Apocarotenoid biosynthesis in arbuscular mycorrhizal roots: contributions from methylerythritol phosphate pathway isogenes and tools for its manipulation

During colonization by arbuscular mycorrhizal (AM) fungi plant roots frequently accumulate two types of apocarotenoids (carotenoid cleavage products). Both compounds, C(14) mycorradicin and C(13) cyclohexenone derivatives, are predicted to originate from a common C(40) 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.     

Expression of a xyloglucan endotransglucosylase/hydrolase gene, Mt-XTH1, from Medicago truncatula is induced systemically in mycorrhizal roots

Xyloglucan endotransglucosylase/hydrolases (XTH) are enzymes that catalyze the hydrolysis and transglycosylation of xyloglucan polymers in plant cell walls. Previously, we isolated a cDNA from mycorrhizal roots of Medicago truncatula that is predicted to encode an XTH [van Buuren, M.L., Maldonado-Mendoza, I.E., Trieu, A.T., Blaylock, L.A., Harrison, M.J., 1999. Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme. Mol. Plant-Microb. Interact. 12, 171-181.]. Here, we identified the corresponding XTH gene, designated Mt-XTH1. The Mt-XTH1 gene contains four exons separated by three introns and resides on a 15-kb Xba1 fragment adjacent to a second XTH gene designated Mt-XTH2. Mt-XTH2 shares the same exon-intron structure as Mt-XTH1. Exons 2, 3 and 4 and introns 1 and 2 are identical to Mt-XTH1, while exon 1 and intron 3 are divergent, both in sequence and in length. Mt-XTH1 is induced following colonization of the roots by AM fungi but does not respond to changes in phosphate status. Analysis of transgenic roots expressing an Mt-XTH1 promoterColon, two colonsuidA fusion revealed that the Mt-XTH1 promoter directs expression in cells throughout the root system with significantly higher levels of activity in mycorrhizal roots. Mt-XTH1 expression is elevated not only in the regions of the roots colonized by the fungus, but also at sites distal to the infected regions. These expression patterns are consistent with activation in response to a systemic signal.     

Metabolite profiling of mycorrhizal roots of Medicago truncatula

Metabolite profiling of soluble primary and secondary metabolites, as well as cell wall-bound phenolic compounds from roots of barrel medic (Medicago truncatula) was carried out by GC-MS, HPLC and LC-MS. These analyses revealed a number of metabolic characteristics over 56 days of symbiotic interaction with the arbuscular mycorrhizal (AM) fungus Glomus intraradices, when compared to the controls, i.e. nonmycorrhizal roots supplied with low and high amounts of phosphate. During the most active stages of overall root mycorrhization, elevated levels of certain amino acids (Glu, Asp, Asn) were observed accompanied by increases in amounts of some fatty acids (palmitic and oleic acids), indicating a mycorrhiza-specific activation of plastidial metabolism. In addition, some accumulating fungus-specific fatty acids (palmitvaccenic and vaccenic acids) were assigned that may be used as markers of fungal root colonization. Stimulation of the biosynthesis of some constitutive isoflavonoids (daidzein, ononin and malonylononin) occurred, however, only at late stages of root mycorrhization. Increase of the levels of saponins correlated AM-independently with plant growth. Only in AM roots was the accumulation of apocarotenoids (cyclohexenone and mycorradicin derivatives) observed. The structures of the unknown cyclohexenone derivatives were identified by spectroscopic methods as glucosides of blumenol C and 13-hydroxyblumenol C and their corresponding malonyl conjugates. During mycorrhization, the levels of typical cell wall-bound phenolics (e.g. 4-hydroxybenzaldehyde, vanillin, ferulic acid) did not change; however, high amounts of cell wall-bound tyrosol were exclusively detected in AM roots. Principal component analyses of nonpolar primary and secondary metabolites clearly separated AM roots from those of the controls, which was confirmed by an hierarchical cluster analysis. Circular networks of primary nonpolar metabolites showed stronger and more frequent correlations between metabolites in the mycorrhizal roots. The same trend, but to a lesser extent, was observed in nonmycorrhizal roots supplied with high amounts of phosphate. These results indicate a tighter control of primary metabolism in AM roots compared to control plants. Network correlation analyses revealed distinct clusters of amino acids and sugars/aliphatic acids with strong metabolic correlations among one another in all plants analyzed; however, mycorrhizal symbiosis reduced the cluster separation and enlarged the sugar cluster size. The amino acid clusters represent groups of metabolites with strong correlations among one another (cliques) that are differently composed in mycorrhizal and nonmycorrhizal roots. In conclusion, the present work shows for the first time that there are clear differences in development- and symbiosis-dependent primary and secondary metabolism of M. truncatula roots.     

A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.     

丛枝菌根真菌对植物次生代谢的影响

丛枝菌根(AM)是自然界中分布最为广泛、最为重要的一类菌根,许多研究已经观察到丛枝菌根真菌与植物次生代谢的相关性,丛枝菌根真菌能够直接或间接地影响植物的次生代谢过程。植物的次生代谢产物主要分为萜类物质、酚类物质和含氮化合物(主要是生物碱)三大类群,该文简要介绍了丛枝菌根真菌对这3类植物次生代谢产物的影响。丛枝菌根真菌与萜类物质代谢关系的研究比较细致和深入,有些工作已经从细胞及分子水平探讨其间的作用机制,如Blumenin、类胡萝卜素等。丛枝菌根真菌与酚类物质代谢关系的研究也比较深入,其中具有特殊功能的酚类物质——植保素、细胞壁酚酸、类黄酮/异类黄酮等倍受关注。目前有关丛枝菌根真菌与生物碱关系的研究相对较少,不过现有的研究表明,菌根的形成有助于生物碱积累。     

Suppression of allene oxide cyclase in hairy roots of Medicago truncatula reduces jasmonate levels and the degree of mycorrhization with Glomus intraradices

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35SuidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.