Development and analysis of de novo transcriptome assemblies of multiple genotypes of Cymbopogon spp. reveal candidate genes involved in the biosynthesis of aromatic monoterpenes.

Despite the high economic value of the monoterpene-rich essential oils from different genotypes of Cymbopogon, the knowledge about the genes and metabolic route(s) involved in the biosynthesis of aromatic monoterpenes in this genus is limited. In the present study, a comprehensive transcriptome analysis of four genotypes of Cymbopogon, displaying diverse quantitative and qualitative profiles of volatile monoterpenes in their essential oils has been carried out. The comparative analysis of the deduced protein sequences corresponding to the transcriptomes of the four genotypes revealed 4609 genotype-specific orthogroups, which might contribute in defining genotype-specific phenotypes. The transcriptome data mining led to the identification of unigenes involved in the isoprenogenesis. The homology searches, combined with the phylogenetic and expression analyses provided information about candidate genes concerning the biosynthesis of monoterpene aldehyde, monoterpene alcohol, and monoterpene esters. In addition, the present study suggests a potential role of geranial reductase like enzyme in the biosynthesis of monoterpene aldehyde in Cymbopogon spp. The detailed analysis of the candidate pathway genes suggested that multiple enzymatic routes might be involved in the biosynthesis of aromatic monoterpenes in the genus Cymbopogon. The present study provides deeper insights into the biosynthesis of monoterpenes, which will be useful for the genetic improvement of these aromatic grasses.

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.

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.

CstMYB14 links ROS signaling, apocarotenoid metabolism, and stress response in Crocus sativus L.

Reactive oxygen species (ROS) behave as signaling molecules and induce biosynthesis of many secondary metabolites, including apocarotenoids, which play critical roles in stress tolerance through radical scavenging. However, the mechanism that regulates ROS responsive apocarotenoid metabolism and subsequent stress response is unknown. In this study, an R2R3-MYB transcription factor (CstMYB14) was identified from Crocus sativus L., which acts as a regulator of apocarotenoid biosynthesis. CstMYB14 expression increases in response to H2 O2 in a concentration and time-dependent manner. CstMYB14 localizes to the nucleus and acts as a transcriptional activator. Over-expression of CstMYB14 in Crocus stigmas enhanced apocarotenoid biosynthesis. Yeast-one-hybrid demonstrated binding of CstMYB14 to promoters of two apocarotenoid pathway genes (phytoene synthase and carotenoid cleavage dioxygenase 2). Nicotiana benthamiana plants overexpressing CstMYB14 showed better growth and higher stress tolerance than wild type plants. Higher antioxidant activity in CstMYB14-Ox plants indicated that stress tolerance might be due to ROS scavenging. These results establish a molecular link between ROS signaling, apocarotenoid metabolism and stress tolerance. Further, CstMYB14 is shown to act as a key regulator which modulates ROS responsive biosynthesis of apocarotenoids which in turn impart stress tolerance through ROS scavenging.

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.