A non-canonical Aux/IAA gene MsIAA32 regulates peltate glandular trichome development in spearmint.

Phytohormone auxin controls various aspects of plant growth and development. The typical auxin signalling involves the degradation of canonical Aux/IAA proteins upon auxin perception releasing the auxin response factors (ARF) to activate auxin-regulated gene expression. Extensive research has been pursued in deciphering the role of canonical Aux/IAAs, however, the function of non-canonical Aux/IAA genes remains elusive. Here we identified a non-canonical Aux/IAA gene, MsIAA32 from spearmint (Mentha spicata), which lacks the TIR1-binding domain and shows its involvement in the development of peltate glandular trichomes (PGT), which are the sites for production and storage of commercially important essential oils. Using yeast two-hybrid studies, two canonical Aux/IAAs, MsIAA3, MsIAA4 and an ARF, MsARF3 were identified as the preferred binding partners of MsIAA32. Expression of a R2R3-MYB gene MsMYB36 and a cyclin gene MsCycB2-4 was altered in MsIAA32 suppressed plants indicating that these genes are possible downstream targets of MsIAA32 mediated signalling. Ectopic expression of MsIAA32 in Arabidopsis affected non-glandular trichome formation along with other auxin related developmental traits. Our findings establish the role of non-canonical Aux/IAA mediated auxin signalling in PGT development and reveal species-specific functionalization of Aux/IAAs.

Single-Crystal 2D Covalent Organic Frameworks for Plant Biotechnology.

Molecules chemically synthesized as periodic two-dimensional (2D) frameworks via covalent bonds can form some of the highest-surface area and -charge density particles possible. There is significant potential for applications such as nanocarriers in life sciences if biocompatibility can be achieved; however, significant synthetic challenges remain in avoiding kinetic traps from disordered linking during 2D polymerization of compatible monomers, resulting in isotropic polycrystals without a long-range order. Here, we establish thermodynamic control over dynamic control on the 2D polymerization process of biocompatible imine monomers by minimizing the surface energy of nuclei. As a result, polycrystal, mesocrystal, and single-crystal 2D covalent organic frameworks (COFs) are obtained. We achieve COF single crystals by exfoliation and minification methods, forming high-surface area nanoflakes that can be dispersed in aqueous medium with biocompatible cationic polymers. We find that these 2D COF nanoflakes with high surface area are excellent plant cell nanocarriers that can load bioactive cargos, such as the plant hormone abscisic acid (ABA) via electrostatic attraction, and deliver them into the cytoplasm of intact living plants, traversing through the cell wall and cell membrane due to their 2D geometry. This synthetic route to high-surface area COF nanoflakes has promise for life science applications including plant biotechnology.

Metabolic Engineering of Nicotiana benthamiana to Produce Cannabinoid Precursors and Their Analogues.

In recent years, the perspective towards the use of cannabis has slowly shifted from being an illicit drug to a medicinal plant. The pathway and enzymes involved in the production of cannabinoids are known; however, studies evaluating the production of cannabinoids in heterologous plants and cell cultures are still limited. In this study, we assessed the potential use of N. benthamiana (Nicotiana benthamiana) plants as a heterologous host for producing natural and novel cannabinoids. Transgenic N. benthamiana plants expressing genes encoding cannabis acyl-activating enzyme and olivetol synthase were generated, which were then used for transiently expressing other downstream pathway genes. Production of olivetolic acid and divarinic acid, the universal precursors for major and minor cannabinoids, respectively, was observed in transgenic N. benthamiana plants. To produce novel cannabinoid precursors with different side chains, various fatty acids were infiltrated into the transgenic N. benthamiana plants and the production of novel derivatives was observed. Although we were not able to derive the core intermediate, cannabigerolic acid, from our transgenic plants, possibly due to the low production levels of the precursors, our transgenics plants still serve as a high-potential platform for further development and exploring the N. benthamiana chemical space for generating novel cannabinoids.

Comparative Transcriptome Analysis Reveals Coordinated Transcriptional Regulation of Central and Secondary Metabolism in the Trichomes of Cannabis Cultivars.

Cannabis is one of the few plant genera capable of producing cannabinoids, the effects of which are synergized by terpene interactions. As the biosynthesis of both metabolite classes requires the same intracellular feedstocks, this work describes the coordinated regulation of global metabolic pathways that allows for their joint copious production in vivo. To this end, a transcriptomics-based approach to characterize the glandular trichomes of five Cannabis cultivars was pursued. Besides revealing metabolic traits that enhanced and proportionated the supply of critical carbon precursors, in-depth analysis showed significantly increased gene expression of two particular enzymes to meet the huge nicotinamide adenine dinucleotide phosphate (NADPH) demand of secondary metabolite production. Furthermore, it led to a hypothesis that the methyl-d-erythritol 4-phosphate pathway might be utilized more than the mevalonic acid pathway in Cannabis trichomes. While both pathways were found to be activated in a modular and calibrated way that reflected their broad participation in physiological processes, the genes for hexanoate, cannabinoid, and terpene biosynthesis were, in contrast, up-regulated in an en bloc and multi-loci manner due to their specific roles in secondary metabolite production. In addition, three new terpene synthases were characterized based on both in silico and experimental assays. Altogether, the study enhances the current understanding of secondary metabolite production in Cannabis cultivars, which may assist in their characterization and development.

Sweet Basil Has Distinct Synthases for Eugenol Biosynthesis in Glandular Trichomes and Roots with Different Regulatory Mechanisms.

Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.

Characterization of a sweet basil acyltransferase involved in eugenol biosynthesis.

Sweet basil (Ocimum basilicum) plants produce its characteristic phenylpropene-rich essential oil in specialized structures known as peltate glandular trichomes (PGTs). Eugenol and chavicol are the major phenylpropenes produced by sweet basil varieties whose synthetic pathways are not fully elucidated. Eugenol is derived from coniferyl acetate by a reaction catalysed by eugenol synthase. An acyltransferase is proposed to convert coniferyl alcohol to coniferyl acetate which is the first committed step towards eugenol synthesis. Here, we perform a comparative next-generation transcriptome sequencing of different tissues of sweet basil, namely PGT, leaf, leaf stripped of PGTs (leaf-PGT), and roots, to identify differentially expressed transcripts specific to PGT. From these data, we identified a PGT-enriched BAHD acyltransferase gene ObCAAT1 and functionally characterized it. In vitro coupled reaction of ObCAAT1 with eugenol synthase in the presence of coniferyl alcohol resulted in eugenol production. Analysis of ObCAAT1-RNAi transgenic lines showed decreased levels of eugenol and accumulation of coniferyl alcohol and its derivatives. Coniferyl alcohol acts as a common substrate for phenylpropene and lignin biosynthesis. No differences were found in total lignin content of PGTs and leaves of transgenic lines, indicating that phenylpropene biosynthesis is not coupled to lignification in sweet basil.

Comparative transcriptome analysis of oil palm flowers reveals an EAR-motif-containing R2R3-MYB that modulates phenylpropene biosynthesis.

BACKGROUND: Oil palm is the most productive oil crop and the efficiency of pollination has a direct impact on the yield of oil. Pollination by wind can occur but maximal pollination is mediated by the weevil E. kamerunicus. These weevils complete their life cycle by feeding on male flowers. Attraction of weevils to oil palm flowers is due to the emission of methylchavicol by both male and female flowers. In search for male flowers, the weevils visit female flowers by accident due to methylchavicol fragrance and deposit pollen. Given the importance of methylchavicol emission on pollination, we performed comparative transcriptome analysis of oil palm flowers and leaves to identify candidate genes involved in methylchavicol production in flowers. RESULTS: RNA sequencing (RNA-Seq) of male open flowers, female open flowers and leaves was performed using Illumina HiSeq 2000 platform. Analysis of the transcriptome data revealed that the transcripts of methylchavicol biosynthesis genes were strongly up-regulated whereas transcripts encoding genes involved in lignin production such as, caffeic acid O-methyltransferase (COMT) and Ferulate-5-hydroxylase (F5H) were found to be suppressed in oil palm flowers. Among the transcripts encoding transcription factors, an EAR-motif-containing R2R3-MYB transcription factor (EgMYB4) was found to be enriched in oil palm flowers. We determined that EgMYB4 can suppress the expression of a monolignol pathway gene, EgCOMT, in vivo by binding to the AC elements present in the promoter region. EgMYB4 was further functionally characterized in sweet basil which also produces phenylpropenes like oil palm. Transgenic sweet basil plants showed significant reduction in lignin content but produced more phenylpropenes. CONCLUSIONS: Our results suggest that EgMYB4 possibly restrains lignin biosynthesis in oil palm flowers thus allowing enhanced carbon flux into the phenylpropene pathway. This study augments our understanding of the diverse roles that EAR-motif-containing MYBs play to fine tune the metabolic flux along the various branches of core phenylpropanoid pathway. This will aid in metabolic engineering of plant aromatic compounds.

Spearmint R2R3-MYB transcription factor MsMYB negatively regulates monoterpene production and suppresses the expression of geranyl diphosphate synthase large subunit (MsGPPS.LSU).

Many aromatic plants, such as spearmint, produce valuable essential oils in specialized structures called peltate glandular trichomes (PGTs). Understanding the regulatory mechanisms behind the production of these important secondary metabolites will help design new approaches to engineer them. Here, we identified a PGT-specific R2R3-MYB gene, MsMYB, from comparative RNA-Seq data of spearmint and functionally characterized it. Analysis of MsMYB-RNAi transgenic lines showed increased levels of monoterpenes, and MsMYB-overexpressing lines exhibited decreased levels of monoterpenes. These results suggest that MsMYB is a novel negative regulator of monoterpene biosynthesis. Ectopic expression of MsMYB, in sweet basil and tobacco, perturbed sesquiterpene- and diterpene-derived metabolite production. In addition, we found that MsMYB binds to cis-elements of MsGPPS.LSU and suppresses its expression. Phylogenetic analysis placed MsMYB in subgroup 7 of R2R3-MYBs whose members govern phenylpropanoid pathway and are regulated by miR858. Analysis of transgenic lines showed that MsMYB is more specific to terpene biosynthesis as it did not affect metabolites derived from phenylpropanoid pathway. Further, our results indicate that MsMYB is probably not regulated by miR858, like other members of subgroup 7.

Metabolic engineering of terpene biosynthesis in plants using a trichome-specific transcription factor MsYABBY5 from spearmint (Mentha spicata).

In many aromatic plants including spearmint (Mentha spicata), the sites of secondary metabolite production are tiny specialized structures called peltate glandular trichomes (PGT). Having high commercial values, these secondary metabolites are exploited largely as flavours, fragrances and pharmaceuticals. But, knowledge about transcription factors (TFs) that regulate secondary metabolism in PGT remains elusive. Understanding the role of TFs in secondary metabolism pathway will aid in metabolic engineering for increased yield of secondary metabolites and also the development of new production techniques for valuable metabolites. Here, we isolated and functionally characterized a novel MsYABBY5 gene that is preferentially expressed in PGT of spearmint. We generated transgenic plants in which MsYABBY5 was either overexpressed or silenced using RNA interference (RNAi). Analysis of the transgenic lines showed that the reduced expression of MsYABBY5 led to increased levels of terpenes and that overexpression decreased terpene levels. Additionally, ectopic expression of MsYABBY5 in Ocimum basilicum and Nicotiana sylvestris decreased secondary metabolite production in them, suggesting that the encoded transcription factor is probably a repressor of secondary metabolism.