An Engineered Heat-Inducible Expression System for the Production of Casbene in Nicotiana benthamiana.

Plants respond to heat stress by producing heat-shock proteins. These are regulated by heat-shock promoters containing regulatory elements, which can be harnessed to control protein expression both temporally and spatially. In this study, we designed heat-inducible promoters to produce the diterpene casbene in Nicotiana benthamiana, through a multi-step metabolic pathway. To potentially increase gene transcription, we coupled heat-shock elements from Arabidopsis thaliana Hsp101 or Glycine max GmHsp17.3-B promoters, CAAT and TATA boxes from CaMV 35S, and the 5'UTR from the tobacco mosaic virus. The resulting four chimeric promoters fused to a green fluorescent protein (GFP) reporter showed that the variant Ara2 had the strongest fluorescent signal after heat shock. We next created a 4-gene cassette driven by the Ara2 promoter to allow for exogenous synthesis of casbene and transformed this multigene construct along with a selectable marker gene into Nicotiana benthamiana. Metabolic analysis on the transgenic lines revealed that continuous heat outperforms heat shock, with up to 1 µg/mg DW of casbene detected after 32 h of uninterrupted 40 °C heat. These results demonstrate the potential of heat-inducible promoters as synthetic biology tools for metabolite production in plants.

3'Nucleotidase/nuclease is required for Leishmania infantum clinical isolate susceptibility to miltefosine.

BACKGROUND: Miltefosine treatment failure in visceral leishmaniasis in Brazil has been associated with deletion of the miltefosine susceptibility locus (MSL) in Leishmania infantum. The MSL comprises four genes, 3'-nucleotidase/nucleases (NUC1 and NUC2); helicase-like protein (HLP); and 3,2-trans-enoyl-CoA isomerase (TEI). METHODS: In this study CRISPR-Cas9 was used to either epitope tag or delete NUC1, NUC2, HLP and TEI, to investigate their role in miltefosine resistance mechanisms. Additionally, miltefosine transporter genes and miltefosine-mediated reactive oxygen species homeostasis were assessed in 26 L. infantum clinical isolates. A comparative lipidomic analysis was also performed to investigate the molecular basis of miltefosine resistance. FINDINGS: Deletion of both NUC1, NUC2 from the MSL was associated with a significant decrease in miltefosine susceptibility, which was restored after re-expression. Metabolomic analysis of parasites lacking the MSL or NUC1 and NUC2 identified an increase in the parasite lipid content, including ergosterol; these lipids may contribute to miltefosine resistance by binding the drug in the membrane. Parasites lacking the MSL are more resistant to lipid metabolism perturbation caused by miltefosine and NUC1 and NUC2 are involved in this pathway. Additionally, L. infantum parasites lacking the MSL isolated from patients who relapsed after miltefosine treatment were found to modulate nitric oxide accumulation in host macrophages. INTERPRETATION: Altogether, these data indicate that multifactorial mechanisms are involved in natural resistance to miltefosine in L. infantum and that the absence of the 3'nucleotidase/nuclease genes NUC1 and NUC2 contributes to the phenotype. FUNDING: MRC GCRF and FAPES.

Artemisia annua L. plants lacking Bornyl diPhosphate Synthase reallocate carbon from monoterpenes to sesquiterpenes except artemisinin.

The monoterpene camphor is produced in glandular secretory trichomes of the medicinal plant Artemisia annua, which also produces the antimalarial drug artemisinin. We have found that, depending on growth conditions, camphor can accumulate at levels ranging from 1- 10% leaf dry weight (LDW) in the Artemis F1 hybrid, which has been developed for commercial production of artemisinin at up to 1% LDW. We discovered that a camphor null (camphor-0) phenotype segregates in the progeny of self-pollinated Artemis material. Camphor-0 plants also show reduced levels of other less abundant monoterpenes and increased levels of the sesquiterpene precursor farnesyl pyrophosphate plus sesquiterpenes, including enzymatically derived artemisinin pathway intermediates but not artemisinin. One possible explanation for this is that high camphor concentrations in the glandular secretory trichomes play an important role in generating the hydrophobic conditions required for the non-enzymatic conversion of dihydroartemisinic acid tertiary hydroperoxide to artemisinin. We established that the camphor-0 phenotype associates with a genomic deletion that results in loss of a Bornyl diPhosphate Synthase (AaBPS) gene candidate. Functional characterization of the corresponding enzyme in vitro confirmed it can catalyze the first committed step in not only camphor biosynthesis but also in a number of other monoterpenes, accounting for over 60% of total volatiles in A. annua leaves. This in vitro analysis is consistent with loss of monoterpenes in camphor-0 plants. The AaBPS promoter drives high reporter gene expression in A. annua glandular secretory trichomes of juvenile leaves with expression shifting to non-glandular trichomes in mature leaves, which is consistent with AaBPS transcript abundance.

A functionally conserved STORR gene fusion in Papaver species that diverged 16.8 million years ago.

The STORR gene fusion event is considered essential for the evolution of the promorphinan/morphinan subclass of benzylisoquinoline alkaloids (BIAs) in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline essential for their biosynthesis. Here, we show that of the 12 Papaver species analysed those containing the STORR gene fusion also contain promorphinans/morphinans with one important exception. P. californicum encodes a functionally conserved STORR but does not produce promorphinans/morphinans. We also show that the gene fusion event occurred only once, between 16.8-24.1 million years ago before the separation of P. californicum from other Clade 2 Papaver species. The most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs, raising the possibility that STORR, once evolved, contributes to the biosynthesis of more than just the promorphinan/morphinan subclass of BIAs in the Papaveraceae.

Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.

Plant metabolic gene clusters in the multi-omics era.

Secondary metabolism in plants gives rise to a vast array of small-molecule natural products. The discovery of operon-like gene clusters in plants has provided a new perspective on the evolution of specialized metabolism and the opportunity to rapidly advance the metabolic engineering of natural product production. Here, we review historical aspects of the study of plant metabolic gene clusters as well as general strategies for identifying plant metabolic gene clusters in the multi-omics era. We also emphasize the exploration of their natural variation and evolution, as well as new strategies for the prospecting of plant metabolic gene clusters and a deeper understanding of how their structure influences their function.

Impact on Arabidopsis growth and stress resistance of depleting the Maf1 repressor of RNA polymerase III.

Maf1 is a transcription factor that is conserved in sequence and structure between yeasts, animals and plants. Its principal molecular function is also well conserved, being to bind and repress RNA polymerase (pol) III, thereby inhibiting synthesis of tRNAs and other noncoding RNAs. Restrictions on tRNA production and hence protein synthesis can provide a mechanism to preserve resources under conditions that are suboptimal for growth. Accordingly, Maf1 is found in some organisms to influence growth and/or stress survival. Because of their sessile nature, plants are especially vulnerable to environmental changes and molecular adaptations that enhance growth under benign circumstances can increase sensitivity to external challenges. We tested if Maf1 depletion in the model plant Arabidopsis affects growth, pathogen resistance and tolerance of drought or soil salinity, a common physiological challenge that imposes both osmotic and ionic stress. We find that disruption of the Maf1 gene or RNAi-mediated depletion of its transcript is well-tolerated and confers a modest growth advantage without compromising resistance to common biotic and abiotic challenges.

Engineering Production of a Novel Diterpene Synthase Precursor in Nicotiana benthamiana.

Diterpene biosynthesis commonly originates with the methylerythritol phosphate (MEP) pathway in chloroplasts, leading to the C20 substrate, geranylgeranyl pyrophosphate (GGPP). The previous work demonstrated that over-expression of genes responsible for the first and last steps in the MEP pathway in combination with GERANYLGERANYL PYROPHOSPHATE SYNTHASE (GGPPS) and CASBENE SYNTHASE (CAS) is optimal for increasing flux through to casbene in Nicotiana benthamiana. When the gene responsible for the last step in the MEP pathway, 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE (HDR), is removed from this combination, casbene is still produced but at lower amounts. Here, we report the unexpected finding that this reduced gene combination also results in the production of 16-hydroxy-casbene (16-OH-casbene), consistent with the presence of 16-hydroxy-geranylgeranyl phosphate (16-OH-GGPP) in the same material. Indirect evidence suggests the latter is formed as a result of elevated levels of 4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) caused by a bottleneck at the HDR step responsible for conversion of HMBPP to dimethylallyl pyrophosphate (DMAPP). Over-expression of a GERANYLLINALOOL SYNTHASE from Nicotiana attenuata (NaGLS) produces 16-hydroxy-geranyllinalool (16-OH-geranyllinalool) when transiently expressed with the same reduced combination of MEP pathway genes in N. benthamiana. This work highlights the importance of pathway flux control in metabolic pathway engineering and the possibility of increasing terpene diversity through synthetic biology.

A chromosome-level Amaranthus cruentus genome assembly highlights gene family evolution and biosynthetic gene clusters that may underpin the nutritional value of this traditional crop.

Traditional crops have historically provided accessible and affordable nutrition to millions of rural dwellers but have been neglected, with most modern agricultural systems over-reliant on a small number of internationally traded crops. Traditional crops are typically well-adapted to local agro-ecological conditions and many are nutrient-dense. They can play a vital role in local food systems through enhanced nutrition (particularly where diets are dominated by starch crops), food security and livelihoods for smallholder farmers, and a climate-resilient and biodiverse agriculture. Using short-read, long-read and phased sequencing technologies, we generated a high-quality chromosome-level genome assembly for Amaranthus cruentus, an under-researched crop with micronutrient- and protein-rich leaves and gluten-free seed, but lacking improved varieties, with respect to productivity and quality traits. The 370.9 Mb genome demonstrates a shared whole genome duplication with a related species, Amaranthus hypochondriacus. Comparative genome analysis indicates chromosomal loss and fusion events following genome duplication that are common to both species, as well as fission of chromosome 2 in A. cruentus alone, giving rise to a haploid chromosome number of 17 (versus 16 in A. hypochondriacus). Genomic features potentially underlying the nutritional value of this crop include two A. cruentus-specific genes with a likely role in phytic acid synthesis (an anti-nutrient), expansion of ion transporter gene families, and identification of biosynthetic gene clusters conserved within the amaranth lineage. The A. cruentus genome assembly will underpin much-needed research and global breeding efforts to develop improved varieties for economically viable cultivation and realization of the benefits to global nutrition security and agrobiodiversity.

Superoxide is promoted by sucrose and affects amplitude of circadian rhythms in the evening.

Plants must coordinate photosynthetic metabolism with the daily environment and adapt rhythmic physiology and development to match carbon availability. Circadian clocks drive biological rhythms which adjust to environmental cues. Products of photosynthetic metabolism, including sugars and reactive oxygen species (ROS), are closely associated with the plant circadian clock, and sugars have been shown to provide metabolic feedback to the circadian oscillator. Here, we report a comprehensive sugar-regulated transcriptome of Arabidopsis and identify genes associated with redox and ROS processes as a prominent feature of the transcriptional response. We show that sucrose increases levels of superoxide (O2-), which is required for transcriptional and growth responses to sugar. We identify circadian rhythms of O2--regulated transcripts which are phased around dusk and find that O2- is required for sucrose to promote expression of TIMING OF CAB1 (TOC1) in the evening. Our data reveal a role for O2- as a metabolic signal affecting transcriptional control of the circadian oscillator in Arabidopsis.

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co-transformed with the optimal set of precursor-pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an 'alkenal reductase'-like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13-double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.

Over 100 Million Years of Enzyme Evolution Underpinning the Production of Morphine in the Papaveraceae Family of Flowering Plants.

Phylogenomic analysis of whole genome sequences of five benzylisoquinoline alkaloid (BIA)-producing species from the Ranunculales and Proteales orders of flowering plants revealed the sequence and timing of evolutionary events leading to the diversification of these compounds. (S)-Reticuline is a pivotal intermediate in the synthesis of many BIAs and our analyses revealed parallel evolution between the two orders, which diverged ∼122 million years ago (MYA). Berberine is present in species across the entire Ranunculales, and we found co-evolution of genes essential for production of the protoberberine class. The benzophenanthridine class, which includes the antimicrobial compound sanguinarine, is specific to the Papaveraceae family of Ranunculales, and biosynthetic genes emerged after the split with the Ranunculaceae family ∼110 MYA but before the split of the three Papaveraceae species used in this study at ∼77 MYA. The phthalideisoquinoline noscapine and morphinan class of BIAs are exclusive to the opium poppy lineage. Ks estimation of paralogous pairs indicates that morphine biosynthesis evolved more recently than 18 MYA in the Papaver genus. In the preceding 100 million years gene duplication, neofunctionalization and recruitment of additional enzyme classes, combined with gene clustering, gene fusion, and gene amplification, resulted in emergence of medicinally valuable BIAs including morphine and noscapine.

Impact of osmotic stress on the growth and root architecture of introgression lines derived from a wild ancestor of rice and a modern cultivar.

Many modern rice varieties have been intensively selected for high-yielding performance under irrigated conditions, reducing their genetic diversity and potentially increasing their susceptibility to abiotic stresses such as drought. In this study, we tested benefits for stress tolerance of introducing DNA segments from wild ancestor Oryza rufipogon to the modern cultivar O. sativa cv Curinga (CUR) by applying a gradient of osmotic stress to both parents and seven introgressed lines. Shoot growth of O. rufipogon had a high tolerance to osmotic stress, and the number of total root tips increased under mild osmotic stress. One introgression line showed greater shoot growth, root growth, and higher number of total root tips than the parent line CUR under osmotic stress. Abscisic acid (ABA) is a key hormone mediating plant responses to abiotic stresses. Both root and shoot growth of O. rufipogon were much more sensitive to ABA than CUR. Introgression lines varied in the extent to which the sensitivity of their growth responses to ABA and some lines correlated with their sensitivity to osmotic stress. Our results suggest that rice responses to ABA and osmotic stress are genotype dependent, and growth responses of rice to ABA are not a consistent indicator of resilience to abiotic stress in introgression lines.

Combining GAL4 GFP enhancer trap with split luciferase to measure spatiotemporal promoter activity in Arabidopsis.

In multicellular organisms different types of tissues have distinct gene expression profiles associated with specific function or structure of the cell. Quantification of gene expression in whole organs or whole organisms can give misleading information about levels or dynamics of expression in specific cell types. Tissue- or cell-specific analysis of gene expression has potential to enhance our understanding of gene regulation and interactions of cell signalling networks. The Arabidopsis circadian oscillator is a gene network which orchestrates rhythmic expression across the day/night cycle. There is heterogeneity between cell and tissue types of the composition and behaviour of the oscillator. In order to better understand the spatial and temporal patterns of gene expression, flexible tools are required. By combining a Gateway®-compatible split luciferase construct with a GAL4 GFP enhancer trap system, we describe a tissue-specific split luciferase assay for non-invasive detection of spatiotemporal gene expression in Arabidopsis. We demonstrate the utility of this enhancer trap-compatible split luciferase assay (ETSLA) system to investigate tissue-specific dynamics of circadian gene expression. We confirm spatial heterogeneity of circadian gene expression in Arabidopsis leaves and describe the resources available to investigate any gene of interest.

Flavonoid Versus Artemisinin Anti-malarial Activity in Artemisia annua Whole-Leaf Extracts.

Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for uncomplicated malaria caused by Plasmodium falciparum parasites. Other metabolites in A. annua or related species, particularly flavonoids, have been proposed to either act as antimalarials on their own or act synergistically with artemisinin to enhance antimalarial activity. We identified a mutation that disrupts the CHALCONE ISOMERASE 1 (CHI1) enzyme that is responsible for the second committed step of flavonoid biosynthesis. Detailed metabolite profiling revealed that chi1-1 lacks all major flavonoids but produces wild-type artemisinin levels, making this mutant a useful tool to test the antiplasmodial effects of flavonoids. We used whole-leaf extracts from chi1-1 and mutant lines impaired in artemisinin production in bioactivity in vitro assays against intraerythrocytic P. falciparum Dd2. We found that chi1-1 extracts did not differ from wild-type extracts in antiplasmodial efficacy nor initial rate of cytocidal action. Furthermore, extracts from the A. annua cyp71av1-1 mutant and RNAi lines impaired in amorpha-4,11-diene synthase gene expression, which are both severely compromised in artemisinin biosynthesis but unaffected in flavonoid metabolism, showed very low or no antiplasmodial activity. These results demonstrate that in vitro bioactivity against P. falciparum of flavonoids is negligible when compared to that of artemisinin.

cis-12-Oxo-phytodienoic acid represses Arabidopsis seed germination in shade conditions.

Light-dependent seed germination is induced by gibberellins (GA) and inhibited by abscisic acid (ABA). The widely accepted view of the GA/ABA ratio controlling germination does not, however, explain the fact that seeds deficient in ABA still germinate poorly under shade conditions that repress germination. In Arabidopsis, MOTHER-OF-FT-AND-TFL1 (MFT) acts as a key negative regulator of germination, modulating GA and ABA responses under shade conditions. Under full light the oxylipin cis-12-oxo-phytodienoic acid (OPDA), a precursor of the stress-related phytohormone jasmonic acid, interacts with ABA and MFT to repress germination. Here, we show that under shade conditions both OPDA and ABA repress germination to varying extents. We demonstrate that the level of shade-induced MFT expression influences the ability of OPDA and/or ABA to fully repress germination. We also found that MFT expression decreases with seed age and this again correlates with the response of seeds to OPDA and ABA. We conclude that OPDA plays an essential role alongside ABA in repressing germination in response to shade and the combined effect of these phytohormones is integrated to a significant extent through MFT.