Emerging mechanistic insights into the regulation of specialized metabolism in plants.

Plants biosynthesize a broad range of natural products through specialized and species-specific metabolic pathways that are fuelled by core metabolism, together forming a metabolic network. Specialized metabolites have important roles in development and adaptation to external cues, and they also have invaluable pharmacological properties. A growing body of evidence has highlighted the impact of translational, transcriptional, epigenetic and chromatin-based regulation and evolution of specialized metabolism genes and metabolic networks. Here we review the forefront of this research field and extrapolate to medicinal plants that synthetize rare molecules. We also discuss how this new knowledge could help in improving strategies to produce useful plant-derived pharmaceuticals.

The Vinca minor genome highlights conserved evolutionary traits in monoterpene indole alkaloid synthesis.

Vinca minor, also known as the lesser periwinkle, is a well-known species from the Apocynaceae, native to central and southern Europe. This plant synthesizes monoterpene indole alkaloids, which are a class of specialized metabolites displaying a wide range of bioactive- and pharmacologically important properties. Within the almost 50 monoterpene indole alkaloids it produces, V. minor mainly accumulates vincamine, which is commercially used as a nootropic. Using a combination of Oxford Nanopore Technologies long read- and Illumina short-read sequencing, a 679,098 Mb V. minor genome was assembled into 296 scaffolds with an N50 scaffold length of 6 Mb, and encoding 29,624 genes. These genes were functionally annotated and used in a comparative genomic analysis to establish gene families and to investigate gene family expansion and contraction across the phylogenetic tree. Furthermore, homology-based monoterpene indole alkaloid gene predictions together with a metabolic analysis across 4 different V. minor tissue types guided the identification of candidate monoterpene indole alkaloid genes. These candidates were finally used to identify monoterpene indole alkaloid gene clusters, which combined with synteny analysis allowed for the discovery of a functionally validated vincadifformine-16-hydroxylase, reinforcing the potential of this dataset for monoterpene indole alkaloids gene discovery. It is expected that access to these resources will facilitate the elucidation of unknown monoterpene indole alkaloid biosynthetic routes with the potential of transferring these pathways to heterologous expression systems for large-scale monoterpene indole alkaloid production.

You shall not pass! A Chromatin barrier story in plants.

As in other eukaryotes, the plant genome is functionally organized in two mutually exclusive chromatin fractions, a gene-rich and transcriptionally active euchromatin, and a gene-poor, repeat-rich, and transcriptionally silent heterochromatin. In Drosophila and humans, the molecular mechanisms by which euchromatin is preserved from heterochromatin spreading have been extensively studied, leading to the identification of insulator DNA elements and associated chromatin factors (insulator proteins), which form boundaries between chromatin domains with antagonistic features. In contrast, the identity of factors assuring such a barrier function remains largely elusive in plants. Nevertheless, several genomic elements and associated protein factors have recently been shown to regulate the spreading of chromatin marks across their natural boundaries in plants. In this minireview, we focus on recent findings that describe the spreading of chromatin and propose avenues to improve the understanding of how plant chromatin architecture and transitions between different chromatin domains are defined.

Topoisomerase VI participates in an insulator-like function that prevents H3K9me2 spreading.

The organization of the genome into transcriptionally active and inactive chromatin domains requires well-delineated chromatin boundaries and insulator functions in order to maintain the identity of adjacent genomic loci with antagonistic chromatin marks and functionality. In plants that lack known chromatin insulators, the mechanisms that prevent heterochromatin spreading into euchromatin remain to be identified. Here, we show that DNA Topoisomerase VI participates in a chromatin boundary function that safeguards the expression of genes in euchromatin islands within silenced heterochromatin regions. While some transposable elements are reactivated in mutants of the Topoisomerase VI complex, genes insulated in euchromatin islands within heterochromatic regions of the Arabidopsis thaliana genome are specifically down-regulated. H3K9me2 levels consistently increase at euchromatin island loci and decrease at some transposable element loci. We further show that Topoisomerase VI physically interacts with S-adenosylmethionine synthase methionine adenosyl transferase 3 (MAT3), which is required for H3K9me2. A Topoisomerase VI defect affects MAT3 occupancy on heterochromatic elements and its exclusion from euchromatic islands, thereby providing a possible mechanistic explanation to the essential role of Topoisomerase VI in the delimitation of chromatin domains.

More than a Catharanthus plant: A multicellular and pluri-organelle alkaloid-producing factory.

Plants represent a huge reservoir of natural products. A broad series of these compounds now find application for human health. In this respect, the monoterpene indole alkaloids (MIAs), particularly from Madagascar periwinkle, are a prominent example of plant specialized metabolites with an important therapeutic potential. However, the supply of MIA drugs has always been a challenge since the low-yield accumulation in planta. This mainly results from the complex architecture of the MIA biosynthetic pathway that involves several organs, tissue types and subcellular organelles. Here, we describe the most recent advances towards the elucidation of this pathway route as well as its spatial organization in planta. Besides allowing a better understanding of the MIA biosynthetic flux in the whole plant, such knowledge will also probably pave the way for the development of metabolic engineering strategies to sustain the MIA supply.

In vivo stabilization of endogenous chloroplast RNAs by customized artificial pentatricopeptide repeat proteins.

Pentatricopeptide repeat (PPR) proteins are helical repeat-proteins that bind RNA in a modular fashion with a sequence-specificity that can be manipulated by the use of an amino acid code. As such, PPR repeats are promising scaffolds for the design of RNA binding proteins for synthetic biology applications. However, the in vivo functional capabilities of artificial PPR proteins built from consensus PPR motifs are just starting to be explored. Here, we report in vivo functions of an artificial PPR protein, dPPRrbcL, made of consensus PPR motifs that were designed to bind a sequence near the 5' end of rbcL transcripts in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing processed rbcL mRNA. We targeted a second protein of analogous design to the petL 5' UTR, where it substituted for the native stabilizing PPR protein PGR3, albeit inefficiently. These results showed that artificial PPR proteins can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases.

Plant to Insect Horizontal Gene Transfer: Empowering Whiteflies.

Horizontal gene transfer (HGT) is a well-documented evolutionary driving phenomenon in prokaryotes and eukaryotes, but its impact on the plant kingdom has remained elusive. A recent study provides compelling evidences, which support the idea that a plant-derived gene allows for the detoxification of plant defense metabolites in a polyphagous arthropod herbivore.

Arabidopsis mTERF9 protein promotes chloroplast ribosomal assembly and translation by establishing ribonucleoprotein interactions in vivo.

The mitochondrial transcription termination factor proteins are nuclear-encoded nucleic acid binders defined by degenerate tandem helical-repeats of ∼30 amino acids. They are found in metazoans and plants where they localize in organelles. In higher plants, the mTERF family comprises ∼30 members and several of these have been linked to plant development and response to abiotic stress. However, knowledge of the molecular basis underlying these physiological effects is scarce. We show that the Arabidopsis mTERF9 protein promotes the accumulation of the 16S and 23S rRNAs in chloroplasts, and interacts predominantly with the 16S rRNA in vivo and in vitro. Furthermore, mTERF9 is found in large complexes containing ribosomes and polysomes in chloroplasts. The comprehensive analysis of mTERF9 in vivo protein interactome identified many subunits of the 70S ribosome whose assembly is compromised in the null mterf9 mutant, putative ribosome biogenesis factors and CPN60 chaperonins. Protein interaction assays in yeast revealed that mTERF9 directly interact with these proteins. Our data demonstrate that mTERF9 integrates protein-protein and protein-RNA interactions to promote chloroplast ribosomal assembly and translation. Besides extending our knowledge of mTERF functional repertoire in plants, these findings provide an important insight into the chloroplast ribosome biogenesis.

The Arabidopsis mTERF-repeat MDA1 protein plays a dual function in transcription and stabilization of specific chloroplast transcripts within the psbE and ndhH operons.

The mTERF gene family encodes for nucleic acid binding proteins that are predicted to regulate organellar gene expression in eukaryotes. Despite the implication of this gene family in plant development and response to abiotic stresses, a precise molecular function was assigned to only a handful number of its c. 30 members in plants. Using a reverse genetics approach in Arabidopsis thaliana and combining molecular and biochemical techniques, we revealed new functions for the chloroplast mTERF protein, MDA1. We demonstrated that MDA1 associates in vivo with components of the plastid-encoded RNA polymerase and transcriptional active chromosome complexes. MDA1 protein binds in vivo and in vitro with specificity to 27-bp DNA sequences near the 5'-end of psbE and ndhA chloroplast genes to stimulate their transcription, and additionally promotes the stabilization of the 5'-ends of processed psbE and ndhA messenger (m)RNAs. Finally, we provided evidence that MDA1 function in gene transcription likely coordinates RNA folding and the action of chloroplast RNA-binding proteins on mRNA stabilization. Our results provide examples for the unexpected implication of DNA binding proteins and gene transcription in the regulation of mRNA stability in chloroplasts, blurring the boundaries between DNA and RNA metabolism in this organelle.

Translatome analysis of an NB-LRR immune response identifies important contributors to plant immunity in Arabidopsis.

An important branch of plant immunity involves the recognition of pathogens by nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. However, signaling events downstream of NB-LRR activation are poorly understood. We have analysed the Arabidopsis translatome using ribosome affinity purification and RNA sequencing. Our results show that the translational status of hundreds of transcripts is differentially affected upon activation of the NB-LRR protein RPM1, showing an overall pattern of a switch away from growth-related activities to defense. Among these is the central translational regulator and growth promoter, Target of Rapamycin (TOR) kinase. Suppression of TOR expression leads to increased resistance to pathogens while overexpression of TOR results in increased susceptibility, indicating an important role for translational control in the switch from growth to defense. Furthermore, we show that several additional genes whose mRNAs are translationally regulated, including BIG, CCT2, and CIPK5, are required for both NB-LRR-mediated and basal plant innate immunity, identifying novel actors in plant defense.

De novo computational identification of stress-related sequence motifs and microRNA target sites in untranslated regions of a plant translatome.

Gene regulation at the transcriptional and translational level leads to diversity in phenotypes and function in organisms. Regulatory DNA or RNA sequence motifs adjacent to the gene coding sequence act as binding sites for proteins that in turn enable or disable expression of the gene. Whereas the known DNA and RNA binding proteins range in the thousands, only a few motifs have been examined. In this study, we have predicted putative regulatory motifs in groups of untranslated regions from genes regulated at the translational level in Arabidopsis thaliana under normal and stressed conditions. The test group of sequences was divided into random subgroups and subjected to three de novo motif finding algorithms (Seeder, Weeder and MEME). In addition to identifying sequence motifs, using an in silico tool we have predicted microRNA target sites in the 3' UTRs of the translationally regulated genes, as well as identified upstream open reading frames located in the 5' UTRs. Our bioinformatics strategy and the knowledge generated contribute to understanding gene regulation during stress, and can be applied to disease and stress resistant plant development.