PtaRHE1, a Populus tremula × Populus alba RING‐H2 protein of the ATL family,has a regulatory role in secondary phloem fibre development

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING‐H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild‐type plants. Following an RNA‐seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development.     

Metabolic diversification of nitrogen‐containing metabolites by the expression of a heterologous lysine decarboxylase gene in Arabidopsis

Lysine decarboxylase converts l ‐lysine to cadaverine as a branching point for the biosynthesis of plant Lys‐derived alkaloids. Although cadaverine contributes towards the biosynthesis of Lys‐derived alkaloids, its catabolism, including metabolic intermediates and the enzymes involved, is not known. Here, we generated transgenic Arabidopsis lines by expressing an exogenous lysine/ornithine decarboxylase gene from Lupinus angustifolius (La‐L/ODC) and identified cadaverine‐derived metabolites as the products of the emerged biosynthetic pathway. Through untargeted metabolic profiling, we observed the upregulation of polyamine metabolism, phenylpropanoid biosynthesis and the biosynthesis of several Lys‐derived alkaloids in the transgenic lines. Moreover, we found several cadaverine‐derived metabolites specifically detected in the transgenic lines compared with the non‐transformed control. Among these, three specific metabolites were identified and confirmed as 5‐aminopentanal, 5‐aminopentanoate and δ‐valerolactam. Cadaverine catabolism in a representative transgenic line (DC29) was traced by feeding stable isotope‐labeled [α‐¹⁵N]‐ or [ε‐¹⁵N]‐l ‐lysine. Our results show similar ¹⁵N incorporation ratios from both isotopomers for the specific metabolite features identified, indicating that these metabolites were synthesized via the symmetric structure of cadaverine. We propose biosynthetic pathways for the metabolites on the basis of metabolite chemistry and enzymes known or identified through catalyzing specific biochemical reactions in this study. Our study shows that this pool of enzymes with promiscuous activities is the driving force for metabolite diversification in plants. Thus, this study not only provides valuable information for understanding the catabolic mechanism of cadaverine but also demonstrates that cadaverine accumulation is one of the factors to expand plant chemodiversity, which may lead to the emergence of Lys‐derived alkaloid biosynthesis.     

Personal model‐assisted identification of NAD+ and glutathione metabolism as intervention target in NAFLD

To elucidate the molecular mechanisms underlying non‐alcoholic fatty liver disease (NAFLD), we recruited 86 subjects with varying degrees of hepatic steatosis (HS). We obtained experimental data on lipoprotein fluxes and used these individual measurements as personalized constraints of a hepatocyte genome‐scale metabolic model to investigate metabolic differences in liver, taking into account its interactions with other tissues. Our systems level analysis predicted an altered demand for NAD⁺ and glutathione (GSH) in subjects with high HS. Our analysis and metabolomic measurements showed that plasma levels of glycine, serine, and associated metabolites are negatively correlated with HS, suggesting that these GSH metabolism precursors might be limiting. Quantification of the hepatic expression levels of the associated enzymes further pointed to altered de novo GSH synthesis. To assess the effect of GSH and NAD⁺ repletion on the development of NAFLD, we added precursors for GSH and NAD⁺ biosynthesis to the Western diet and demonstrated that supplementation prevents HS in mice. In a proof‐of‐concept human study, we found improved liver function and decreased HS after supplementation with serine (a precursor to glycine) and hereby propose a strategy for NAFLD treatment.     

The assembly of (+)‐vincadifformine‐ and (−)‐tabersonine‐derived monoterpenoid indole alkaloids in Catharanthus roseus involves separate branch pathways

The biological activity of monoterpenoid indole alkaloids (MIAs) has led to their use in cancer treatment and other medical applications. Their biosynthesis has involved the formation of reactive intermediates by responsible enzymes to elaborate several different chemical scaffolds. Modification of scaffolds through different substitution reactions has produced chemically diverse MIAs and related biological activities. The present study characterizes the three‐step pathway involved in the formation of (+)‐echitovenine, the major O‐acetylated MIA of Catharanthus roseus roots, and differentiates it from a parallel pathway involved in the formation of hörhammericine. Separate hydrolases convert a common reactive MIA intermediate to aspidosperma skeletons of opposite specific rotations, that is (+)‐vincadifformine and (?)‐tabersonine, respectively. The formation of (+) minovincinine from (+) vincadifformine 19‐hydroxylase (V19H) is catalyzed by a root‐specific cytochrome P450 with high amino acid sequence similarity to the leaf‐specific tabersonine‐3‐hydroxylase involved in vindoline biosynthesis. Similarly, O‐acetylation of (+)‐minovincinine to form (+) echitovenine involves minovincinine‐O‐acetytransferase. The substrate specificity of V19H and MAT for their respective (+)‐enantiomers defines the separate enantiomer‐specific pathway involved in (+)‐echitovenine biosynthesis and differentiates it from a parallel (?)‐enantiomer‐specific pathway involved in the formation of hörhammericine from (?)‐tabersonine.     

Two polyketide synthases are necessary for 4‐hydroxy‐5‐methylcoumarin biosynthesis in Gerbera hybrida

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4‐hydroxy‐5‐methylcoumarin (HMC). Unlike most coumarins, 5‐methylcoumarins have been suggested to be derived through the acetate‐malonate pathway. All of these polyketide‐derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2‐pyrone synthases (G2PS1–3). The enzymes are chalcone synthase‐like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4‐hydroxy‐6‐methyl‐2‐pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7‐dihydroxy‐5‐methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site‐directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.     

Time‐specific and pleiotropic quantitative trait loci coordinately modulate stem growth in Populus

In perennial woody plants, the coordinated increase of stem height and diameter during juvenile growth improves competitiveness (i.e. access to light); however, the factors underlying variation in stem growth remain unknown in trees. Here, we used linkage‐linkage disequilibrium (linkage‐LD) mapping to decipher the genetic architecture underlying three growth traits during juvenile stem growth. We used two Populus populations: a linkage mapping population comprising a full‐sib family of 1,200 progeny and an association mapping panel comprising 435 unrelated individuals from nearly the entire natural range of Populus tomentosa. We mapped 311 quantitative trait loci (QTL) for three growth traits at 12 timepoints to 42 regions in 17 linkage groups. Of these, 28 regions encompassing 233 QTL were annotated as 27 segmental homology regions (SHRs). Using SNPs identified by whole‐genome re‐sequencing of the 435‐member association mapping panel, we identified significant SNPs (P ≤ 9.4 × 10^?7) within 27 SHRs that affect stem growth at nine timepoints with diverse additive and dominance patterns, and these SNPs exhibited complex allelic epistasis over the juvenile growth period. Nineteen genes linked to potential causative alleles that have time‐specific or pleiotropic effects, and mostly overlapped with significant signatures of selection within SHRs between climatic regions represented by the association mapping panel. Five genes with potential time‐specific effects showed species‐specific temporal expression profiles during the juvenile stages of stem growth in five representative Populus species. Our observations revealed the importance of considering temporal genetic basis of complex traits, which will facilitate the molecular design of tree ideotypes.     

The effects of age and dietary restriction on the tissue‐specific metabolome of Drosophila

Dietary restriction (DR) is a robust intervention that extends lifespan and slows the onset of age‐related diseases in diverse organisms. While significant progress has been made in attempts to uncover the genetic mechanisms of DR, there are few studies on the effects of DR on the metabolome. In recent years, metabolomic profiling has emerged as a powerful technology to understand the molecular causes and consequences of natural aging and disease‐associated phenotypes. Here, we use high‐resolution mass spectroscopy and novel computational approaches to examine changes in the metabolome from the head, thorax, abdomen, and whole body at multiple ages in Drosophila fed either a nutrient‐rich ad libitum (AL) or nutrient‐restricted (DR) diet. Multivariate analysis clearly separates the metabolome by diet in different tissues and different ages. DR significantly altered the metabolome and, in particular, slowed age‐related changes in the metabolome. Interestingly, we observed interacting metabolites whose correlation coefficients, but not mean levels, differed significantly between AL and DR. The number and magnitude of positively correlated metabolites was greater under a DR diet. Furthermore, there was a decrease in positive metabolite correlations as flies aged on an AL diet. Conversely, DR enhanced these correlations with age. Metabolic set enrichment analysis identified several known (e.g., amino acid and NAD metabolism) and novel metabolic pathways that may affect how DR effects aging. Our results suggest that network structure of metabolites is altered upon DR and may play an important role in preventing the decline of homeostasis with age.