Physiological Ecology of Plants
Research Activities of AG Requena
The mycorrhizal association between the Glomeromycota and the roots
of vascular plants represent for both partners a whole set of morphological
and metabolic adaptations to the life in symbiosis. However,
it is the fungal partner who depends absolutely on the plant to complete
its life cycle. In the absence of it, the arbuscular mycorrhizal spores can
only germinate and have a very restricted saprotrophic growth,
mainly upon its own carbon expenses. This growth is very limited in extension
but not in time, since the AM fungi have the ability to re-germinate
several times. For that, the hyphae expanding out of the spore arrest growth
after a certain time, about 2 to 3 weeks for G. mosseae, and suffer
apical septation with retrieval of protoplasm towards the spore
(Mosse, 1959). This rare mechanism is understood as a survival strategy of
the AM fungi under conditions where to find a root partner
becomes difficult or impossible. These spores and growing hyphae have therefore
a very different role as the extraradical symbiotic hyphae coming out of
the root. The first ones are using their stored energy in form
of triacylglyerides, glycerol and trehalose to grow enough to find a compatible
root, and if not, to arrest growth. In contrast, the extraradical
hyphae have as a main mission to scavenge mineral nutrients from the
soil (mainly phosphate) for nursing its symbiotic partner and, eventually,
also to colonize other roots.
A: Glomus mosseae spores crushed under the dissecting microscope showing its funnel like shape germination tube. B: Extraradical hyphae from G. mosseae stained with DAPI and showing the nuclei distribution along the hyphae
Symbiosis formation in AM fungi is induced upon a
partner recognition event that switches the developmental programme of the
fungus and triggers appressorium formation. In the absence
of host root recognition the fungus retracts back its protoplasm and suffers
a temporary growth arrest. This developmental switch is the subject of our
study, which tries to dissect the responsible molecular components.
We took a comparative molecular approach and studied the changes on gene
expression that occurred to the fungus upon induction with a host root. We
induced appressorium development on water agar medium by bringing
into contact parsley seedlings with germinated spores or sporocarps of AM
Glomus mosseae appressoria formed in the rhizodermis of parsley plants and visualized after staining with trypan blue.
A time course of appressorium development has shown that first induction takes place around 120 h, while at 168 h a plateau in the number of appressoria is achieved. We investigated both, early gene expression and later gene expression in order to selectively search for genes involved in signaling and recognition, or for genes responsible for structural changes. A novel gene GmGIN1, exclusively expressed during the out planta phase in G. mosseae is currently under close investigation. The full-length GmGIN1 cDNA encodes a protein of 429 amino acids with two-domain structure and a putative self-splicing activity. The N-terminal domain shares sequence similarity to a novel family of GTP binding proteins while the C-terminus has a striking homology to the C-terminal part of the hedgehog protein family from metazoa. A role of the GmGIN protein in the early signaling events is proposed (Requena et al., 2002).
Plants roots are sink tissues that rely on the assimilated carbon fixed during photosynthesis at source tissues such as leaves. In nature, most roots usually live intimately associated to heterotrophic fungi, forming the so-called arbuscular mycorrhizal symbiosis. The fungus is hence dependent on the fixed carbon allocated to the root and in turn, it provides the plant with mineral nutrients, mainly phosphate, located beyond the depletion area around the root. This association is therefore a key player for the maintenance of the nutrient cycling in the terrestrial ecosystem. This equilibrium, required for a sustainable development, is however strongly affected by anthropogenic stresses such as intensive agriculture, overgrazing, deforestation or soil contamination. Although the way in which mycorrhizal fungi benefit plants is well studied from physiological and ecological perspectives, the precise molecular mechanisms by which the nutrient transfer takes place are largely unknown. It is also uncertain how the impact of man-made stresses might affect this nutrient cycling. The aim of our project is to gain knowledge about the genes and the proteins involved in the nutrient transfer between plant and fungus with especial emphasis on the carbon transport towards the rhizosphere and its regulation in response to ecosystem disturbance.
State of the art
The arbuscular mycorrhizal (AM) symbiosis formed between a subgroup of fungi from the Zygomycota, the Glomalean, and the root of most vascular plants is mainly characterized by the reciprocal nutrient exchange between both symbiotic partners where the fungus receives from the plant part of the fixed carbon allocated to the root, calculated to be up to 20% of the photoassimilated carbon. This carbon enters in that way into the soil system where it can be later used by the rest of soil microbiota. In turn, the plant improves its mineral supply (mainly in phosphate) due to the ability of the fungal external mycelium to overcome the nutrient depletion area surrounding the root. A major question in the study of the arbuscular mycorrhizal symbiosis is where and how this nutrient exchange takes place. The fungus forms inside of the inner cortical cells of the root specialized haustoria called arbuscules. These are branched hyphae with a very reduced cell wall, surrounded by apoplastic space and by the so-called periarbuscular membrane of plant origin formed by invagination of the plant plasma membrane.
1. Effect of the mycorrhizal inoculation on plant growth. Anthyllis cytisoides plants were inoculated with Glomus deserticola and grown for 4 months. 2. Arbuscules formed in the inner cortical cells of the root. 3. A coil of a fungal hyphae formed inside cortical cells near the epidermis. 4. Detailed structure of two arbuscules stained with calcofluor to make the cell wall visible (courtesy of Prof. P. Bonfante, University of Torino).
While there is increasing evidence that phosphate translocated from the soil through the fungus is downloaded at the arbuscule interface where it is taken up by plant transporters, little is known about the place where the exchange of carbon takes place. Besides the arbuscule, inter- or intra-cellular hyphae (i.e. coils) formed in upper cortical cells cannot be ruled out as locations for the nutrient exchange.
Role of H+-ATPases in the nutrient exchange and regulation during the AM symbiosis
A main issue is of course, what form of carbon could be transported at these interfaces. Recent papers indicate that glucose or fructose could be the main form of carbohydrate that the fungus imports from the apoplastic space. It is known that both, phosphate as well as hexoses, are usually translocated by means of symporters which are supported by an electrochemical gradient in the plasma membrane created by H+ATPase enzymes. Therefore, these H+ATPases are likely to play an important role both at the plant and at the fungal symbiotic interfaces where either phosphate or carbon are translocated. In plants, H+ATPases form a large gene family whose members are either transcriptionally or/and posttranscriptionally regulated at different stages of plant development. A recent paper has shown that at least two of these plant H+ATPase isoforms are involved in the interaction with symbiotic mycorrhizal fungi. Using GUS-fused promoters the authors showed gene induction for these two isoforms in arbuscule-containing cells. In our laboratory we have identified two fungal H+ATPases which are developmental and nutrient regulated at the transcriptional level. One of these H+ATPase genes is preferentially expressed during pre-symbiotic growth and in intraradical hyphae, while the other is mainly expressed during the in planta phase. Our results indicate that, similarly to what plants, arbuscular mycorrhizal fungi recruit different proton pumps at different developmental or nutritional stages.
Role of neutral trehalase in the carbohydrate metabolism of the symbiotic arbuscular mycorrhizal fungi
Trehalose (alpha-D-glucopyranosyl, alpha-D-glucopyranoside) is a non-reducing disaccharide found in a wide variety of organisms including fungi, bacteria, protozoa, nematodes, insects and plants. In fungi, trehalose is considered to be a storage carbohydrate in vegetative cells and spores, but also to have protectant role against several adverse conditions (heat, desiccation, freezing or drying). In arbuscular mycorrhizal (AM) fungi, the dominant storage carbohydrates detected in hyphae and spores are trehalose and glycogen. During asymbiotic growth, in the firsts days after the germ tube emergence, part of the trehalose stored in spores is broken down, possibly indicating that this carbohydrate sustains fungal growth at the very early stages of germination. The enzyme responsible of the intracellular breakdown of trehalose in several fungi is the neutral trehalase, a cytosolic enzyme with a optimum pH of 7.0. The aim of our work is to investigate the role of this enzyme in the carbohydrate metabolism of AM fungi. For that, the neutral trehalase gene from the fungus Glomus mosseae has been isolated by PCR using degenerated primers. The expression of the gene is being studied during different developmental stages of the life cycle of the fungus and its function analysed by functional complementation in yeast.