Alfonso Navarro-Ródenas

Expression Analysis of Aquaporins from Desert Truffle Mycorrhizal Symbiosis Reveals a Fine-Tuned Regulation Under Drought

We have performed the isolation, functional characterization, and expression analysis of aquaporins in roots and leaves of Helianthemum almeriense, in order to evaluate their roles in tolerance to water deficit. Five cDNAs, named HaPIP1;1, HaPIP1;2, HaPIP2;1, HaPIP2;2, and HaTIP1;1, were isolated from H. almeriense. A phylogenetic analysis of deduced proteins confirmed that they belong to the water channel proteins family. The HaPIP1;1, HaPIP2;1, and HaTIP1;1 genes encode functional water channel proteins, as indicated by expression assays in Saccharomyces cerevisiae, showing divergent roles in the transport of water, CO2, and NH3. The expression patterns of the genes isolated from H. almeriense and of a previously described gene from Terfezia claveryi (TcAQP1) were analyzed in mycorrhizal and nonmycorrhizal plants cultivated under well-watered or drought-stress conditions. Some of the studied aquaporins were subjected to fine-tuned expression only under drought-stress conditions. A beneficial effect on plant physiological parameters was observed in mycorrhizal plants with respect to nonmycorrhizal ones. Moreover, stress induced a change in the mycorrhizal type formed, which was more intracellular under drought stress. The combination of a high intracellular colonization, together with the fine-tuned expression of aquaporins could result in a morphophysiological adaptation of this symbiosis to drought conditions.

Terfezia Cultivation in Arid and Semiarid Soils

Since the first plantation of Terfezia mycorrhizal plants was established in 1999 in Murcia (Spain), an increasing demand for this crop, not only in Spain but also in other countries, has prompted research into new strategies and aspects that will enable us to pass from the experimental scale to medium- to large-scale cultivation. As a consequence of this leap, a new photoautotrophic Helianthemum micropropagation system has been developed. This system reduces the time needed to obtain mycorrhizal plants to 3 months since fungal inoculation is carried out at the moment plants are transferred from in vitro to ex vitro conditions, so that plant acclimatization and mycorrhization occur at the same time.

Partial purification, characterisation and histochemical localisation of alkaline phosphatase from ascocarps of the edible desert truffle Terfezia claveryi Chatin.

In the present paper, we confirmed that alkaline phosphatase (ALP) is the main phosphatase present in ascocarps of the edible mycorrhizal fungus Terfezia claveryi. The enzyme was partially purified by precipitation with polyethylene glycol. The purification achieved from a crude extract was fivefold, with 53% of the activity recovered, and acid phosphatase, most of the lipids and phenolic compounds were eliminated. Alkaline phosphatase was kinetically characterised at pH 10.0, the optimum for this enzyme, using p-nitrophenyl phosphate as substrate. The V(max) and K(m) values were 0.3 micromol.min(-1).mg(-1) protein and 9.0 mM, respectively. Orthovanadate was a competitive inhibitor of ALP, with a K(i) of 42.5 microM. The enzyme was histochemically localised in the peridium, the hypothecium and in the ascogenic hyphae of the gleba using both colour and fluorescent reactions. The results presented suggest that the ascocarp of T. claveryi, at some stages of its development, may become nutritionally autonomous and independent of the host plant.

Significance of oxygen transport through aquaporins

Aquaporins are membrane integral proteins responsible for the transmembrane transport of water and other small neutral molecules. Despite their well-acknowledged importance in water transport, their significance in gas transport processes remains unclear. Growing evidence points to the involvement of plant aquaporins in CO2 delivery for photosynthesis. The role of these channel proteins in the transport of O2 and other gases may also be more important than previously envisioned. In this study, we examined O2 permeability of various human, plant, and fungal aquaporins by co-expressing heterologous aquaporin and myoglobin in yeast. Two of the most promising O2-transporters (Homo sapiens AQP1 and Nicotiana tabacum PIP1;3) were confirmed to facilitate O2 transport in the spectrophotometric assay using yeast protoplasts. The over-expression of NtPIP1;3 in yeasts significantly increased their O2 uptake rates in suspension culture. In N. tabacum roots subjected to hypoxic hydroponic conditions, the transcript levels of the O2-transporting aquaporin NtPIP1;3 significantly increased after the seven-day hypoxia treatment, which was accompanied by the increase of ATP levels in the apical root segments. Our results suggest that the functional significance of aquaporin-mediated O2 transport and the possibility of controlling the rate of transmembrane O2 transport should be further explored.

Types of Mycorrhizal Association

The following types of mycorrhizas are distinguished: Ectomycorrhizas, which are characterized by a Hartig net and may or may not have a mantle Endomycorrhizas, which have no Hartig net and may or may not have a mantle but are characterized by undifferentiated coil-shaped intracellular hyphae Ectendomycorrhizas, which display a Hartig net with or without a mantle alongside various forms of intracellular coiled or spherical hyphae

Basic and Applied Research for Desert Truffle Cultivation

This chapter summarizes the latest basic and applied advances in desert truffle research carried out to improve our knowledge of the biodiversity, physiology, biotechnology, and cultivation of these hypogeous and edible fungi. ITS-rDNA sequences in phylo-geographic studies and host plant and soil pH characteristics have been the key to describing eight new desert truffle species. The production of desert truffle mycorrhizal plants has been improved by using β-cyclodextrin and bioreactors for mycelium culture and native beneficial bacteria (PGPR and MHB) to increase seedling survival and mycorrhization. Some fungal enzymes have also been characterized in Terfezia claveryi ascocarps. The presence of alkaline phosphatase both in mycelia and ascocarps indicates that this enzyme plays an important role during the life cycle of T. claveryi, while acid phosphatase might be involved in a process that takes place during the ascocarp stage. Numerous desert truffle plantations have been established in Spain in the last 10 years. A high density of mycorrhizal plants combined with a proper irrigation are two important factors to stimulate ascocarp production. The combination of a high rate of intracellular colonization together with the fine-tuned expression of fungal and plant aquaporins could result in a morpho-physiological adaptation of this symbiosis in drought conditions. Moreover, desert truffle sylviculture is proposed for improving truffle production and for conserving the natural areas where desert truffle grow.

Identification of an Alternative rRNA Post-transcriptional Maturation of 26S rRNA in the Kingdom Fungi

Despite of the integrity of their RNA, some desert truffles present a non-canonical profile of rRNA where 3.3 kb is absent, 1.8 kb is clear and a band of 1.6 kb is observed. A similar rRNA profile was identified in organisms belonging to different life kingdoms, with the exception of the Kingdom Fungi, as a result of a split LSU rRNA called hidden gap. rRNA profiles of desert truffles were analyzed to verify the presence of the non-canonical profile. The RNA of desert truffles and yeast were blotted and hybridized with probes complementary to LSU extremes. RACE of LSU rRNA was carried out to determine the LSU rRNA breakage point. LSU rRNA of desert truffles presents a post-transcriptional cleavage of five nucleotides that generates a hidden gap located in domain D7. LSU splits into two molecules of 1.6 and 1.8 kb. Similar to other organisms, a UAAU tract, downstream of the breakage point, was identified. Phylogenetic comparison suggests that during fungi evolution mutations were introduced in the hypervariable D7 domain, resulting in a sequence that is specifically post-transcriptionally cleaved in some desert truffles.