Changes in carbon allocation and expression of carbon transporter genes in Betula pendula Roth. colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr.

Comparative analyses of aspects of the carbon (C) physiology and the expression of C transporter genes in birch (Betula pendula Roth.) colonized by the ectomycorrhizal fungus Paxillus involutus (Batsch) Fr. were performed using mycorrhizal (M) and non‐mycorrhizal (NM) plants of similar foliar nutrient status. After six months of growth, the biomass of M plants was significantly lower than that of NM plants. Diurnal C budgets of both sets of plants revealed that M plants exhibited higher rates of photosynthesis and root respiration expressed per unit dry weight. However, the diurnal net C gain of M and NM plants remained similar. Ectomycorrhizal roots contained higher soluble carbohydrate pools and increased activity of cell wall invertase, suggesting that additional C was allocated to these roots and their ectomycorrhizal fungi consistent with an increased sink demand for C due to the presence of the mycobiont. In M roots, the expression of two hexose and one sucrose transporter genes of birch were reduced to less than one‐third of the expression level observed in NM roots. Analysis using a probe against the birch ribosomal internal transcribed spacer region revealed that M roots contained 22% less plant RNA than NM roots. As the expression of birch hexose and sucrose transporter genes was reduced to a much greater extent, this suggests that these specific genes were down‐regulated in response to alterations in C metabolism within M roots.     

Uptake and transfer of nutrients in ectomycorrhizal associations: interactions between photosynthesis and phosphate nutrition

Energy-dispersive X-ray microanalytical investigations and microautoradiographic studies were carried out to examine whether the uptake and transfer of phosphate (P) by an ectomycorrhizal fungus is affected by the carbohydrate supply of its host plant. For this purpose, non-mycorrhizal seedlings of Pinus sylvestris L. and plants inoculated with the ectomycorrhizal basidiomycete Suillus bovinus (L. ex Fr.) Kuntze were placed in the dark for 7 days in advance of a P supply. The subcellular element distribution and the uptake and distribution of (33)P was analyzed in non-mycorrhizal and mycorrhizal roots of these plants and compared with plants kept constantly under normal light conditions (control plants). The results show that placing non-mycorrhizal plants in the dark in advance of the nutrient supply led to (1) a reduction of the subcellular contents of P, S and K, but to an increase in the cytoplasmic Na content, and (2) an increase of (33)P absorption and translocation to the shoot. It can be assumed that this increased inflow of (33)P in non-mycorrhizal plants was due to P starvation after suppressed photosynthesis and reduced respiration of these plants. The suppression of photosynthesis by an ectomycorrhizal host plant and the resulting lower carbohydrate supply conditions for the ectomycorrhizal fungus led to (1) a decrease of P absorption by the mycobiont, (2) a change of the P allocation in the fungal cell compartments of an ectomycorrhizal root, and (3) a reduction of P transfer to the host plant. However, microautoradiographic studies revealed that, under these conditions, P was also absorbed by the mycorrhizal fungus and translocated via the Hartig net to the host plant. In mycorrhizal roots of plants placed in the dark in advance of the nutrient supply, the cytoplasmic P content of the Hartig net was reduced and, instead, a high number of polyphosphate granules could be detected within the hyphae. The results indicate that the exchange processes between the symbionts in a mycorrhiza are possibly linked and that P uptake and translocation by an ectomycorrhizal fungus is also regulated by the carbohydrate supply of its host plant.     

Root colonization by Piriformospora indica enhances grain yield in barley under diverse nutrient regimes by accelerating plant development

The basidiomycete fungus Piriformospora indica colonizes roots of a broad range of mono- and dicotyledonous plants. It confers enhanced growth, improves resistance against biotic and tolerance to abiotic stress, and enhances grain yield in barley. To analyze mechanisms underlying P. indica-induced improved grain yield in a crop plant, the influence of different soil nutrient levels and enhanced biotic stress were tested under outdoor conditions. Higher grain yield was induced by the fungus independent of different phosphate and nitrogen fertilization levels. In plants challenged with the root rot-causing fungus Fusarium graminearum, P. indica was able to induce a similar magnitude of yield increase as in unchallenged plants. In contrast to the arbuscular mycorrhiza fungus Glomus mosseae, total phosphate contents of host plant roots and shoots were not significantly affected by P. indica. On the other hand, barley plants colonised with the endophyte developed faster, and were characterized by a higher photosynthetic activity at low light intensities. Together with the increased root formation early in development these factors contribute to faster development of ears as well as the production of more tillers per plant. The results indicate that the positive effect of P. indica on grain yield is due to accelerated growth of barley plants early in development, while improved phosphate supply—a central mechanism of host plant fortification by arbuscular mycorrhizal fungi—was not observed in the P. indica-barley symbiosis.     

A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment

The majority of vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. These symbioses, termed arbuscular mycorrhizas, are mutually beneficial, and the fungus delivers phosphate to the plant while receiving carbon. In these symbioses, phosphate uptake by the arbuscular mycorrhizal fungus is the first step in the process of phosphate transport to the plant. Previously, we cloned a phosphate transporter gene involved in this process. Here, we analyze the expression and regulation of a phosphate transporter gene (GiPT) in the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices during mycorrhizal association with carrot or Medicago truncatula roots. These analyses reveal that GiPT expression is regulated in response to phosphate concentrations in the environment surrounding the extra-radical hyphae and modulated by the overall phosphate status of the mycorrhiza. Phosphate concentrations, typical of those found in the soil solution, result in expression of GiPT. These data imply that G. intraradices can perceive phosphate levels in the external environment but also suggest the presence of an internal phosphate sensing mechanism.     

Carbon Cost of the Fungal Symbiont Relative to Net Leaf P Accumulation in a Split-Root VA Mycorrhizal Symbiosis

Translocation of ¹⁴C-photosynthates to mycorrhizal (+ +), half mycorrhizal (0+), and nonmycorrhizal (00) split-root systems was compared to P accumulation in leaves of the host plant. Carrizo citrange seedlings (Poncirus trifoliata [L.] Raf. × Citrus sinensis [L.] Osbeck) were inoculated with the vesicular-arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith. Plants were exposed to ¹⁴ CO₂ for 10 minutes and ambient air for 2 hours. Three to 4% of recently labeled photosynthate was allocated to metabolism of the mycorrhiza in each inoculated root half independent of shoot P concentration, growth response, and whether one or both root halves were colonized. Nonmycorrhizal roots respired more of the label translocated to them than did mycorrhizal roots. Label recovered in the potting medium due to exudation or transport into extraradical hyphae was 5 to 6 times greater for (+ +) versus (00) plants. In low nutrient media, roots of (0+) and (+ +) plants transported more P to leaves per root weight than roots of (00) plants. However, when C translocated to roots utilized for respiration, exudation, etc., as well as growth is considered, (00) plant roots were at least as efficient at P uptake (benefit) per C utilized (cost) as (0+) and (+ +) plants. Root systems of (+ +) plants did not supply more P to leaves than (0+) plants in higher nutrient media, yet they still allocated twice the ¹⁴C-photosynthate to the mycorrhiza as did (0+) root systems. This indicates there is an optimal level of mycorrhizal colonization above which the plant receives no enhanced P uptake yet continues to partition photosynthates to metabolism of the mycorrhiza.     

Gene expression in mycorrhizal orchid protocorms suggests a friendly plant–fungus relationship

Main conclusion

Orchid mycorrhiza has been often interpreted as an antagonistic relationship. Our data on mycorrhizal protocorms do not support this view as plant defence genes were not induced, whereas some nodulin-like genes were significantly up-regulated. Orchids fully depend on symbiotic interactions with specific soil fungi for seed germination and early development. Germinated seeds give rise to a protocorm, a heterotrophic organ that acquires nutrients, including organic carbon, from the mycorrhizal partner. It has long been debated if this interaction is mutualistic or antagonistic. To investigate the molecular bases of the orchid response to mycorrhizal invasion, we developed a symbiotic in vitro system between Serapias vomeracea, a Mediterranean green meadow orchid, and the rhizoctonia-like fungus Tulasnella calospora. 454 pyrosequencing was used to generate an inventory of plant and fungal genes expressed in mycorrhizal protocorms, and plant genes could be reliably identified with a customized bioinformatic pipeline. A small panel of plant genes was selected and expression was assessed by real-time quantitative PCR in mycorrhizal and non-mycorrhizal protocorm tissues. Among these genes were some markers of mutualistic (e.g. nodulins) as well as antagonistic (e.g. pathogenesis-related and wound/stress-induced) genes. None of the pathogenesis or wound/stress-related genes were significantly up-regulated in mycorrhizal tissues, suggesting that fungal colonization does not trigger strong plant defence responses. In addition, the highest expression fold change in mycorrhizal tissues was found for a nodulin-like gene similar to the plastocyanin domain-containing ENOD55. Another nodulin-like gene significantly more expressed in the symbiotic tissues of mycorrhizal protocorms was similar to a sugar transporter of the SWEET family. Two genes coding for mannose-binding lectins were significantly up-regulated in the presence of the mycorrhizal fungus, but their role in the symbiosis is unclear.     

European and African maize cultivars differ in their physiological and molecular responses to mycorrhizal infection

Physiological and molecular responses to phosphorus (P) supply and mycorrhizal infection by Glomus intraradices were compared in European (River) and African (H511) maize (Zea mays) cultivars to examine the extent to which these responses differed between plants developed for use in high- and low-nutrient-input agricultural systems. Biomass, photosynthetic rates, nutrient and carbohydrate contents, mycorrhizal colonization and nutrient-responsive phosphate transporter gene expression were measured in nonmycorrhizal and mycorrhizal plants grown at different inorganic phosphorus (P(i)) supply rates. Nonmycorrhizal River plants grew poorly at low P(i) but were highly responsive to mycorrhizal infection; there were large increases in biomass, tissue P content and the rate of photosynthesis and a decline in the expression of phosphate transporter genes. Nonmycorrhizal H511 plants grew better than River plants at low P(i), and had a higher root : shoot ratio. However, the responses of H511 plants to higher P(i) supplies and mycorrhizal infection were much more limited than those of River plants. The adaptations that allowed nonmycorrhizal H511 plants to perform well in low-P soils limited their ability to respond to higher nutrient supply rates and mycorrhizal infection. The European variety had not lost the ability to respond to mycorrhizas and may have traits useful for low-nutrient agriculture where mycorrhizal symbioses are established.     

Phosphate uptake, transport and transfer by the arbuscular mycorrhizal fungus Glomus intraradices is stimulated by increased carbohydrate availability

* The influence of carbohydrate availability to mycorrhizal roots on uptake, metabolism and translocation of phosphate (P) by the fungus was examined in axenic cultures of transformed carrot (Daucus carota) roots in symbiosis with Glomus intraradices. * 14C-labelled carbohydrates, 33P-phosphate and energy dispersive X-ray microanalysis were used to follow the uptake and transfer of C and P in the arbuscular mycorrhizal (AM) symbiosis. * The uptake of P by the extraradical mycelium (ERM) and its translocation to the mycorrhizal roots was stimulated and the metabolic and spatial distribution of P within the fungus were altered in response to increased carbohydrate availability. Sucrose supply resulted in a decrease of polyphosphates and an increased incorporation into phospholipids and other growth-related P pools and also caused elevated cytoplasmic P levels in the intraradical mycelium (IRM) within the root and higher cytoplasmic P levels in the root cortex. * These findings indicate that the uptake of P by the fungus and its transfer to the host is also stimulated by the transfer of carbon from plant to fungus across the mycorrhizal interface.     

Photochemical processes, carbon assimilation and RNA accumulation of sucrose transporter genes in tomato arbuscular mycorrhiza

Arbuscular mycorrhizal fungi enhance CO₂ assimilation of their hosts which ensure the demand for carbohydrates of these obligate biotrophic microorganisms. Photosynthetic parameters were measured in tomato colonised or not by the arbuscular mycorrhizal fungus Glomus mosseae. In addition, carbohydrate contents and mRNA accumulation of three sucrose transporter genes were analysed. Mycorrhizal plants showed increased opening of stomata and assimilated significant more CO₂. A higher proportion of the absorbed light was used for photochemical processes, while non-photochemical quenching and the content of photoprotective pigments were lower. Analysis of sugar contents showed no significant differences in leaves but enhanced levels of sucrose and fructose in roots, while glucose amounts stayed constant. The three sucrose transporter encoding genes of tomato SlSUT1, SlSUT2 and SlSUT4 were up-regulated providing transport capacities to transfer sucrose into the roots. It is proposed that a significant proportion of sugars is used by the mycorrhizal fungus, because only amounts of fructose were increased, while levels of glucose, which is mainly transferred towards the fungus, were nearly constant.     

Conservation and divergence of both phosphate- and mycorrhiza-regulated physiological responses and expression patterns of phosphate transporters in solanaceous species

Here, orthologous genes of six phosphate transporter (PiT) genes, which are members of the Pht1 and Pht2 families in tomato and potato, have been cloned from the solanaceous species pepper, eggplant and tobacco. Overall, expressions of these genes in pepper, eggplant and tobacco showed similar patterns to those in tomato and potato: P-starvation enhancement in both leaves and roots for Pht1;1, P-depletion induction exclusively in roots for Pht1;2, mycorrhizal enhancement for Pht1;3, and mycorrhizal induction for both Pht1;4 and Pht1;5. In the roots of nonmycorrhizal eggplant, SmPht1;3, SmPht1;4 and SmPht1;5 were also expressed under extreme P starvation. Mycorrhizal symbiosis under high-P supply conditions reduced plant growth, with concurrent enhancement of Pht1;2 expression in the roots of pepper as well as eggplant. In addition, the mycorrhizal symbiosis down-regulated the expression of Pht2;1 genes greatly in the leaves of pepper and tobacco. The discrepancies between the evolutionary distances of the PiT genes and their expression patterns among the five species suggest greater complexity in function of PiT in plants than previously expected.     

Polyamines in humic acid and their effect on radical growth of lettuce seedlings

Linum usitatissimum, Sorghum bicolor and Triticum aestivum plants were further colonised by the arbuscular mycorrhizal fungus, Glomus mosseae, during a four week period of hydroponic culture after a pre-culture period of three weeks with the fungus in perlite substrate. The viability of mycorrhizal colonisation of T. aestivum was indicated by an initial experiment where G. mosseae from mycorrhizal plants colonised non-mycorrhizal plants when the plants were grown together in the same hydroponic container using modified Long Ashton nutrient solution. Intermittant aeration of the plant roots (2 h periods, four times per day) provided a compromise between adequate aeration and minimal disturbance of the fungus. In a second experiment, two nutrient media, modified Long Ashton and modified Knop plus Hoagland medium were compared for culturing G. mosseae on T. aestivum. A significantly higher root dry weight was found for the mycorrhizal versus the non-mycorrhizal wheat plants in modified Long Ashton nutrient medium, which contained 10 µM P and an organic buffer. Modified Knop plus Hoagland nutrient medium contained a high P concentration (0.9 mM) and did not produce viable cultures of mycorrhizal colonisation. In a third experiment, modified Long Ashton medium was used for hydroponic culture of mycorrhizal L. usitatissimum, S. bicolor and T. aestivum. The root colonisation percentages for T. aestivum (73%), S. bicolor (36%) and L. usitatissimum (65%) were within the range of colonisation rates obtained with solid substrate culture in perlite. Viability of the mycorrhizal structures in hydroponic culture was assessed by monitoring activity of fungal succinate dehydrogenase and found to be similar to cultures in perlite. No difference in the P concentration of mycorrhizal and non-mycorrhizal plants was observed, possibly owing to the lack of diffusion limits for P in hydroponic solution. This report describes a system for the viable culture of G. mosseae with different plant species where a high mycorrhizal colonisation rate was produced under conditions of a short culture period using intermittent aeration, a low concentration of P supply and an organic buffer.     

Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g^?1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD-malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non-mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea-Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non-VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.     

Cloning of a Picea abies monosaccharide transporter gene and expression – analysis in plant tissues and ectomycorrhizas

Ectomycorrhizas are formed between certain soil fungi and fine roots of woody plants. An important feature of this symbiosis is the supply of photoassimilates to the fungus. Hexoses, formed from sucrose in the common apoplast at the root/fungus interface, can be taken up by both plant and fungal monosaccharide transporters. Recently we characterised a monosaccharide transporter from the ectomycorrhizal fungus Amanita muscaria. This transporter was up-regulated in mycorrhizas, thus increasing the hexose uptake capacity of the fungal partner in symbiosis. In order to characterise host (Picea abies) root monosaccharide transporters, degenerate oligonucleotide primers, designed to match conserved regions from known plant hexose transporters, were used to isolate a cDNA fragment of a transporter by PCR. This fragment was used to identify a presumably full length clone (PaMST1) in a P. abies/A. muscaria mycorrhizal cDNA library. The entire cDNA code for an open reading frame of 513 amino acids, revealing best homology to H⁺/monosaccharide transporters from Ara- bidopsis, Saccharum and Ricinus. PaMST1 was highly expressed in the hypocotyl and in roots of P. abies seedlings, but not in needles. Mycorrhiza formation led to a slight reduction of PaMST1 expression. The results are discussed with special reference to carbon allocation in ectomycorrhizas. Received: 9 October 1999 / Accepted: 22 December 1999