A 7-Deoxyloganetic Acid Glucosyltransferase Contributes a Key Step in Secologanin Biosynthesis in Madagascar Periwinkle

Iridoids form a broad and versatile class of biologically active molecules found in thousands of plant species. In addition to the many hundreds of iridoids occurring in plants, some iridoids, such as secologanin, serve as key building blocks in the biosynthesis of thousands of monoterpene indole alkaloids (MIAs) and many quinoline alkaloids. This study describes the molecular cloning and functional characterization of three iridoid glucosyltransfeases (UDP-SUGAR GLYCOSYLTRANSFERASE6 [UGT6], UGT7, and UGT8) from Madagascar periwinkle (Catharanthus roseus) with remarkably different catalytic efficiencies. Biochemical analyses reveal that UGT8 possessed a high catalytic efficiency toward its exclusive iridoid substrate, 7-deoxyloganetic acid, making it better suited for the biosynthesis of iridoids in periwinkle than the other two iridoid glucosyltransfeases. The role of UGT8 in the fourth to last step in secologanin biosynthesis was confirmed by virus-induced gene silencing in periwinkle plants, which reduced expression of this gene and resulted in a large decline in secologanin and MIA accumulation within silenced plants. Localization studies of UGT8 using a carborundum abrasion method for RNA extraction show that its expression occurs preferentially within periwinkle leaves rather than in epidermal cells, and in situ hybridization studies confirm that UGT8 is preferentially expressed in internal phloem associated parenchyma cells of periwinkle species.     

Improved Expression of His6‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3α(S)‐strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme‐mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His₆‐STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co‐expression of chaperones pG‐Tf2 and pG‐LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG‐Tf2 alone. His₆‐STR1 immobilized on Ni‐NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co‐expressed His₆‐STR1, novel 3α(S)‐12‐azastrictosidine was obtained by enzymatic catalysis of 7‐azatryptamine and secologanin. The results obtained are of significant importance for application to chemo‐enzymatic approaches leading to diversification of alkaloids with novel improved structures.     

Evolution and diversity of the 2–oxoglutarate‐dependent dioxygenase superfamily in plants

The 2–oxoglutarate‐dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes – DOXA, DOXB and DOXC – based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4–hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC‐class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm‐specific clades were found to be involved in various lineage‐specific specialized metabolisms, but 31 of the 57 DOXC‐class clades were only found in a single species. Therefore, the evolution and diversification of DOXC‐class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.     

Virus‐induced gene silencing in Catharanthus roseus by biolistic inoculation of tobacco rattle virus vectors

Catharanthus roseus constitutes the unique source of several valuable monoterpenoid indole alkaloids, including the antineoplastics vinblastine and vincristine. These alkaloids result from a complex biosynthetic pathway encompassing between 30 and 50 enzymatic steps whose characterisation is still underway. The most recent identifications of genes from this pathway relied on a tobacco rattle virus‐based virus‐induced gene silencing (VIGS) approach, involving an Agrobacterium‐mediated inoculation of plasmids encoding the two genomic components of the virus. As an alternative, we developed a biolistic‐mediated approach of inoculation of virus‐encoding plasmids that can be easily performed by a simple bombardment of young C. roseus plants. After optimisation of the transformation conditions, we showed that this approach efficiently silenced the phytoene desaturase gene, leading to strong and reproducible photobleaching of leaves. This biolistic transformation was also used to silence a previously characterised gene from the alkaloid biosynthetic pathway, encoding iridoid oxidase. Plant bombardment caused down‐regulation of the targeted gene (70%), accompanied by a correlated decreased in MIA biosynthesis (45–90%), similar to results obtained via agro‐transformation. Thus, the biolistic‐based VIGS approach developed for C. roseus appears suitable for gene function elucidation and can readily be used instead of the Agrobacterium‐based approach, e.g. when difficulties arise with agro‐inoculations or when Agrobacterium‐free procedures are required to avoid plant defence responses.     

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.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐phenylalanine‐CoA ligase

Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.     

Alteration of cell wall xylan acetylation triggers defense responses that counterbalance the immune deficiencies of plants impaired in the β‐subunit of the heterotrimeric G‐protein

Arabidopsis heterotrimeric G‐protein complex modulates pathogen‐associated molecular pattern‐triggered immunity (PTI) and disease resistance responses to different types of pathogens. It also plays a role in plant cell wall integrity as mutants impaired in the Gβ‐ (agb1‐2) or Gγ‐subunits have an altered wall composition compared with wild‐type plants. Here we performed a mutant screen to identify suppressors of agb1‐2 (sgb) that restore susceptibility to pathogens to wild‐type levels. Out of the four sgb mutants (sgb10–sgb13) identified, sgb11 is a new mutant allele of ESKIMO1 (ESK1), which encodes a plant‐specific polysaccharide O‐acetyltransferase involved in xylan acetylation. Null alleles (sgb11/esk1‐7) of ESK1 restore to wild‐type levels the enhanced susceptibility of agb1‐2 to the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM), but not to the bacterium Pseudomonas syringae pv. tomato DC3000 or to the oomycete Hyaloperonospora arabidopsidis. The enhanced resistance to PcBMM of the agb1‐2 esk1‐7 double mutant was not the result of the re‐activation of deficient PTI responses in agb1‐2. Alteration of cell wall xylan acetylation caused by ESK1 impairment was accompanied by an enhanced accumulation of abscisic acid, the constitutive expression of genes encoding antibiotic peptides and enzymes involved in the biosynthesis of tryptophan‐derived metabolites, and the accumulation of disease resistance‐related secondary metabolites and different osmolites. These esk1‐mediated responses counterbalance the defective PTI and PcBMM susceptibility of agb1‐2 plants, and explain the enhanced drought resistance of esk1 plants. These results suggest that a deficient PTI‐mediated resistance is partially compensated by the activation of specific cell‐wall‐triggered immune responses.     

Chemical Traits of Hemiparasitism in Odontites luteus

The study of the monoterpene glycosides content of Odontites luteus has shown the presence of a total of fifteen iridoid glucosides. The presence of compounds 1  –  5 and 7  –  10 is perfectly on‐line with both the biogenetic pathway for iridoids biosynthesis in Lamiales and the current botanical classification of the species. On the other side, the presence of compounds like agnuside ( 6 ), adoxosidic acid ( 11 ), monotropein ( 12 ), 6,7‐dihydromonotropein ( 13 ), methyl oleoside ( 14 ) and methyl glucooleoside ( 15 ) is of high interest because, first of all, they have never been reported before in Lamiales. In second instance, the majority of the last compounds are formally derived from a different biogenetic pathway which involves deoxyloganic acid/loganin and led to the formation of decarboxylated iridoid showing the 8β‐configuration. Furthermore, a second abnormality was found during our study and this regards compounds 14 and 15 which are seco‐iriodids and thus not typical for this family. The presence of these unusual compounds, biogenetically not related to species belonging to Lamiales, is a clear evidence of the metabolites transfer from the hosts. In fact, the collection area is also populated by species belonging to Oleaceae and Ericaceae which could be the possible hosts since the biosynthesis of seco‐iridoids and or iridoids related to deoxyloganic acid/loganin pathway, with the 8β‐configuration, is well documented in these species.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Herbivore perception decreases photosynthetic carbon assimilation and reduces stomatal conductance by engaging 12‐oxo‐phytodienoic acid,mitogen‐activated protein kinase 4 and cytokinin perception

Herbivory‐induced changes in photosynthesis have been documented in many plant species; however, the complexity of photosynthetic regulation and analysis has thwarted progress in understanding the mechanism involved, particularly those elicited by herbivore‐specific elicitors. Here, we analysed the early photosynthetic gas exchange responses in Nicotiana attenuata plants after wounding and elicitation with Manduca sexta oral secretions and the pathways regulating these responses. Elicitation with M. sexta oral secretions rapidly decreased photosynthetic carbon assimilation (A_C) in treated and systemic (untreated, vascularly connected) leaves, which were associated with changes in stomatal conductance, rather than with changes in Rubisco activity and 1‐5 ribulose‐1,5‐bisphosphate turnover. Phytohormone profiling and gas exchange analysis of oral secretion‐elicited transgenic plants altered in phytohormone regulation, biosynthesis and perception, combined with micrografting techniques, revealed that the local photosynthetic responses were mediated by 12‐oxo‐phytodienoic acid, while the systemic responses involved interactions among jasmonates, cytokinins and abscisic acid signalling mediated by mitogen‐activated protein kinase 4. The analysis also revealed a role for cytokinins interacting with mitogen‐activated protein kinase 4 in CO₂‐mediated stomatal regulation. Hence, oral secretions, while eliciting jasmonic acid‐mediated defence responses, also elicit 12‐oxo‐phytodienoic acid‐mediated changes in stomatal conductance and A_C, an observation illustrating the complexity and economy of the signalling that regulates defence and carbon assimilation pathways in response to herbivore attack.