SummaryIn higher plants, isopentenyl diphosphate (IPP) is synthesised both from the plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) and from the cytosolic mevalonate (MVA) pathways. Primary metabolites, such as phytol group of chlorophylls, carotenoids and the plant hormones abscisic acid (ABA) and gibberellins (GAs) are derived directly from the MEP pathway. Many secondary metabolites, such as monoterpene indole alkaloids (MIAs) in Catharanthus roseus, are also synthesised from this source of IPP. Using Northern blot and in situ hybridisation experiments, we show that three MEP pathway genes (1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR ) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MECS)) and the gene encoding geraniol 10-hydroxylase (G10H ), a cytochrome P450 monooxygenase involved in the ®rst committed step in the formation of iridoid monoterpenoids display identical cell-speci®c expression patterns. The co-localisation of these four transcripts to internal phloem parenchyma of young aerial organs of C. roseus adds a new level of complexity to the multicellular nature of MIA biosynthesis. We predict the translocation of pathway intermediates from the internal phloem parenchyma to the epidermis and, ultimately, to laticifers and idioblasts during MIA biosynthesis. Similarly, the translocation of intermediates from the phloem parenchyma is probably also required during the biosynthesis of hormones and photosynthetic primary metabolites derived from the MEP pathway.
The Apocynaceae Catharanthus roseus accumulates a number of monoterpene indole alkaloids (MIAs) that originate from the coupling of the indole and the iridoid pathways. The latter pathway is usually considered as limiting for MIA biosynthesis, but evidence is now strong that the precursors tryptamine (from the indole pathway) and secologanin (from the iridoid pathway) have to be provided within the cells in a concerted manner for sustained MIA synthesis. Secologanin is formed from isopentenyl diphosphate (IPP) in a number of steps, some of which are still unknown. However significant progress has been obtained recently with the characterisation of cDNAs encoding secologanin synthase and the two constituents of geraniol 10-hydroxylase (G10H). IPP itself is formed through both the plastidial methyl-erythritol phosphate (MEP) pathway and the cytosolic mevalonate (MVA) pathway. The MEP pathway comprises 7 steps of which 4 have been identified at the molecular level in C. roseus. This pathway plays a major role in the production of MIAs, but there is now evidence that the MVA pathway serves as a minor source of precursors for iridoid biosynthesis and/or contributes (through protein prenylation) to a fine regulation of the MEP gene expression. G10H is one of the key enzymes of the MIA pathway and the upregulation of the gene activity concomitantly with an increase in G10H activity and MIA production have been reported with various hormones and elicitors. Since regulatory genes encoding transcription factors acting on several genes of the MEP and terpenoid pathways are beginning to be characterised, metabolic engineering of the iridoid pathway could be a promising approach to control the metabolite flux towards secologanin and MIA production through biotechnological applications in the future.Keywords Madagascar periwinkle Á Cross talk Á Methyl-erythritol phosphate pathway Á Mevalonate pathway Á Secologanin-derived monoterpene indole alkaloids Á Terpenoid pathway Abbreviations AACT Acetoacetyl-CoA thiolase ADH (monoterpene primary) Alcohol dehydrogenase CDP-ME 4-Diphosphocytidyl-2-C-methyl-D-erythritol CMK 4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase A. Oudin (&) Á M. Courtois Á M. Rideau Á M. Clastre EA 2106,
The effects of hypoxia and reoxygenation on action potentials (AP), contractions, and certain biochemical parameters were studied in isolated rat ventricular myocytes in monolayer culture in the presence and absence of glucose. Substrate deprivation alone had no influence on the basal properties. In the presence of glucose, a 4-h hypoxic treatment caused only a moderate decrease in AP amplitude and rate. In substrate-free conditions, hypoxia induced a gradual decline in plateau potential level and in AP duration and rate, followed by rhythm abnormalities and a failure of the electromechanical coupling. Spontaneous AP generation then ceased, and the resting potential decreased with increased duration of hypoxia. These alterations were associated with a decrease in ATP content, an increase in the lactate production, and a leakage of about 50% of the total cellular lactate dehydrogenase (LDH). Cells reoxygenated after 150 min hypoxia recovered near-normal function, while the ATP depletion ceased and the rate of lactate and LDH loss was diminished. Conversely, cells reoxygenated after 4 h hypoxia exhibited a further decrease of the residual resting polarization and no change in the decline of intracellular ATP and in the efflux of cytosolic lactate and LDH. The results of this study indicate that (1) the sequence and the extent of functional alterations are dependent on the duration of hypoxia in the absence of exogenous substrate and (2) ATP depletion and the amount of lactate and LDH released during hypoxia are related to the shift from reversibly to irreversibly damaged cells.
The tagging-via-substrate approach designed for the capture of mammal prenylated proteins was adapted to Arabidopsis cell culture. In this way, proteins are in vivo tagged with an azide-modified farnesyl moiety and captured thanks to biotin alkyne Click-iT® chemistry with further streptavidin-affinity chromatography. Mass spectrometry analyses identified four small GTPases and ASG2 (ALTERED SEED GERMINATION 2), a protein previously associated to the seed germination gene network. ASG2 is a conserved protein in plants and displays a unique feature that associates WD40 domains and tetratricopeptide repeats. Additionally, we show that ASG2 has a C-terminal CaaX-box that is farnesylated in vitro. Protoplast transfections using CaaX prenyltransferase mutants show that farnesylation provokes ASG2 nucleus exclusion. Moreover, ASG2 interacts with DDB1 (DAMAGE DNA BINDING protein 1), and the subcellular localization of this complex depends on ASG2 farnesylation status. Finally, germination and root elongation experiments reveal that asg2 and the farnesyltransferase mutant era1 (ENHANCED RESPONSE TO ABSCISIC ACID (ABA) 1) behave in similar manners when exposed to ABA or salt stress. To our knowledge, ASG2 is the first farnesylated DWD (DDB1 binding WD40) protein related to ABA response in Arabidopsis that may be linked to era1 phenotypes.
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