From isolation to application: a case study of arbuscular mycorrhizal fungi of the Arabian Peninsula.

The vegetation in the Arabian Peninsula experiences drought, heat, soil salinity, and low fertility, mainly due to low phosphorus (P) availability. The beneficial mycorrhizal symbiosis between plants and arbuscular mycorrhizal fungi (AMF) is a key factor supporting plant growth under such environmental conditions. Therefore, AMF strains isolated from these soils might be useful as biotechnological tools for agriculture and revegetation practices in the region. Here we present a pioneering program to isolate, identify, and apply AMF isolated from rhizosphere soils of agricultural and natural habitats, namely date palm plantations and five native desert plants, respectively in the Southern Arabian Peninsula. We established taxonomically unique AMF species as single-spore cultures as part of an expanding collection of AMF strains adapted to arid ecosystems. Preliminary experiments were conducted to evaluate the abilities of these AMF strains to promote seedling growth of a main crop Phoenix dactylifera L. and a common plant Prosopis cineraria L. (Druce) in the Arabian Peninsula. The results showed that inoculation with certain AMF species enhanced the growth of both plants, highlighting the potential of these fungi as part of sustainable land use practices in this region.

Effects of two contrasted arbuscular mycorrhizal fungal isolates on nutrient uptake by Sorghum bicolor under drought.

Drought is a limiting factor for crop production, especially in arid and semi-arid climates. In this study, Sorghum bicolor plants were inoculated, or not, with Rhizophagus irregularis, an arbuscular mycorrhizal (AM) strain typical for temperate climates, or Rhizophagus arabicus, a strain endemic to hyper-arid ecosystems. Plants were grown under well-watered or drought conditions in compartmented microcosms. Transpiration rates, plant growth, and nutrient uptake (using 15N as a tracer) were determined to assess the impact of drought stress on sorghum plants in AM symbiosis. Although AM colonization did not affect the bulk biomass of host plants, R. arabicus improved their transpiration efficiency and drought tolerance more than R. irregularis. Moreover, R. arabicus was able to extract more 15N from the soil under both water regimes, and AM-driven enhancement of the nitrogen and phosphorus content of sorghum, especially when water was limiting, was greater for R. arabicus-inoculated plants than for R. irregularis-inoculated plants. Our work demonstrates close links between AM hyphal phosphorus and nitrogen transport and uptake by AM plants for both AM fungal species. It also underscores that, under the drought stress conditions we applied, R. arabicus transfers significantly more nitrogen to sorghum than R. irregularis.

Phylogenetic, structural, and functional characterization of AMT3;1, an ammonium transporter induced by mycorrhization among model grasses.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both 15N and 33P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.

Options of partners improve carbon for phosphorus trade in the arbuscular mycorrhizal mutualism.

The mutualism between plants and arbuscular mycorrhizal fungi (AMF) is widespread and has persisted for over 400 million years. Although this mutualism depends on fair resource exchange between plants and fungi, inequality exists among partners despite mechanisms that regulate trade. Here, we use (33) P and (14) C isotopes and a split-root system to test for preferential allocation and reciprocal rewards in the plant-AMF symbiosis by presenting a plant with two AMF that differ in cooperativeness. We found that plants received more (33) P from less cooperative AMF in the presence of another AMF species. This increase in (33) P resulted in a reduced (14) C cost per unit of (33) P from less cooperative AMF when alternative options were available. Our results indicate that AMF diversity promotes cooperation between plants and AMF, which may be an important mechanism maintaining the evolutionary persistence of and diversity within the plant-AMF mutualism.

Plant phosphorus acquisition in a common mycorrhizal network: regulation of phosphate transporter genes of the Pht1 family in sorghum and flax.

In a preceding microcosm study, we found huge differences in phosphorus (P) acquisition in sorghum (Sorghum bicolor) and flax (Linum usitatissimum) sharing a common mycorrhizal network (CMN). Is the transcriptional regulation of arbuscular mycorrhizal (AM)-induced inorganic orthophosphate (Pi) transporters responsible for these differences? We characterized and analyzed the expression of Pi transporters of the Pht1 family in both plant species, and identified two new AM-inducible Pi transporters in flax. Mycorrhizal Pi acquisition was strongly affected by the combination of plant and AM fungal species. A corresponding change in the expression of two AM-inducible Pht1 transporters was noticed in both plants (SbPT9, SbPT10, LuPT5 and LuPT8), but the effect was very weak. Overall, the expression level of these genes did not explain why flax took up more Pi from the CMN than did sorghum. The post-transcriptional regulation of the transporters and their biochemical properties may be more important for their function than the fine-tuning of their gene expression.

Impact of water regimes on an experimental community of four desert arbuscular mycorrhizal fungal (AMF) species, as affected by the introduction of a non-native AMF species.

Field studies have revealed the impact of changing water regimes on the structure of arbuscular mycorrhizal fungal (AMF) communities, but it is not known what happens to the abundance of individual AMF species within the community when the water conditions in the rhizosphere change. The behavior of four AMF species isolated from the Arabian desert (Diversispora aurantia, Diversispora omaniana, Septoglomus africanum, and an undescribed Paraglomus species) was investigated when assembled in microcosms containing Sorghum bicolor as host plant, and treated with various water regimes. Furthermore, the impact of invasion of these assemblages by Rhizophagus irregularis, an AMF species widely used in commercial inocula, was studied. The abundance of each AMF species in sorghum roots was measured by determining the transcript numbers of their large ribosomal subunit (rLSU) by real-time PCR, using cDNA and species-specific primers. Plant biomass and length of AMF extraradical hyphae were also measured. The abundance of each AMF species within the sorghum roots was influenced by both the water regime and the introduction of R. irregularis. Under dry conditions, the introduction of R. irregularis reduced the total abundance of all native AMF species in roots and also led to a reduction in the amount of extraradical mycelium, as well as to a partial decrease in plant biomass. The results indicate that both water regime and the introduction of an invasive AMF species can strongly alter the structure of an AMF native assemblage with a consequent impact on the entire symbiotic mycorrhizal relationship.

Three new species of arbuscular mycorrhizal fungi discovered at one location in a desert of Oman: Diversispora omaniana, Septoglomus nakheelum and Rhizophagus arabicus.

Three new species of arbuscular mycorrhizal (AM) fungi (Glomeromycota) were isolated from soil samples collected from a hyperarid sandy plain of South Arabia. Morphological characteristics of the spores clearly differentiated them from closely related AM species. Molecular analyses were performed on rDNA sequences obtained from single spores including a ~1700 bp region comprising partial SSU, ITS, partial LSU and the ~600 bp ITS region only. The phylogenetic trees based on these regions showed that the three species belong to well described genera but are clearly distinct from known species. Consequently, we describe them here as Diversispora omaniana, Septoglomus nakheelum and Rhizophagus arabicus spp. nov. D. omaniana and R. arabicus were isolated from the native, arid habitat, while S. nakheelum was isolated from a nearby irrigated date palm plantation. The discovery of three new species of AM fungi from this location suggests that a number of additional undescribed AM taxa may be present in such desert ecosystems. Further work to understand the diversity and functional significance of these new AM taxa may offer new opportunities for conservation, re-vegetation, and sustainable agriculture in extremely arid environments.

The family of ammonium transporters (AMT) in Sorghum bicolor: two AMT members are induced locally, but not systemically in roots colonized by arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi contribute to plant nitrogen (N) acquisition. Recent studies demonstrated the transport of N in the form of ammonium during AM symbiosis. Here, we hypothesize that induction of specific ammonium transporter (AMT) genes in Sorghum bicolor during AM colonization might play a key role in the functionality of the symbiosis. For the first time, combining a split-root experiment and microdissection technology, we were able to assess the precise expression pattern of two AM-inducible AMTs, SbAMT3;1 and SbAMT4. Immunolocalization was used to localize the protein of SbAMT3;1. The expression of SbAMT3;1 and SbAMT4 was greatly induced locally in root cells containing arbuscules and in adjacent cells. However, a split-root experiment revealed that this induction was not systemic. By contrast, a strictly AM-induced phosphate transporter (SbPt11) was expressed systemically in the split-root experiment. However, a gradient of expression was apparent. Immunolocalization analyses demonstrated that SbAMT3;1 was present only in cells containing developing arbuscules. Our results show that the SbAMT3;1 and SbAMT4 genes are expressed in root cortical cells, which makes them ready to accommodate arbuscules, a process of considerable importance in view of the short life span of arbuscules. Additionally, SbAMT3;1 might play an important role in N transfer during AM symbiosis.

Mycorrhizal networks: common goods of plants shared under unequal terms of trade.

Plants commonly live in a symbiotic association with arbuscular mycorrhizal fungi (AMF). They invest photosynthetic products to feed their fungal partners, which, in return, provide mineral nutrients foraged in the soil by their intricate hyphal networks. Intriguingly, AMF can link neighboring plants, forming common mycorrhizal networks (CMNs). What are the terms of trade in such CMNs between plants and their shared fungal partners? To address this question, we set up microcosms containing a pair of test plants, interlinked by a CMN of Glomus intraradices or Glomus mosseae. The plants were flax (Linum usitatissimum; a C(3) plant) and sorghum (Sorghum bicolor; a C(4) plant), which display distinctly different (13)C/(12)C isotope compositions. This allowed us to differentially assess the carbon investment of the two plants into the CMN through stable isotope tracing. In parallel, we determined the plants' "return of investment" (i.e. the acquisition of nutrients via CMN) using (15)N and (33)P as tracers. Depending on the AMF species, we found a strong asymmetry in the terms of trade: flax invested little carbon but gained up to 94% of the nitrogen and phosphorus provided by the CMN, which highly facilitated growth, whereas the neighboring sorghum invested massive amounts of carbon with little return but was barely affected in growth. Overall biomass production in the mixed culture surpassed the mean of the two monocultures. Thus, CMNs may contribute to interplant facilitation and the productivity boosts often found with intercropping compared with conventional monocropping.

Carbon and nitrogen metabolism in mycorrhizal networks and mycoheterotrophic plants of tropical forests: a stable isotope analysis.

Most achlorophyllous mycoheterotrophic (MH) plants obtain carbon (C) from mycorrhizal networks and indirectly exploit nearby autotrophic plants. We compared overlooked tropical rainforest MH plants associating with arbuscular mycorrhizal fungi (AMF) to well-reported temperate MH plants associating with ectomycorrhizal basidiomycetes. We investigated (13)C and (15)N abundances of MH plants, green plants, and AMF spores in Caribbean rainforests. Whereas temperate MH plants and fungi have higher δ(13)C than canopy trees, these organisms displayed similar δ(13)C values in rainforests, suggesting differences in C exchanges. Although temperate green and MH plants differ in δ(15)N, they display similar (15)N abundances, and likely nitrogen (N) sources, in rainforests. Contrasting with the high N concentrations shared by temperate MH plants and their fungi, rainforest MH plants had lower N concentrations than AMF, suggesting differences in C/N of exchanged nutrients. We provide a framework for isotopic studies on AMF networks and suggest that MH plants in tropical and temperate regions evolved different physiologies to adapt in diverging environments.

Towards a better understanding of the generation of fructan structure diversity in plants: molecular and functional characterization of a sucrose:fructan 6-fructosyltransferase (6-SFT) cDNA from perennial ryegrass (Lolium perenne).

The main storage compounds in Lolium perenne are fructans with prevailing ß(2-6) linkages. A cDNA library of L. perenne was screened using Poa secunda sucrose:fructan 6-fructosyltransferase (6-SFT) as a probe. A full-length Lp6-SFT clone was isolated as shown by heterologous expression in Pichia pastoris. High levels of Lp6-SFT transcription were found in the growth zone of elongating leaves and in mature leaf sheaths where fructans are synthesized. Upon fructan synthesis induction, Lp6-SFT transcription was high in mature leaf blades but with no concomitant accumulation of fructans. In vitro studies with the recombinant Lp6-SFT protein showed that both 1-kestotriose and 6G-kestotriose acted as fructosyl acceptors, producing 1- and 6-kestotetraose (bifurcose) and 6G,6-kestotetraose, respectively. Interestingly, bifurcose formation ceased and 6G,6-kestotetraose was formed instead, when recombinant fructan:fructan 6G-fructosyltransferase (6G-FFT) of L. perenne was introduced in the enzyme assay with sucrose and 1-kestotriose as substrates. The remarkable absence of bifurcose in L. perenne tissues might be explained by a higher affinity of 6G-FFT, as compared with 6-SFT, for 1-kestotriose, which is the first fructan formed. Surprisingly, recombinant 6-SFT from Hordeum vulgare, a plant devoid of fructans with internal glucosyl residues, also produced 6G,6-kestotetraose from sucrose and 6G-kestotriose. In the presence of recombinant L. perenne 6G-FFT, it produced 6G,6-kestotetraose from 1-kestotriose and sucrose, like L. perenne 6-SFT. Thus, we demonstrate that the two 6-SFTs have close catalytic properties and that the distinct fructans formed in L. perenne and H. vulgare can be explained by the presence of 6G-FFT activity in L. perenne and its absence in H. vulgare.

Unique arbuscular mycorrhizal fungal communities uncovered in date palm plantations and surrounding desert habitats of Southern Arabia.

The main objective of this study was to shed light on the previously unknown arbuscular mycorrhizal fungal (AMF) communities in Southern Arabia. We explored AMF communities in two date palm (Phoenix dactylifera) plantations and the natural vegetation of their surrounding arid habitats. The plantations were managed traditionally in an oasis and according to conventional guidelines at an experimental station. Based on spore morphotyping, the AMF communities under the date palms appeared to be quite diverse at both plantations and more similar to each other than to the communities under the ruderal plant, Polygala erioptera, growing at the experimental station on the dry strip between the palm trees, and to the communities uncovered under the native vegetation (Zygophyllum hamiense, Salvadora persica, Prosopis cineraria, inter-plant area) of adjacent undisturbed arid habitat. AMF spore abundance and species richness were higher under date palms than under the ruderal and native plants. Sampling in a remote sand dune area under Heliotropium kotschyi yielded only two AMF morphospecies and only after trap culturing. Overall, 25 AMF morphospecies were detected encompassing all study habitats. Eighteen belonged to the genus Glomus including four undescribed species. Glomus sinuosum, a species typically found in undisturbed habitats, was the most frequently occurring morphospecies under the date palms. Using molecular tools, it was also found as a phylogenetic taxon associated with date palm roots. These roots were associated with nine phylogenetic taxa, among them eight from Glomus group A, but the majority could not be assigned to known morphospecies or to environmental sequences in public databases. Some phylogenetic taxa seemed to be site specific. Despite the use of group-specific primers and efficient trapping systems with a bait plant consortium, surprisingly, two of the globally most frequently found species, Glomus intraradices and Glomus mosseae, were not detected neither as phylogenetic taxa in the date palm roots nor as spores under the date palms, the intermediate ruderal plant, or the surrounding natural vegetation. The results highlight the uniqueness of AMF communities inhabiting these diverse habitats exposed to the harsh climatic conditions of Southern Arabia.

Nutrient use efficiency and arbuscular mycorrhizal root colonisation of winter wheat cultivars in different farming systems of the DOK long-term trial.

BACKGROUND: For organic farming, cultivars are required with high nutrient use efficiency under nutrient limited conditions. Arbuscular mycorrhizal fungi (AMF) are known to contribute to nutrient uptake under low input conditions. We compared nutrient use efficiency (NUE) of old and modern organically and conventionally bred cultivars in organic and conventional systems and assessed AMF-root colonisation (AMF-RC) in relation to nutrient concentrations. RESULTS: Cultivars and systems had a statistically significant effect on nitrogen (N) and phosphorus (P) concentrations and NUE parameters, whereas no genotype x environment interactions appeared. In contrast to N and P uptake, the NUE parameters were higher under organic than under conventional conditions. NUE for N increased with the year of release of cultivars. In the organic systems, the organically bred cultivars could not outperform the conventionally bred cultivars in grain yield and NUE parameters. AMF-RC was higher in the organic than in the conventional system, but did not differ among cultivars. CONCLUSION: Cultivars achieving high NUE in the organic systems were found among modern cultivars, irrespective of the breeding programme. Nutrient conditions during the breeding programme did not affect AMF-RC. No clear evidence was found that AMF symbiosis contributed more to nutrient concentrations under low input than under high input conditions.

Protein phosphatase activity and sucrose-mediated induction of fructan synthesis in wheat.

In this work, we analyze protein phosphatase (PP) involvement in the sucrose-mediated induction of fructan metabolism in wheat (Triticum aestivum). The addition of okadaic acid (OA), a PP-inhibitor, to sucrose-fed leaves reduced fructosylsucrose-synthesizing activity (FSS) induction in a dose-dependent manner. The expression of the two enzymes that contribute to FSS activity, 1-SST (1-sucrose:sucrose fructosyltransferase, E.C. 2.4.1.99) and 6-SFT (6-sucrose:fructan fructosyltransferase, E.C. 2.4.1.10), was blocked by 1 microM OA. These results suggest the involvement of a PP type 2A in sucrose signaling leading to fructan synthesis. OA addition to the feeding medium impaired both sucrose accumulation in leaves and the expression of sucrose-H+ symporter (SUT1). It is known that sucrose concentration must exceed a threshold for the induction of fructan metabolism; hence PP2A inhibition may result in lower sucrose levels than required for this induction. OA also induced the vacuolar acid invertase (acid INV) transcript levels suggesting that PP activity might play a role in carbon partitioning. Total extractable PP2A activity decreased during 24 h of treatment with sucrose, in parallel with declining sugar uptake into leaf tissues. In conclusion, our results suggest that PP2A is involved in sucrose-induction of fructan metabolism and may play a role in regulating sucrose uptake, but do not rule out that further steps in sucrose signaling pathway may be affected.

Promiscuous arbuscular mycorrhizal symbiosis of yam (Dioscorea spp.), a key staple crop in West Africa.

Yam (Dioscorea spp.) is a tuberous staple food crop of major importance in the sub-Saharan savannas of West Africa. Optimal yields commonly are obtained only in the first year following slash-and-burn in the shifting cultivation systems. It appears that the yield decline in subsequent years is not merely caused by soil nutrient depletion but might be due to a loss of the beneficial soil microflora, including arbuscular mycorrhizal fungi (AMF), associated with tropical "tree-aspect" savannas and dry forests that are the natural habitats of the wild relatives of yam. Our objective was to study the AMF communities of natural savannas and adjacent yam fields in the Southern Guinea savanna of Benin. AMF were identified by morphotyping spores in the soil from the field sites and in AMF trap cultures with Sorghum bicolor and yam (Dioscorea rotundata and Dioscorea cayenensis) as bait plants. AMF species richness was higher in the savanna than in the yam-field soils (18-25 vs. 11-16 spp.), but similar for both ecosystems (29-36 spp.) according to the observations in trap cultures. Inoculation of trap cultures with soil sampled during the dry season led to high AMF root colonization, spore production, and species richness (overall 45 spp.) whereas inoculation with wet-season soil was inefficient (two spp. only). The use of D. cayenensis and D. rotundata as baits yielded 28 and 29 AMF species, respectively, and S. bicolor 37 species. AMF root colonization, however, was higher in yam than in sorghum (70-95 vs. 11-20%). After 8 months of trap culturing, the mycorrhizal yam had a higher tuber biomass than the nonmycorrhizal controls. The AMF actually colonizing D. rotundata roots in the field were also studied using a novel field sampling procedure for molecular analyses. Multiple phylotaxa were detected that corresponded with the spore morphotypes observed. It is, therefore, likely that the legacy of indigenous AMF from the natural savanna plays a crucial role for yam productivity, particularly in the low-input traditional farming systems prevailing in West Africa.

Impact of antifungals producing rhizobacteria on the performance of Vigna radiata in the presence of arbuscular mycorrhizal fungi.

Plant growth-promoting rhizobacteria (PGPR) that produce antifungal metabolites are potential threats for the arbuscular mycorrhizal (AM) fungi known for their beneficial symbiosis with plants that is crucially important for low-input sustainable agriculture. To address this issue, we used a compartmented container system where test plants, Vigna radiata, could only reach a separate nutrient-rich compartment indirectly via the hyphae of AM fungi associated with their roots. In this system, where plants depended on nutrient uptake via AM symbiosis, we explored the impact of various PGPR. Plants were inoculated with or without a consortium of four species of AM fungi (Glomus coronatum, Glomus etunicatum, Glomus constrictum, and Glomus intraradices), and one or more of the following PGPR strains: phenazine producing (P(+)) and phenazine-less mutant (P(-)), diacetylphloroglucinol (DAPG) producing (G(+)) and DAPG-less mutant (G(-)) strains of Pseudomonas fluorescens, and an unknown antifungal metabolite-producing Alcaligenes faecalis strain, SLHRE425 (D). PGPR exerted only a small if any effect on the performance of AM symbiosis. G(+) enhanced AM root colonization and had positive effects on shoot growth and nitrogen content when added alone, but not in combination with P(+). D negatively influenced AM root colonization, but did not affect nutrient acquisition. Principal component analysis of all treatments indicated correlation between root weight, shoot weight, and nutrient uptake by AM fungus. The results indicate that antifungal metabolites producing PGPR do not necessarily interfere with AM symbiosis and may even promote it thus carefully chosen combinations of such bioinoculants could lead to better plant growth.

Microsatellites for disentangling underground networks: strain-specific identification of Glomus intraradices, an arbuscular mycorrhizal fungus.

The underground network of arbuscular mycorrhizal (AM) fungi is decisive for the above-ground diversity of many plant ecosystems, but tools to investigate the population structure of AM fungi are sorely lacking. Here, we present a bioinformatics approach to identify microsatellite markers in the AM fungus Glomus intraradices. Based on 1958 contigs of this fungus, assembled from public databases, we identified 842 microsatellites. One hundred of them were subjected to closer scrutiny by designing flanking primers and performing an extensive screen to identify polymorphic loci. We obtained 18 polymorphic microsatellite markers, and we found that seven out of eight individual single-spore cultures of G. intraradices could readily be identified by at least five allelic differences, as compared to all other strains. Two single-spore cultures, however, nominally originating from completely different locations, displayed identity at all 18 loci, suggesting with 99.999999% probability that they represent a single clone.

Purification, cloning and functional differences of a third fructan 1-exohydrolase (1-FEHw3) from wheat (Triticum aestivum).

A third fructan exohydrolase isoform (1-FEHw3) was purified from wheat stems by a combination of ammonium sulfate precipitation, ConA affinity and ion-exchange chromatography. Homogeneity of the preparation was indicated by the presence of a single band (70 kDa) after SDS-PAGE. The enzyme hydrolyzed mainly beta2-1 linkages in fructans and was inhibited by sucrose. A cDNA could be obtained after reverse transcriptase polymerase chain reaction (RT-PCR)-based strategies and screening of a cDNA library. Functionality tests of the cDNA performed after heterologous expression in the yeast Pichia pastoris showed that the encoded protein has essentially the same characteristics as the native enzyme. Homology with previously described 1-FEH isoforms from wheat was high (97% identity), and the enzyme showed minor differences to the previously published enzymes. The relative abundance of 1-FEH transcripts in different tissues was investigated by using quantitative RT-PCR.

Sucrose regulated expression of a Ca2+-dependent protein kinase (TaCDPK1) gene in excised leaves of wheat.

Sucrose (Suc) can influence the expression of a large number of genes and thereby regulates many metabolic and developmental processes. However, the Suc sensing and the components of the ensuing signaling transduction pathway leading to the regulation of gene expression are not fully understood. We have shown that protein kinases and phosphatases are involved in the Suc induced expression of fructosyltransferase (FT) genes and fructan accumulation by an hexokinase independent pathway in wheat (Triticum aestivum). In the present study, using an RT-PCR based strategy, we have cloned a calcium-dependent protein kinase (TaCDPK1) cDNA that is upregulated during Suc treatment of excised wheat leaves. The deduced amino-acid sequence of CDPK1 has high sequence similarity (>70%) to known CDPKs from both monocots and dicots. Based on sequence homology, TaCDPK1 sequence shows a variable domain preceding a catalytic domain, an autoinhibitory function domain, and a C-terminal calmodulin-domain containing 4 EF-hand calcium-binding motifs, along with a N-myristoylation motif in the N-terminal variable domain. The recombinant Escherichia coli expressed TaCDPK1 was able to phosphorylate histone III-S in a calcium dependent manner in in vitro assays. The TaCDPK1 gene expression, as determined by quantitative RT-PCR, is induced by Suc and this effect is repressed by the inhibitors of the putative components of the Suc signal transduction pathway (calcium, Ser/Thr protein kinases and protein phosphatases). We propose that TaCDPK1 is involved in the Suc induced signaling pathway in wheat leaves.

Acaulospora alpina, a new arbuscular mycorrhizal fungal species characteristic for high mountainous and alpine regions of the Swiss Alps.

Acaulospora alpina sp. nov. forms small (65-85 microm diam), dark yellow to orange-brown spores laterally on the neck of hyaline to subhyaline sporiferous saccules. The spores have a three-layered outer spore wall, a bi-layered middle wall and a three-layered inner wall. The surface of the second layer of the outer spore wall is ornamented, having regular, circular pits (1.5-2 microm diam) that are as deep as wide and truncated conical. A "beaded" wall layer as found in most other Acaulospora spp. is lacking. The spore morphology of A. alpina resembles that of A. paulinae but can be differentiated easily by the unique ornamentation with the characteristic pits and by the spore color. A key is presented summarizing the morphological differences among Acaulospora species with an ornamented outer spore wall. Partial DNA sequences of the ITS1, 5.8S subunit and ITS2 regions of ribosomal DNA show that A. alpina and A. paulinae are not closely related. Acaulospora lacunosa, which has similar color but has generally bigger spores, also has distinct rDNA sequences. Acaulospora alpina is a characteristic member of the arbuscular mycorrhizal fungal communities in soils with pH 3.5-6.5 in grasslands of the Swiss Alps at altitudes between 1800 and 2700 m above sea level. It is less frequent at 1300-1800 m above sea level, and it so far has not been found in the Alps below 1300 m or in the lowlands of Switzerland.