Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic c... more Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B, were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.
The Bradford-reactive soil protein (BRSP) fraction includes glomalin, a glycoprotein produced by ... more The Bradford-reactive soil protein (BRSP) fraction includes glomalin, a glycoprotein produced by arbuscular mycorrhizal (AM) fungi able to bind some metals, such as copper (Cu), which could promote the bioremediation of Cu-polluted soils. This study aimed to analyze the Cu-binding capacity of BRSP in Oenothera picensis that was inoculated or not inoculated with AM fungi. O. picensis plants were established in a Cu contaminated sterilized soil and treated with the following: i) uninoculated (-M); ii) inoculated with native AM fungal propagules (+M); or iii) inoculated with a Claroideoglomus claroideum (CC) strain isolated from non-contaminated soil. In each case, five Cu levels were applied to the soil (basal level 497.3 mg Cu kg-1): 0 (T1); 75 (T2); 150 (T3); 225 (T4); and 300 mg Cu kg-1 (T5). A high BRSP accumulation in AM inoculated treatments, especially with CC, was observed. A higher Cu-bound-to-BRSP content was found with increasing Cu concentrations, representing up to 20-22% of the total Cu in the soil. Moreover, a higher root Cu concentration in +M was observed. These results suggest a high Cu binding capacity by BRSP, which is a relevant aspect to consider in the design of bioremediation programs together with the selection of endemic metallophytes and AM fungal strains, which are able to produce glomalin at high quantities.
Arbuscular mycorrhizal symbioses that involve most plants and Glomeromycota fungi are integral an... more Arbuscular mycorrhizal symbioses that involve most plants and Glomeromycota fungi are integral and functional parts of plant roots. In these associations, the fungi not only colonize the root cortex but also maintain an extensive network of hyphae that extend out of the root into the surrounding environment. These external hyphae contribute to plant uptake of low mobility nutrients, such as P, Zn, and Cu. Besides improving plant mineral nutrition, arbuscular mycorrhizal fungi (AMF) can alleviate heavy metal (HM) toxicity to their host plants. HMs, such as Cu, Zn, Fe, and Mn, play essential roles in many biological processes but are toxic when present in excess. This makes their transport and homeostatic control of particular importance to all living organisms. AMF play an important role in modulating plant HM acquisition in a wide range of soil metal concentrations and have been considered to be a key element in the improvement of micronutrient concentrations in crops and in the phy...
Many studies have established that arbuscular mycorrhizal fungi transfer N to the host plant. How... more Many studies have established that arbuscular mycorrhizal fungi transfer N to the host plant. However, the role and importance of arbuscular mycorrhiza (AM) in plant N nutrition is still uncertain, as are the C/N interactions within the symbiosis. Published reports provide differing, and often contradictory, results that are difficult to combine in a coherent framework. This review explores questions such as: What makes the difference between a positive and a negative effect of AM on plant N nutrition? Is the mycorrhizal N response (MNR) correlated to the mycorrhizal growth response (MGR), and how or under which conditions? Is the MNR effect on plant growth C mediated? Is plant C investment on fungal growth related to N needs or N benefit? How is the N for C trade between symbionts regulated? The patternless nature of current knowledge is made evident, and possible reasons for this are discussed.
The aim of this Chapter is to review and integrate current knowledge of the impact of mycorrhizal... more The aim of this Chapter is to review and integrate current knowledge of the impact of mycorrhizal symbioses on plant functioning and adaptation with specific emphasis on P acquisition at various levels of cellular organization, from the molecular, biochemical and physiological to the whole plant. Accordingly, the available information will be structured as follows: (i) mycorrhizas as a plant strategy
The plant plasma membrane ATPase has been described as a key regulatory enzyme in cellular : even... more The plant plasma membrane ATPase has been described as a key regulatory enzyme in cellular : events such as adaptation to abiotic stresses and development changes [1,2]; however, the mechanism by which this activity is regulated is still uncertain. The modulation by lipids of ATPases of many sources is a recognized phenomenon which has been discussed [3]. This lipid dependency suggested a possible mode of regulation of plasma membrane H+-ATPase activity in vivo via the modification of its lipid environment [4] . The modulation by lipids may occur via a direct interaction with the enzyme (the annulus hypothesis) or through changes in membrane fluidity, which causes an alteration in the enzyme conformation, and thus in activity.
Abstract Arbuscular mycorrhizal fungi are soilborne microorganisms that form a mutualistic symbio... more Abstract Arbuscular mycorrhizal fungi are soilborne microorganisms that form a mutualistic symbiotic association with most land plants. As obligate biotrophs these fungi are unable to complete their life cycle in the absence of the host plant. This symbiosis is increasingly being recognised as an integral and important part of natural ecosystems throughout the word. Because of the incalcitrance of arbuscular mycorrhizal fungi to grow in pure culture and consequently the difficulties in obtaining sufficiently large quantities of fungal material, the ...
Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic r... more Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon _CaZ+-.ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATPdependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots.
The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncerta... more The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncertain. We propose that this be clarified by using more integrative experimental designs, with the use of a gradient of N supply and the quantification of an extensive array of plant nutrient contents. Using such an experimental design, we investigated AM effects on plant N nutrition, whether the mycorrhizal N response (MNR) determines the mycorrhizal growth response (MGR), and how MNR influences plants' C economy. Oryza sativa plants were inoculated with Rhizophagus irregularis or Funneliformis mossae. AM effects were studied along a gradient of N supplies. Biomass, photosynthesis, nutrient and starch contents, mycorrhizal colonization and OsPT11 gene expression were measured. C investment in fungal growth was estimated. Results showed that, in rice, MGR was dependent on AM nutrient uptake effects, namely on the synergy between N and Zn, and not on C expenditure. The supply of C to the fungus was dependent on the plant's nutrient demand, indicated by high shoot C/N or low %N. We conclude that one of the real reasons for the negative MGR of rice, Zn deficiency of AMF plants, would have remained hidden without an experimental design allowing the observation of plants' response to AM along gradients of nutrient concentrations. Adopting more integrative and comprehensive experimental approaches in mycorrhizal studies seems therefore essential if we are to achieve a true understanding of AM function, namely of the mechanisms of C/N exchange regulation in AM.
Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots ... more Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH þ 4 is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH þ 4 transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH þ 4 supplied via the OsAMT3;1 AM-inducible transporter.
... M. Pilar Rodríguez-Rosales Corresponding Author Contact Information , E-mail The Correspondin... more ... M. Pilar Rodríguez-Rosales Corresponding Author Contact Information , E-mail The Corresponding Author , Loubna Kerkeb , Nuria Ferrol and Juan P. Douaire. ... Received 8 April 1997; accepted 19 August 1997. Available online 20 March 1999. Abstract. ...
In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots.-Phys... more In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots.-Physiol. Plant. 84: 49-54. The effect of boron excess and deficiency on H* efflux from excised roots from sunflower (Helianthus annum L. cv. Enano) seedlings and on plasma membrane H^-ATPase (EC 3.6.1.35) in isolated Kl-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H3BO3 (5 mAf) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H3BO, concentration (0.25 mM). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H+-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsome vesicles. A strong effect of boron on the vanadate-sensitive, ATPdependent H* transport was found, but the vanadate-sensitive phospho-hydrolase activity was not affected. These results suggest that boron could esert an effect on the plasma membrane properties, directly or indirectly regulating proton transport.
To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expressi... more To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expression, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) patterns of crude extracts, soluble and membrane proteins of tomato roots, either mycorrhizal and the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or nonmycorrhizal, have been compared. In the three fractions analysed, AM colonization induced up-regulation with down-regulation of the synthesis of polypeptides already present in tomato roots and induction of some new polypeptides. Separation of root extracts into soluble and membrane fractions allowed us to identify two soluble, and five membrane-bound, newly induced polypeptides in AM roots. Comparison of the protein patterns of AM roots with those of the external mycelium of G. mosseae showed that one of the newly induced polypeptides might correspond to a fungal polypeptide. By using this experimental approach, we have been able to detect 44 polypeptides that are differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis.
A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintAB... more A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two sixtransmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD) 2 topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra-vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.
A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensi... more A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensis, was isolated from soil near the roots of several endemic and endangered plant species (e.g. Plantago nivalis and Alchemilla fontqueri) growing in Sierra Nevada National Park (Granada, Andalucia, Spain). The fungus was propagated in trap cultures on Plantago nivalis and Sorbus hybrida and in pure cultures on Trifolium pratense and Sorghum vulgare. Spores are yellow brown to brown, 90-115 .m diam and form singly in soil, in the neck of adherent sporiferous saccules that form either terminally or intercalary on mycelial hyphae. Spores have two three-layered walls and conspicuous, 6-12 microm long, spiny, thorn-like projections on the outer wall consisting of hyaline to subhyaline, evanescent tips and yellow brown to brown, persistent bases. In aging spores these projections are usually shorter (1-2.8 microm) and dome-shaped or rounded, sometimes with a central pit on top where the evanescent tip has sloughed off. Molecular analysis with partial sequences of the 18S ribosomal gene places the fungus within the Diversisporales. The new fungus was found in soil near plants with different living strategies but growing in high altitude soils with acidic pH, high soil moisture and organic carbon content, and close to streams.
A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora gran... more A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 mm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 mm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 mm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.
Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic c... more Arbuscular mycorrhizal fungi increase fitness of their host plants under Cu deficient and toxic conditions. In this study, we have characterized two Cu transporters of the CTR family (RiCTR1 and RiCTR2) and a CTR-like protein (RiCTR3A) of Rhizophagus irregularis. Functional analyses in yeast revealed that RiCTR1 encodes a plasma membrane Cu transporter, RiCTR2 a vacuolar Cu transporter and RiCTR3A a plasma membrane protein involved in Cu tolerance. RiCTR1 was more highly expressed in the extraradical mycelia (ERM) and RiCTR2 in the intraradical mycelia (IRM). In the ERM, RiCTR1 expression was up-regulated by Cu deficiency and down-regulated by Cu toxicity. RiCTR2 expression increased only in the ERM grown under severe Cu-deficient conditions. These data suggest that RiCTR1 is involved in Cu uptake by the ERM and RiCTR2 in mobilization of vacuolar Cu stores. Cu deficiency decreased mycorrhizal colonization and arbuscule frequency, but increased RiCTR1 and RiCTR2 expression in the IRM, which suggest that the IRM has a high Cu demand. The two alternatively spliced products of RiCTR3, RiCTR3A and RiCTR3B, were more highly expressed in the ERM. Up-regulation of RiCTR3A by Cu toxicity and the yeast complementation assays suggest that RiCTR3A might function as a Cu receptor involved in Cu tolerance.
The Bradford-reactive soil protein (BRSP) fraction includes glomalin, a glycoprotein produced by ... more The Bradford-reactive soil protein (BRSP) fraction includes glomalin, a glycoprotein produced by arbuscular mycorrhizal (AM) fungi able to bind some metals, such as copper (Cu), which could promote the bioremediation of Cu-polluted soils. This study aimed to analyze the Cu-binding capacity of BRSP in Oenothera picensis that was inoculated or not inoculated with AM fungi. O. picensis plants were established in a Cu contaminated sterilized soil and treated with the following: i) uninoculated (-M); ii) inoculated with native AM fungal propagules (+M); or iii) inoculated with a Claroideoglomus claroideum (CC) strain isolated from non-contaminated soil. In each case, five Cu levels were applied to the soil (basal level 497.3 mg Cu kg-1): 0 (T1); 75 (T2); 150 (T3); 225 (T4); and 300 mg Cu kg-1 (T5). A high BRSP accumulation in AM inoculated treatments, especially with CC, was observed. A higher Cu-bound-to-BRSP content was found with increasing Cu concentrations, representing up to 20-22% of the total Cu in the soil. Moreover, a higher root Cu concentration in +M was observed. These results suggest a high Cu binding capacity by BRSP, which is a relevant aspect to consider in the design of bioremediation programs together with the selection of endemic metallophytes and AM fungal strains, which are able to produce glomalin at high quantities.
Arbuscular mycorrhizal symbioses that involve most plants and Glomeromycota fungi are integral an... more Arbuscular mycorrhizal symbioses that involve most plants and Glomeromycota fungi are integral and functional parts of plant roots. In these associations, the fungi not only colonize the root cortex but also maintain an extensive network of hyphae that extend out of the root into the surrounding environment. These external hyphae contribute to plant uptake of low mobility nutrients, such as P, Zn, and Cu. Besides improving plant mineral nutrition, arbuscular mycorrhizal fungi (AMF) can alleviate heavy metal (HM) toxicity to their host plants. HMs, such as Cu, Zn, Fe, and Mn, play essential roles in many biological processes but are toxic when present in excess. This makes their transport and homeostatic control of particular importance to all living organisms. AMF play an important role in modulating plant HM acquisition in a wide range of soil metal concentrations and have been considered to be a key element in the improvement of micronutrient concentrations in crops and in the phy...
Many studies have established that arbuscular mycorrhizal fungi transfer N to the host plant. How... more Many studies have established that arbuscular mycorrhizal fungi transfer N to the host plant. However, the role and importance of arbuscular mycorrhiza (AM) in plant N nutrition is still uncertain, as are the C/N interactions within the symbiosis. Published reports provide differing, and often contradictory, results that are difficult to combine in a coherent framework. This review explores questions such as: What makes the difference between a positive and a negative effect of AM on plant N nutrition? Is the mycorrhizal N response (MNR) correlated to the mycorrhizal growth response (MGR), and how or under which conditions? Is the MNR effect on plant growth C mediated? Is plant C investment on fungal growth related to N needs or N benefit? How is the N for C trade between symbionts regulated? The patternless nature of current knowledge is made evident, and possible reasons for this are discussed.
The aim of this Chapter is to review and integrate current knowledge of the impact of mycorrhizal... more The aim of this Chapter is to review and integrate current knowledge of the impact of mycorrhizal symbioses on plant functioning and adaptation with specific emphasis on P acquisition at various levels of cellular organization, from the molecular, biochemical and physiological to the whole plant. Accordingly, the available information will be structured as follows: (i) mycorrhizas as a plant strategy
The plant plasma membrane ATPase has been described as a key regulatory enzyme in cellular : even... more The plant plasma membrane ATPase has been described as a key regulatory enzyme in cellular : events such as adaptation to abiotic stresses and development changes [1,2]; however, the mechanism by which this activity is regulated is still uncertain. The modulation by lipids of ATPases of many sources is a recognized phenomenon which has been discussed [3]. This lipid dependency suggested a possible mode of regulation of plasma membrane H+-ATPase activity in vivo via the modification of its lipid environment [4] . The modulation by lipids may occur via a direct interaction with the enzyme (the annulus hypothesis) or through changes in membrane fluidity, which causes an alteration in the enzyme conformation, and thus in activity.
Abstract Arbuscular mycorrhizal fungi are soilborne microorganisms that form a mutualistic symbio... more Abstract Arbuscular mycorrhizal fungi are soilborne microorganisms that form a mutualistic symbiotic association with most land plants. As obligate biotrophs these fungi are unable to complete their life cycle in the absence of the host plant. This symbiosis is increasingly being recognised as an integral and important part of natural ecosystems throughout the word. Because of the incalcitrance of arbuscular mycorrhizal fungi to grow in pure culture and consequently the difficulties in obtaining sufficiently large quantities of fungal material, the ...
Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic r... more Previously, a partial-length cDNA and a complete genomic clone encoding a putative sarcoplasmic reticulum-type Ca2+-ATPase (LCA, Lycopersicon _CaZ+-.ATPase) were isolated from tomato. To determine the subcellular localization of this Ca2+-ATPase, specific polyclonal antibodies raised against a fusion protein encoding a portion of the LCA polypeptide were generated. Based on hybridization of the LCA cDNA and of the nucleotide sequence encoding the fusion protein to genomic DNA, it appears that LCA and the fusion protein domain are encoded by a single gene in tomato. Antibodies raised against the LCA domain fusion protein reacted specifically with two polypeptides of 116 and 120 kD that are localized in the vacuolar and plasma membranes, respectively. The distribution of vanadate-sensitive ATP-dependent Ca2+ transport activities in sucrose gradients coincided with the distribution of the immunodetected proteins. The ATPdependent Ca2+ transport activities associated with tonoplast and plasma membrane fractions shared similar properties, because both fractions were inhibited by vanadate but insensitive to carbonyl cyanide m-chlorophenylhydrazone, nitrate, and calmodulin. Moreover, antibodies raised against the LCA domain fusion protein inhibited ATP-dependent Ca2+ uptake activity associated with both the tonoplast and plasma membrane fractions. These data suggest that a single gene (LCA) may encode two P-type Ca2+-ATPase isoforms that are differentially localized in the tonoplast and plasma membrane of tomato roots.
The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncerta... more The role and importance of arbuscular mycorrhizae (AM) in plant nitrogen (N) nutrition is uncertain. We propose that this be clarified by using more integrative experimental designs, with the use of a gradient of N supply and the quantification of an extensive array of plant nutrient contents. Using such an experimental design, we investigated AM effects on plant N nutrition, whether the mycorrhizal N response (MNR) determines the mycorrhizal growth response (MGR), and how MNR influences plants' C economy. Oryza sativa plants were inoculated with Rhizophagus irregularis or Funneliformis mossae. AM effects were studied along a gradient of N supplies. Biomass, photosynthesis, nutrient and starch contents, mycorrhizal colonization and OsPT11 gene expression were measured. C investment in fungal growth was estimated. Results showed that, in rice, MGR was dependent on AM nutrient uptake effects, namely on the synergy between N and Zn, and not on C expenditure. The supply of C to the fungus was dependent on the plant's nutrient demand, indicated by high shoot C/N or low %N. We conclude that one of the real reasons for the negative MGR of rice, Zn deficiency of AMF plants, would have remained hidden without an experimental design allowing the observation of plants' response to AM along gradients of nutrient concentrations. Adopting more integrative and comprehensive experimental approaches in mycorrhizal studies seems therefore essential if we are to achieve a true understanding of AM function, namely of the mechanisms of C/N exchange regulation in AM.
Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots ... more Arbuscular mycorrhizal (AM) fungi play a key role in the nutrition of many land plants. AM roots have two pathways for nutrient uptake, directly through the root epidermis and root hairs and via AM fungal hyphae into root cortical cells, where arbuscules or hyphal coils provide symbiotic interfaces. Recent studies demonstrated that the AM symbiosis modifies the expression of plant transporter genes and that NH þ 4 is the main form of N transported in the symbiosis. The aim of the present work was to get insights into the mycorrhizal N uptake pathway in Oryza sativa by analysing the expression of genes encoding ammonium transporters (AMTs), glutamine synthase (GS) and glutamate synthase (GOGAT) in roots colonized by the AM fungus Rhizophagus irregularis and grown under two N regimes. We found that the AM symbiosis down-regulated OsAMT1;1 and OsAMT1;3 expression at low-N, but not at high-N conditions, and induced, independently of the N status of the plant, a strong up-regulation of OsAMT3;1 expression. The AM-inducible NH þ 4 transporter OsAMT3;1 belongs to the family 2 of plant AMTs and is phylogenetically related to the AM-inducible AMTs of other plant species. Moreover, for the first time we provide evidence of the specific induction of a GOGAT gene upon colonization with an AM fungus. These data suggest that OsAMT3;1 is likely involved in the mycorrhizal N uptake pathway in rice roots and that OsGOGAT2 plays a role in the assimilation of the NH þ 4 supplied via the OsAMT3;1 AM-inducible transporter.
... M. Pilar Rodríguez-Rosales Corresponding Author Contact Information , E-mail The Correspondin... more ... M. Pilar Rodríguez-Rosales Corresponding Author Contact Information , E-mail The Corresponding Author , Loubna Kerkeb , Nuria Ferrol and Juan P. Douaire. ... Received 8 April 1997; accepted 19 August 1997. Available online 20 March 1999. Abstract. ...
In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots.-Phys... more In vivo and in vitro effects of boron on the plasma membrane proton pump of sunflower roots.-Physiol. Plant. 84: 49-54. The effect of boron excess and deficiency on H* efflux from excised roots from sunflower (Helianthus annum L. cv. Enano) seedlings and on plasma membrane H^-ATPase (EC 3.6.1.35) in isolated Kl-washed microsomes has been investigated. When seedlings were grown in media with toxic levels of H3BO3 (5 mAf) or without added boron and exposed to light conditions, an inhibition of the capacity for external acidification by excised roots was observed as compared to roots from seedlings grown with optimal H3BO, concentration (0.25 mM). Toxic and deficient boron conditions also inhibited the vanadate-sensitive H+-ATPase of microsomes isolated from the roots. The mechanism of boron toxicity was investigated in vitro with microsome vesicles. A strong effect of boron on the vanadate-sensitive, ATPdependent H* transport was found, but the vanadate-sensitive phospho-hydrolase activity was not affected. These results suggest that boron could esert an effect on the plasma membrane properties, directly or indirectly regulating proton transport.
To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expressi... more To analyse the effect of arbuscular mycorrhizal (AM) colonization on tomato gene expression, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) patterns of crude extracts, soluble and membrane proteins of tomato roots, either mycorrhizal and the AM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe or nonmycorrhizal, have been compared. In the three fractions analysed, AM colonization induced up-regulation with down-regulation of the synthesis of polypeptides already present in tomato roots and induction of some new polypeptides. Separation of root extracts into soluble and membrane fractions allowed us to identify two soluble, and five membrane-bound, newly induced polypeptides in AM roots. Comparison of the protein patterns of AM roots with those of the external mycelium of G. mosseae showed that one of the newly induced polypeptides might correspond to a fungal polypeptide. By using this experimental approach, we have been able to detect 44 polypeptides that are differentially displayed in tomato roots as a consequence of the establishment of the AM symbiosis.
A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintAB... more A full-length cDNA sequence putatively encoding an ATP-binding cassette (ABC) transporter (GintABC1) was isolated from the extraradical mycelia of the arbuscular mycorrhizal fungus Glomus intraradices. Bioinformatic analysis of the sequence indicated that GintABC1 encodes a 1513 amino acid polypeptide, containing two sixtransmembrane clusters (TMD) intercalated with sequences characteristics of the nucleotide binding domains (NBD) and an extra N-terminus extension (TMD0). GintABC1 presents a predicted TMD0-(TMD-NBD) 2 topology, typical of the multidrug resistance-associated protein subfamily of ABC transporters. Gene expression analyses revealed no difference in the expression levels of GintABC1 in the extra-vs the intraradical mycelia. GintABC1 was up-regulated by Cd and Cu, but not by Zn, suggesting that this transporter might be involved in Cu and Cd detoxification. Paraquat, an oxidative agent, also induced the transcription of GintABC1. These data suggest that redox changes may be involved in the transcriptional regulation of GintABC1 by Cd and Cu.
A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensi... more A new fungal species in the arbuscular mycorrhiza-forming Glomeromycetes, Entrophospora nevadensis, was isolated from soil near the roots of several endemic and endangered plant species (e.g. Plantago nivalis and Alchemilla fontqueri) growing in Sierra Nevada National Park (Granada, Andalucia, Spain). The fungus was propagated in trap cultures on Plantago nivalis and Sorbus hybrida and in pure cultures on Trifolium pratense and Sorghum vulgare. Spores are yellow brown to brown, 90-115 .m diam and form singly in soil, in the neck of adherent sporiferous saccules that form either terminally or intercalary on mycelial hyphae. Spores have two three-layered walls and conspicuous, 6-12 microm long, spiny, thorn-like projections on the outer wall consisting of hyaline to subhyaline, evanescent tips and yellow brown to brown, persistent bases. In aging spores these projections are usually shorter (1-2.8 microm) and dome-shaped or rounded, sometimes with a central pit on top where the evanescent tip has sloughed off. Molecular analysis with partial sequences of the 18S ribosomal gene places the fungus within the Diversisporales. The new fungus was found in soil near plants with different living strategies but growing in high altitude soils with acidic pH, high soil moisture and organic carbon content, and close to streams.
A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora gran... more A new dimorphic fungal species in the arbuscular mycorrhiza-forming Glomeromycota, Ambispora granatensis, was isolated from an agricultural site in the province of Granada (Andalucía, Spain) growing in the rhizosphere of Asparagus officinalis. It was propagated in pot cultures with Trifolium pratense and Sorghum vulgare. The fungus also colonized Ri T-DNA transformed Daucus carota roots but did not form spores in these root organ cultures. The spores of the acaulosporoid morph are 90-150 mm diam and hyaline to white to pale yellow. They have three walls and a papillae-like rough irregular surface on the outer surface of the outer wall. The irregular surface might become difficult to detect within a few hours in lactic acid-based mountings but are clearly visible in water. The structural central wall layer of the outer wall is only 0.8-1.5 mm thick. The glomoid spores are formed singly or in small, loose spore clusters of 2-10 spores. They are hyaline to pale yellow, (25)40-70 mm diam and have a bilayered spore wall without ornamentation. Nearly full length sequences of the 18S and the ITS regions of the ribosomal gene place the new fungus in a separate clade next to Ambispora fennica and Ambispora gerdemannii. The acaulosporoid spores of the new fungus can be distinguished easily from all other spores in genus Ambispora by the conspicuous thin outer wall.
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