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Glycosylation is one of the most prevalent molecular modifications in nature. Single or multiple sugars can decorate a wide range of acceptors from proteins to lipids, cell wall glycans and small molecules, dramatically affecting their activity. Here, we discovered that by ‘hijacking’ an enzyme of the cellulose synthesis machinery involved in cell wall assembly, plants evolved cellulose synthase-like enzymes (Csls) and acquired the capacity to glucuronidate specialized metabolites, that is, triterpenoid saponins. Apparently, endoplasmic reticulum-membrane localization of Csls and of other pathway proteins was part of evolving a new glycosyltransferase function, as plant metabolite glycosyltransferases typically act in the cytosol. Discovery of glucuronic acid transferases across several plant orders uncovered the long-pursued enzymatic reaction in the production of a low-calorie sweetener from licorice roots. Our work opens the way for engineering potent saponins through microbial fermentation and plant-based systems. Evolution of a group of plant cellulose synthase-like enzymes into specialized glycosyltransferases in the endoplasmic reticulum membrane confers the ability to glucuronidate triterpenoid saponins and other specialized metabolites.

Plants are a rich source of specialized metabolites with a broad range of bioactivities and many applications in human daily life. Over the past decades significant progress has been made in identifying many such metabolites in different plant species and in elucidating their biosynthetic pathways. However, the biological roles of plant specialized metabolites remain elusive and proposed functions lack an identified underlying molecular mechanism. Understanding the roles of specialized metabolites frequently is hampered by their dynamic production and their specific spatiotemporal accumulation within plant tissues and organs throughout a plant’s life cycle. In this review, we propose the employment of strategies from the field of Synthetic Biology to construct and optimize genetically encoded biosensors that can detect individual specialized metabolites in a standardized and high-throughput manner. This will help determine the precise localization of specialized metabolites at the tissue and single-cell levels. Such information will be useful in developing complete system-level models of specialized plant metabolism, which ultimately will demonstratehowthe biosynthesis of specialized metabolites is integrated with the core processes of plant growth and development.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Glucocorticoids are steroid hormones that regulate inflammation, growth, metabolism, and apoptosis via their cognate receptor, the glucocorticoid receptor (GR). GR, acting mainly as a transcription factor, activates or represses the expression of a large number of target genes, among them, many genes of anti-inflammatory and pro-inflammatory molecules, respectively. Transrepression activity of glucocorticoids also accounts for their anti-inflammatory activity, rendering them the most widely prescribed drug in medicine. However, chronic and high-dose use of glucocorticoids is accompanied with many undesirable side effects, attributed predominantly to GR transactivation activity. Thus, there is a high need for selective GR agonist, capable of dissociating transrepression from transactivation activity. Protopanaxadiol and protopanaxatriol are triterpenoids that share structural and functional similarities with glucocorticoids. The molecular mechanism of their actions is unclear. In this study applying induced-fit docking analysis, luciferase assay, immunofluorescence, and Western blot analysis, we showed that protopanaxadiol and more effectively protopanaxatriol are capable of binding to GR to activate its nuclear translocation, and to suppress the nuclear factor-kappa beta activity in GR-positive HeLa and HEK293 cells, but not in GR-low level COS-7 cells. Interestingly, no transactivation activity was observed, whereas suppression of the dexamethasone-induced transactivation of GR and induction of apoptosis in HeLa and HepG2 cells were observed. Thus, our results indicate that protopanaxadiol and protopanaxatriol could be considered as potent and selective GR agonist.

Satureja khuzistanica is a medicinal herb endemic to Iran which can serve as a source of rosmarinic acid (RA). In the present study, the effect of different concentrations of methyl jasmonate (MeJA) and multi-walled carbon nanotubes (MWCNTs) on the rosmarinic acid accumulation and the expression of genes involved in its biosynthetic pathway was evaluated in nodal segment cultures of S. khuzistanica . The concentration of RA varied in plant extracts derived from the field, plants obtained under in vitro solid media, calli, suspension cultures and nodal segment cultures in liquid medium, with the highest amount recorded in nodal segment cultures. Phenylalanine ammonia-lyase ( PAL ), tyrosine aminotransferase ( TAT ), 4-hydroxyphenylpyruvate reductase ( HPPR ) and RA synthase ( RAS ) cDNA clones as key biosynthetic genes of RA production were identified and expression patterns were assayed in response to MeJA and MWCNTs, exogenously applied at a range of 0, 50, 100, 250 mg L −1 . The expression levels of HPPR, PAL and TAT were up-regulated at 100 mg L −1 MWCNTs, whereas down-regulated levels were observed at 250 mg L −1 . RAS was up-regulated in all MWCNTs treatments. HPPR, PAL and TAT expression increased sharply at 250 mg L −1 MeJA. The highest levels of RAS transcripts were observed at 100 mg L −1 MeJA. The highest HPPR, PAL and TAT expression levels were observed at 250 mg L −1 MeJA. In accordance, HPLC analysis showed a high amount of RA under MeJA and MWCNT elicitors. Our results provide helpful information on the expression profiles of biosynthetic genes in connection with RA accumulation.

Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8–RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8–RFP co-expressed with the actinbinding protein GFP–Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and A rabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume L otus japonicus . In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P 450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in N icotiana benthamiana . The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2 . A new plant cytochrome P 450, CYP 71 D 353, which catalyses the formation of 20‐hydroxybetulinic acid in a sequential three‐step oxidation of 20‐hydroxylupeol was characterized. The genes within the cluster are highly co‐expressed during root and nodule development, in hormone‐treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co‐regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development. See also the Commentary by Weng

Understanding the internal organization of cells from the molecular up to organelle level is a current challenge for biology if we are to better comprehend the mechanisms by which cellular processes occur. A prevailing view of the cell interior is that biochemical reactions and molecular movements are dominated by random diffusion. However, in addition to being compartmented into organelles, cells may well be organized at a finer level. Proteins may localise to specific areas within sub-cellular compartments, by associating with each other, the cytoskeleton and organelle membranes. Thus giving rise to distinct microcompartments. Considerable in vitro evidence exists for such interactions between enzymes and larger cellular components. This strengthens the idea that cells may be microcompartmented. However, little in vivo evidence supports this hypothesis, especially in plants. As a test-case for the concept of microcompartmentation this project investigated the sub-cellular distribution of the glycolytic enzyme fructose-bisphosphate aldolase in Arabidopsis thaliana; the ultimate aim being to establish whether it is microcompartmented in vivo and, further, to test the potential function(s) of such microcompartmentation.