Publications - Cell and Metabolic Biology

Colonisation of maize roots by arbuscular mycorrhizal (AM) fungi leads to the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives). Other root apocarotenoids (strigolactones) are involved in signalling during early steps of the AM symbiosis but also in stimulation of germination of parasitic plant seeds. Both apocarotenoid classes are predicted to originate from cleavage of a carotenoid substrate by a carotenoid cleavage dioxygenase (CCD), but the precursors and cleavage enzymes are unknown. A Zea mays CCD (ZmCCD1) was cloned by RT-PCR and characterised by expression in carotenoid accumulating E. coli strains and analysis of cleavage products using GC–MS. ZmCCD1 efficiently cleaves carotenoids at the 9, 10 position and displays 78% amino acid identity to Arabidopsis thaliana CCD1 having similar properties. ZmCCD1 transcript levels were shown to be elevated upon root colonisation by AM fungi. Mycorrhization led to a decrease in seed germination of the parasitic plant Striga hermonthica as examined in a bioassay. ZmCCD1 is proposed to be involved in cyclohexenone and mycorradicin formation in mycorrhizal maize roots but not in strigolactone formation.

Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N ‐methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli , and shown to encode S‐ adenosyl‐l ‐methionine‐dependent anthranilate N ‐methyltransferase. SDS–PAGE of the purified enzyme revealed a mass of 40 ± 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O ‐methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity (∼20%) to N ‐methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O ‐methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3‐ and 4‐aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N ‐methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT‐PCR amplifications and Western blotting revealed expression of the N ‐methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.

During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C14 mycorradicin and C13 cyclohexenone derivatives, are predicted to originate from a common C40 carotenoid precursor. Mycorradicin is the chromophore of the “yellow pigment” responsible for the long-known yellow discoloration of colonized roots. The biosynthesis of apocarotenoids has been investigated with a focus on the two first steps of the methylerythritol phosphate (MEP) pathway catalyzed by 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR). In Medicago truncatula and other plants the DXS2 isogene appears to be specifically involved in the AM-mediated accumulation of apocarotenoids, whereas in the case of DXR a single gene contributes to both housekeeping and mycorrhizal (apo)carotenoid biosynthesis. Immunolocalization of DXR in mycorrhizal maize roots indicated an arbuscule-associated protein deposition, which occurs late in arbuscule development and accompanies arbuscule degeneration and breakdown. The DXS2 isogene is being developed as a tool to knock-down apocarotenoid biosynthesis in mycorrhizal roots by an RNAi strategy. Preliminary results from this approach provide starting points to suggest a new kind of function for apocarotenoids in mycorrhizal roots.

In livingstone daisy (Dorotheanthus bellidiformis ), betanidin 5‐O‐glucosyltransferase (UGT73A5) is involved in the regiospecific glucosylation of betanidin and various flavonols. Based on sequence alignments several amino acid candidates which might be essential for catalysis were identified. The selected amino acids of the functionally expressed protein, suggested to be involved in substrate binding and turnover, were substituted via site‐directed mutagenesis. The substitution of two highly conserved amino acids, Glu378, located in the proposed UDP‐glucose binding site, and His22, located close to the N‐terminus, led to the complete loss of enzyme activity. A 3D model of this regiospecific betanidin and flavonoid glucosyltransferase was constructed and the active site modelled. This model was based on the crystallographic structure of a bacterial UDP‐glucose‐dependent glucosyltransferase from Amycolatopsis orientalis used as a template and the generated null mutations. To explain the observed inversion in the configuration of the bound sugar, semiempirical calculations favour an SN‐1 reaction, as one plausible alternative to the generally proposed SN‐2 mechanism discussed for plant natural product glucosyltransferases. The calculated structural data do not only explain the abstraction of a proton from the acceptor betanidin, but further imply that the reaction mechanism might also involve a catalytic triad, with similarities described for the serine protease family.

Colonization of plant roots by symbiotic arbuscular mycorrhizal fungi frequently leads to the accumulation of several apocarotenoids. The corresponding carotenoid precursors originate from the plastidial 2-C-methyl-d-erythritol 4-phosphate pathway. We have cloned and characterized 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), catalyzing the first committed step of the pathway, from maize (Zea mays). Functional identification was accomplished by heterologous expression of sequences coding for the mature protein in Escherichia coli. DXR is up-regulated in maize roots during mycorrhization as shown at transcript and protein levels, but is also abundant in leaves and young seedlings. Inspection of sequenced genomes and expressed sequence tag (EST) databases argue for a single-copy DXR gene. Immunolocalization studies in mycorrhizal roots using affinity-purified antibodies revealed a DXR localization in plastids around the main symbiotic structures, the arbuscules. DXR protein accumulation is tightly correlated with arbuscule development. The highest level of DXR protein is reached around maturity and initial senescence of these structures. We further demonstrate the formation of a DXR-containing plastidial network around arbuscules, which is highly interconnected in the mature, functional state of the arbuscules. Our findings imply a functional role of a still unknown nature for the apocarotenoids or their respective carotenoid precursors in the arbuscular life cycle.

Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1‐deoxy‐d ‐xylulose 5‐phosphate synthase (DXS), which was previously thought to be encoded by a single‐copy gene. We have identified two distinct classes of DXS‐like cDNAs from the model legume Medicago truncatula . The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid‐targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula , maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene‐synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.