The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.

Background: Oxidized derivatives of the plant hormone jasmonoyl-isoleucine accumulate in wounded Arabidopsis leaves. Results: Cytochromes P450 CYP94C1 and CYP94B3 cooperate to catalyze the formation of 12OH-JA-Ile and 12COOH-JA-Ile. Conclusion: CYP94C1 and CYP94B3 define a major route for JA-Ile catabolism. Significance: Elucidation of CYP94-mediated JA-Ile oxidation opens new avenues for understanding jasmonate metabolism and signaling.

Background:The plant hormone jasmonoyl-isoleucine (JA-Ile) undergoes oxidative catabolism mediated by cytochrome P450 enzymes. Results: Two amidohydrolases catalyze the cleavage of JA-Ile conjugates and generate 12OH-JA during Arabidopsis wound response. Conclusion: IAR3 and ILL6 define an additional pathway for JA-Ile turnover and establish a biosynthetic route for 12OH-JA. Significance: New enzymatic steps unravel the complexity in jasmonate metabolism.

The cytochrome P450 family encompasses the largest family of enzymes in plant metabolism, and the functions of many of its members in Arabidopsis thaliana are still unknown. Gene coexpression analysis pointed to two P450s that were coexpressed with two monoterpene synthases in flowers and were thus predicted to be involved in monoterpenoid metabolism. We show that all four selected genes, the two terpene synthases (TPS10 and TPS14) and the two cytochrome P450s (CYP71B31 and CYP76C3), are simultaneously expressed at anthesis, mainly in upper anther filaments and in petals. Upon transient expression in Nicotiana benthamiana, the TPS enzymes colocalize in vesicular structures associated with the plastid surface, whereas the P450 proteins were detected in the endoplasmic reticulum. Whether they were expressed in Saccharomyces cerevisiae or in N. benthamiana, the TPS enzymes formed two different enantiomers of linalool: (2)-(R)-linalool for TPS10 and (+)-(S)-linalool for TPS14. Both P450 enzymes metabolize the two linalool enantiomers to form different but overlapping sets of hydroxylated or epoxidized products. These oxygenated products are not emitted into the floral headspace, but accumulate in floral tissues as further converted or conjugated metabolites. This work reveals complex linalool metabolism in Arabidopsis flowers, the ecological role of which remains to be determined.

Metabolome and water homeostasis analysis of Thellungiella salsuginea suggests that dehydration tolerance is a key response to osmotic stress in this halophyte SUMMARYThellungiella salsuginea, a Brassicaceae species closely related to Arabidopsis thaliana, is tolerant to high salinity. The two species were compared under conditions of osmotic stress to assess the relationships between stress tolerance, the metabolome, water homeostasis and growth performance. A broad range of metabolites were analysed by metabolic fingerprinting and profiling, and the results showed that, despite a few notable differences in raffinose and secondary metabolites, the same metabolic pathways were regulated by salt stress in both species. The main difference was quantitative: Thellungiella had much higher levels of most metabolites than Arabidopsis whatever the treatment. Comprehensive quantification of organic and mineral solutes showed a relative stability of the total solute content regardless of the species or treatment, meaning that little or no osmotic adjustment occurred under stress. The reduction in osmotic potential observed in plants under stress was found to result from a passive loss of water. Thellungiella shoots contain less water than Arabidopsis shoots, and have the ability to lose more water, which could contribute to maintain a water potential gradient between soil and plant. Significant differences between Thellungiella and Arabidopsis were also observed in terms of the physicochemical properties of their metabolomes, such as water solubility and polarity. On the whole, the Thellungiella metabolome appears to be more compatible with dehydration. Osmotic stress was also found to impact the metabolome properties in both species, increasing the overall polarity. Together, the results suggest that Thellungiella copes with osmotic stress by tolerating dehydration, with its metabolic configuration lending itself to osmoprotective strategies rather than osmo-adjustment.

The compatible solute hypothesis posits that maintaining osmotic equilibrium under conditions of high salinity requires synthesis of organic compounds, uptake of potassium ions, and partial exclusion of NaCl. To assess whether osmotic adaptation in Limonium latifolium proceeds according to this hypothesis, a comprehensive analysis of solute accumulation during NaCl treatments was conducted. Determination of prevailing inorganic ions and establishment of the metabolic profiles for low M r organic substances revealed that contrary to the mentioned hypothesis the major contributors to osmolarity were constituted by inorganic solutes. Independent of salinity, only 25% of this osmolarity resulted from organic solutes such as Suc and hexoses. Proline (Pro), b-alanine betaine, and choline-O-sulfate were minor contributors to osmolarity. Compatible inositols also occurred, especially chiro-inositol, characterized for the first time in this species, to our knowledge. Principal component analysis showed that only a limited number of metabolic reconfigurations occurred in response to dynamic changes in salinity. Under such conditions only sugars, chiro-inositol, and Pro behave as active osmobalancers. Analysis of metabolic profiles during acclimatization to either mild salinity or nonsaline conditions showed that organic solute accumulation is predominantly controlled by constitutive developmental programs, some of which might be slightly modulated by salinity. Osmolarity provided under such conditions can be sufficient to maintain turgor in salinized seedlings. Compartmental analysis of Pro and b-alanine betaine in leaf tissues demonstrated that these solutes, mainly located in vacuoles under nonsaline conditions, could be partly directed to the cytosol in response to salinization. Thus they did not conform with the predictions of the compatible solute hypothesis.Due to either water loss or induced processes responsible for enhancement of the total number of osmotically active particles, higher plants are able to increase their osmolarity in response to hyperosmotic conditions. Accumulation of such particles, collectively termed osmolytes, contributes to osmotic adjustment (OA), which is needed for survival and resuming of growth at after-stress recovery. Active OA has been recognized as one of the key determinants of tolerance to salinity and other osmotic stresses encountered by higher plants (Jamaux et al., 1997). Inorganic and/or organic solutes could be involved in such function and depending on their origin, their production is associated with different energetic requirements partly responsible for inhibitory effects on growth rate. It is widely believed that osmolytes do not exert, per se, damaging effects on cell compartments where they are located.Primarily OA could be achieved with ions such as K 1 , Na 1 , NO 3 2 , or Cl 2 when available in the root environment (Shabala and Lew, 2002) through stressinduced activation of uptake and translocation of these substances. At the cellular level further accumulation of ...

de Ciência, P-2780-156 Oeiras, Portugal (R.T., G.A.B.M.)Stilbenes are a small family of phenylpropanoids produced in a number of unrelated plant species, including grapevine (Vitis vinifera). In addition to their participation in defense mechanisms in plants, stilbenes, such as resveratrol, display important pharmacological properties and are postulated to be involved in the health benefits associated with a moderate consumption of red wine. Stilbene synthases (STSs), which catalyze the biosynthesis of the stilbene backbone, seem to have evolved from chalcone synthases (CHSs) several times independently in stilbene-producing plants. STS genes usually form small families of two to five closely related paralogs. By contrast, the sequence of grapevine reference genome (cv PN40024) has revealed an unusually large STS gene family. Here, we combine molecular evolution and structural and functional analyses to investigate further the high number of STS genes in grapevine. Our reannotation of the STS and CHS gene families yielded 48 STS genes, including at least 32 potentially functional ones. Functional characterization of nine genes representing most of the STS gene family diversity clearly indicated that these genes do encode for proteins with STS activity. Evolutionary analysis of the STS gene family revealed that both STS and CHS evolution are dominated by purifying selection, with no evidence for strong selection for new functions among STS genes. However, we found a few sites under different selection pressures in CHS and STS sequences, whose potential functional consequences are discussed using a structural model of a typical STS from grapevine that we developed.

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