichomes of N. tabcaum and investigated differential gene expression among different tissues, including trichome-free leaves. We identified a total of 37,269 and 37,371 genes, expressing in trichome free leaf and glandular trichomes, respectively, at a cutoff of FPKM ≥ 1. The analysis revealed that different pathways involved with the primary metabolism are modulated in glandular trichomes of N. tabacum, providing a plausible explanation for the enhanced biosynthesis of secondary metabolism in the glandular trichomes. Further, comparative gene expression analysis revealed several genes, which display preferential expression in the glandular trichomes and thereby seem to be potential candidate genes for future studies in connection to the discovery of novel trichome specific promoters. The present study also led to the comprehensive identification of 1750 transcription factor genes expressing at a cutoff of FPKM ≥ 1 in the glandular trichomes of N. tabacum. The clustering and co-expression analysis suggested that transcription factor genes belonging to HD-ZIP, ERF, WRKY, MYB, TCP, SRS and DOF families may be the major players in the regulation of gene expression in the glandular trichomes of N. tabacum. To the best of our knowledge, the present work is the first effort towards detailed identification of genes, especially regulatory genes expressing in the glandular trichomes of N. tabacum. The data resource and the empirical findings from present work in all probability must, therefore, provide a reference and background context for future work aiming at deciphering molecular mechanism of regulation of secondary metabolism and gene expression in the glandular trichomes of N. tabacum.