At some point during biosynthesis of the antimalarial artemisinin in glandular trichomes of Artemisia annua, the ⌬11(13) double bond originating in amorpha-4,11-diene is reduced. This is thought to occur in artemisinic aldehyde, but other intermediates have been suggested. In an effort to understand double bond reduction in artemisinin biosynthesis, extracts of A. annua flower buds were investigated and found to contain artemisinic aldehyde ⌬11(13) double bond reductase activity. Through a combination of partial protein purification, mass spectrometry, and expressed sequence tag analysis, a cDNA clone corresponding to the enzyme was isolated. The corresponding gene Dbr2, encoding a member of the enoate reductase family with similarity to plant 12-oxophytodienoate reductases, was found to be highly expressed in glandular trichomes. Recombinant Dbr2 was subsequently characterized and shown to be relatively specific for artemisinic aldehyde and to have some activity on small ␣,␤-unsaturated carbonyl compounds. Expression in yeast of Dbr2 and genes encoding four other enzymes in the artemisinin pathway resulted in the accumulation of dihydroartemsinic acid. The relevance of Dbr2 to trichome-specific artemisinin biosynthesis is discussed.Since its discovery in the 1970s (1), the sesquiterpene lactone artemisinin (Fig. 1) from Artemisia annua has become a key factor in the drive to control malaria (2-4). Semi-synthetic derivatives of plant-derived artemisinin form the basis for artemisinin-based combination therapies, which are the treatment of choice for most forms of the disease. Given the pharmaceutical importance of artemisinin and its singular plant source, there is considerable interest in maintaining a reliable and low cost supply (5). A more complete knowledge of artemisinin biosynthesis and the genes involved is likely to provide ways of increasing production and lowering cost through crop improvement or microbial engineering (6, 7).As in other members of the Asteraceae, A. annua has 10-celled biseriate glandular trichomes that appear on the surfaces of aerial parts of the plant (8 -10). Along with other isoprenoids, the sesquiterpene lactone artemisinin accumulates to levels of 0.01-2% dry weight (11). Although progress is being made in understanding the biosynthesis of artemisinin, considerable gaps in our knowledge remain (6, 12). The formation of amorpha-4,11-diene by amorpha-4,11-diene synthase is the first committed step in the pathway. This is followed by oxidation at C-12 of amorpha-4,11-diene by the cytochrome P-450, Cyp71av1 to give artemisinic alcohol. These steps are well supported from biochemical studies and by the molecular cloning of genes encoding the relevant enzymes (12-14). The pathway beyond artemisinic alcohol is somewhat less well established (6, 7, 15). However, there is biochemical evidence supporting a route to dihydroartemsinic acid via artemisinic aldehyde and dihydroartemsinic aldehyde (12). This route includes the proposed reduction of the ⌬11(13) double bond of artemisinic aldeh...

SummaryA highly selective and sensitive method for the simultaneous analysis of several plant hormones and their metabolites is described. The method combines high-performance liquid chromatography (HPLC) with positive and negative electrospray ionization-tandem mass spectrometry (ESI±MS/MS) to quantify a broad range of chemically and structurally diverse compounds. The addition of deuterium-labeled analogs for these compounds prior to sample extraction permits accurate quanti®cation by multiple reaction monitoring (MRM). Endogenous levels of abscisic acid (ABA), abscisic acid glucose ester (ABA-GE), 7H -hydroxyabscisic acid (7 H -OH-ABA), phaseic acid (PA), dihydrophaseic acid (DPA), indole-3-acetic acid (IAA), indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine (2iP), isopentenyladenosine (IPA), and gibberellins (GA) 1 , GA 3 , GA 4 , and GA 7 were determined simultaneously in a single run. Detection limits ranged from 0.682 fmol for Z to 1.53 pmol for ABA. The method was applied to the analysis of plant hormones and hormonal metabolites associated with seed dormancy and germination in lettuce (Lactuca sativa L. cv. Grand Rapids), using extracts from only 50 to 100 mg DW of seed. Thermodormancy was induced by incubating seeds at 338C instead of 238C. Germinating seeds transiently accumulated high levels of ABA-GE. In contrast, thermodormant seeds transiently accumulated high levels of DPA after 7 days at 338C. GA 1 and GA 3 were detected during germination, and levels of GA 1 increased during early postgerminative growth. After several days of incubation, thermodormant seeds exhibited a striking transient accumulation of IAA, which did not occur in seeds germinating at 238C. We conclude that hormone metabolism in thermodormant seeds is surprisingly active and is signi®cantly different from that of germinating seeds.

SummaryIn Arabidopsis thaliana, the etr1-2 mutation confers dominant ethylene insensitivity and results in a greater proportion of mature seeds that exhibit dormancy compared with mature seeds of the wild-type. We investigated the impact of the etr1-2 mutation on other plant hormones by analyzing the profiles of four classes of plant hormones and their metabolites by HPLC-ESI/MS/MS in mature seeds of wild-type and etr1-2 plants. Hormone metabolites were analyzed in seeds imbibed immediately under germination conditions, in seeds subjected to a 7-day moist-chilling (stratification) period, and during germination/early post-germinative growth. Higher than wild-type levels of abscisic acid (ABA) appeared to contribute, at least in part, to the greater incidence of dormancy in mature seeds of etr1-2. The lower levels of abscisic acid glucose ester (ABA-GE) in etr1-2 seeds compared with wild-type seeds under germination conditions (with and without moistchilling treatments) suggest that reduced metabolism of ABA to ABA-GE likely contributed to the accumulation of ABA during germination in the mutant. The mutant seeds exhibited generally higher auxin levels and a large build-up of indole-3-aspartate when placed in germination conditions following moistchilling. The mutant manifested increased levels of cytokinin glucosides through zeatin-O-glucosylation (Z-O-Glu). The resulting increase in Z-O-Glu was the largest and most consistent change associated with the ETR1 gene mutation. There were more gibberellins (GA) and at higher concentrations in the mutant than in wild-type. Our results suggest that ethylene signaling modulates the metabolism of all the other plant hormone pathways in seeds. Additionally, the hormone profiles of etr1-2 seed during germination suggest a requirement for higher than wild-type levels of GA to promote germination in the absence of a functional ethylene signaling pathway.

SummaryA glucosyltransferase (GT) of Arabidopsis, UGT71B6, recognizing the naturally occurring enantiomer of abscisic acid (ABA) in vitro, has been used to disturb ABA homeostasis in planta. Transgenic plants constitutively overexpressing UGT71B6 (71B6-OE) have been analysed for changes in ABA and the related ABA metabolites abscisic acid glucose ester (ABA-GE), phaseic acid (PA), dihydrophaseic acid (DPA), 7¢-hydroxyABA and neo-phaseic acid. Overexpression of the GT led to massive accumulation of ABA-GE and reduced levels of the oxidative metabolites PA and DPA, but had marginal effect on levels of free ABA. The control of ABA homeostasis, as reflected in levels of the different metabolites, differed in the 71B6-OEs whether the plants were grown under standard conditions or subjected to wilt stress. The impact of increased glucosylation of ABA on ABA-related phenotypes has also been assessed. Increased glucosylation of ABA led to phenotypic changes in post-germinative growth. The use of two structural analogues of ABA, known to have biological activity but to differ in their capacity to act as substrates for 71B6 in vitro, confirmed that the phenotypic changes arose specifically from the increased glucosylation caused by overexpression of 71B6. The phenotype and profile of ABA and related metabolites in a knockout line of 71B6, relative to wild type, has been assessed during Arabidopsis development and following stress treatments. The lack of major changes in these parameters is discussed in the context of functional redundancy of the multigene family of GTs in Arabidopsis.

SummaryChanges in gene expression produced by the application of (+)-abscisic acid (ABA) to Arabidopsis thaliana plants were compared with changes produced by the ABA structural analogs ())-ABA, (+)-8¢-acetylene ABA and ())-2¢,3¢-dihydroacetylenic abscisyl alcohol. The maximum expression of many rapidly (+)-ABA-induced genes occurred prior to peak hormone accumulation, suggesting negative feedback regulation that may be mediated by the induction of genes encoding PP2C-type protein phosphatases. For most rapidly (+)-ABA-induced genes, expression was delayed in ABA analog treatments although analogs accumulated to higher levels than did (+)-ABA. For each analog, some genes exhibited a hypersensitive response to the analog and some genes were less sensitive to the analog than to (+)-ABA. Variations in the sensitivity of gene expression to (+)-ABA and analogs reflect the different structural requirements of two or more classes of hormone receptors. By using ABA analogs to reveal and confirm weakly (+)-ABA-regulated genes, we estimate that 14% of Arabidopsis genes are ABA-regulated in aerial tissues. Treatments with the analog (+)-8¢-acetylene ABA (PBI425) led to the identification of new ABA-regulated genes. As an example, the transcription factor MYBR1 was significantly induced by PBI425, but not by (+)-ABA, and is shown to play a role in ABA signaling by phenotypic analysis of gain-of-function and loss-of-function mutants.