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.     

Occurrence and localization of apocarotenoids in arbuscular mycorrhizal plant roots

The core structure of the yellow pigment from arbuscular mycorrhizal (AM) maize roots contains the apocarotenoids mycorradicin (an acyclic C14 polyene) and blumenol C cellobioside (a C13 cyclohexenone diglucoside). The pigment seems to be a mixture of different esterification products of these apocarotenoids. It is insoluble in water and accumulates as hydrophobic droplets in the vacuoles of root cortical cells. Screening 58 species from 36 different plant families, we detected mycorradicin in mycorrhizal roots of all Liliopsida analyzed and of a considerable number of Rosopsida, but also species were found in which mycorradicin was undetectable in mycorrhizal roots. Kinetic experiments and microscopic analyses indicate that accumulation of the yellow pigment is correlated with the concomitant degradation of arbuscules and the extensive plastid network covering these haustorium-like fungal structures. The role of the apocarotenoids in mycorrhizal roots is still unknown. The potential C40 carotenoid precursors, however, are more likely to be of functional importance in the development and functioning of arbuscules.     

"Chromoplast" development in arbuscular mycorrhizal roots

The accumulation of apocarotenoids in arbuscular mycorrhizal (AM) roots suggests a dramatic reorganization of the plastids responsible for the biosynthesis of these compounds. This review describes the cytological and biochemical characterization of this phenomenon. The results presented suggest that plastids are key organelles for the establishment of the symbiotic interface of the AM symbiosis. In addition, a complex interplay of various plant cell components during the different functional phases of this interface is suggested. Arbuscule degradation appears to be of particular interest, as it correlates with the formation of the most extensive plastid structures and with apocarotenoid accumulation.     

Localization of the yellow pigment formed in roots of gramineous plants colonized by arbuscular fungi

Summary Many plants form yellow coloured roots when colonized by arbuscular mycorrhizal (AM) fungi. In maize, a yellow pigment is first visible as small droplets in parenchyma cells of roots in the vicinity of arbuscules, 3–4 weeks after mycorrhizal colonization. During the course of the development of the plants, the yellow pigment spreads all over the cells of the cortex (with the exception of the exodermis) and of the endodermis, whereas the other stelar elements remain uncoloured. Other gramineous plants (wheat, barley, millet) show the same pattern of pigment formation. In contrast, the deposition of this pigment is not detected in roots ofTagetes, garden bean, onion, or leek. Weak yellow fluorescence is also seen in the fungal structures, particularly in the arbuscules of the investigated probes. This is, however, clearly different from the intense yellow colour of the pigment formed in root cells of grasses. The yellow pigment is even detected in such cells which are never colonized by fungal structures (e.g., endodermal cells). A major constituent of the yellow pigment of AM-colonized root cells has been identified as a carotenoid with 14 carbon atoms and two carboxylic groups and termed mycorradicin. This carotenoid is likely deposited in the vacuoles of root cells as a result of the colonization specifically by arbuscular fungi.     

RNA interference-mediated repression of MtCCD1 in mycorrhizal roots of Medicago truncatula causes accumulation of C27 apocarotenoids, shedding light on the functional role of CCD1

Tailoring carotenoids by plant carotenoid cleavage dioxygenases (CCDs) generates various bioactive apocarotenoids. Recombinant CCD1 has been shown to catalyze symmetrical cleavage of C₄₀ carotenoid substrates at 9,10 and 9′,10′ positions. The actual substrate(s) of the enzyme in planta, however, is still unknown. In this study, we have carried out RNA interference (RNAi)-mediated repression of a Medicago truncatula CCD1 gene in hairy roots colonized by the arbuscular mycorrhizal (AM) fungus Glomus intraradices. As a consequence, the normal AM-mediated accumulation of apocarotenoids (C₁₃ cyclohexenone and C₁₄ mycorradicin derivatives) was differentially modified. Mycorradicin derivatives were strongly reduced to 3% to 6% of the controls, while the cyclohexenone derivatives were only reduced to 30% to 47%. Concomitantly, a yellow-orange color appeared in RNAi roots. Based on ultraviolet light spectra and mass spectrometry analyses, the new compounds are C₂₇ apocarotenoic acid derivatives. These metabolic alterations did not lead to major changes in molecular markers of the AM symbiosis, although a moderate shift to more degenerating arbuscules was observed in RNAi roots. The unexpected outcome of the RNAi approach suggests C₂₇ apocarotenoids as the major substrates of CCD1 in mycorrhizal root cells. Moreover, literature data implicate C₂₇ apocarotenoid cleavage as the general functional role of CCD1 in planta. A revised scheme of plant carotenoid cleavage in two consecutive steps is proposed, in which CCD1 catalyzes only the second step in the cytosol (C₂₇ → C₁₄ + C₁₃), while the first step (C₄₀ → C₂₇ + C₁₃) may be catalyzed by CCD7 and/or CCD4 inside plastids.     

A purple acid phosphatase,GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter‐β‐glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule‐containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild‐type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.     

Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

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.     

Comparative differential RNA display analysis of arbuscular mycorrhiza in Pisum sativum wild type and a mutant defective in late stage development

In order to analyse gene expression associated with the late stages of arbuscular mycorrhizal development between Pisum sativum and Glomus mosseae, comparative differential RNA display was carried out using wild-type P. sativum and a mutant, RisNod24, where the fungal partner is not able to form functional arbuscules. Comparison of RNA accumulation patterns between controls, G. mosseae-colonized mutant and wild-type roots resulted in the identification of four differentially occurring cDNA fragments. One of the corresponding genes was from the fungus and three of plant origin. One plant gene, Psam4 (P. sativum arbuscular mycorrhiza-regulated), was analysed in more detail. Sequencing of a cDNA clone showed that Psam4 encodes a proline-rich protein. Northern blot analysis and quantitative RT-PCR revealed a higher basal level of Psam4 RNA accumulation in the mutant compared to the wild type. In both pea genotypes, RNA accumulation was reduced after inoculation with mycorrhiza- or nodule-forming symbiotic microorganisms, but enhanced after infection with a root pathogenic fungus.     

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。