Morphological, phytochemical, and transcriptome analyses provide insights into the biosynthesis of monoterpenes in Monarda citriodora.

MAIN CONCLUSION: Morphological, phytochemical, and transcriptome analyses revealed candidate genes involved in the biosynthesis of volatile monoterpenes and development of glandular trichomes in Monarda citriodora. Monarda citriodora Cerv. ex Lag. is a valuable aromatic plant due to the presence of monoterpenes as major constituents in its essential oil (EO). Thus, it is of sheer importance to gain knowledge about the site of the biosynthesis of these terpenoid compounds in M. citriodora, as well as the genes involved in their biosynthesis. In this study, we studied different types of trichomes and their relative densities in three different developmental stages of leaves, early stage of leaf development (L1), mid-stage of leaf development (L2), and later stage of leaf development (L3) and the histochemistry of trichomes for the presence of lipid and terpenoid compounds. Further, the phytochemical analysis of this plant through GC-MS indicated a higher content of monoterpenes (thymol, thymoquinone, γ-terpinene, p-cymene, and carvacrol) in the L1 stage with a substantial decrease in the L3 stage of leaf development. This considerable decrease in the content of monoterpenes was attributed to the decrease in the trichome density from L1 to L3. Further, we developed a de novo transcriptome assembly by carrying out RNA sequencing of different plant parts of M. citriodora. The transcriptome data revealed several putative unigenes involved in the biosynthesis of specialized terpenoid compounds, as well as regulatory genes involved in glandular trichome development. The data generated in the present study build a strong foundation for further improvement of M. citriodora, in terms of quantity and quality of its essential oil, through genetic engineering.

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.

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.