Root Hair Colonization by Mycorrhizal Fungi

Abstract

Mycorrhizal fungi, i.e., the soil fungi that form mutualistic associations with many land plants, are provided by the host with carbon sources required to complete their life cycle, whereas they assist the plant in nutrient uptake from soil. Such acquisition is also considered to be one of the primary functions of root hairs. The aim of this chapter is to investigate the importance of root hairs in the establishment of mycorrhizal interactions, to verify whether plant (root hairs) and fungal (extraradical hyphae) structures work synergistically to provide efficient mineral nutrition. Evidence from morphological studies, where the mycorrhizal typologies have been compared, point to the direct involvement of root hairs in ecto- and arbuscular mycorrhizas (AM). Root hairs probably play a role during the first stages of ectomycorrhizal development, being sensitive to diffusible factors released by the symbiotic fungi and acting as a preferential anchorage site. During AM establishment, a variety of interactions are reported. In liverworts, AM fungi often penetrate the rhizoids, but the colonization process in higher plants does not usually involve root hairs. The analysis of mutant plants with impaired root hair development has not demonstrated any discernible impact on their mycorrhizal capacities. Confocal microscopy has recently provided important insight into understanding the plant responses upon encountering mycorrhizal fungi. Root hairs respond to the fungal presence with nuclear movements, although fungal penetration most often occurs through atrichoblasts or, occasionally, at the base of trichoblasts. Taken together, experimental evidence points to a strong difference in root hair involvement during AM development and nodulation. Nitrogen-fixing bacteria may have found in root hairs a specific anatomical niche, often neglected by AM fungi, to achieve tissue colonization.

Mineral nutrient acquisition from soil is considered one of the primary functions of root hairs, together with the anchorage of the plant (Gilroy and Jones (2000). However, these crucial structures are not alone in the acquisition of nutrients, since mycorrhizal fungi, i.e., the soil fungi that form mutualistic associations with many land plants, also assist their hosts in this way. The mycorrhizal symbiosis is in fact characterized by reciprocal nutrient exchanges between the symbiotic partners: while the fungus obtains photosynthetically derived carbon compounds, the plant receives mineral nutrients. The fungus receives up to 20% of the photoassimilated carbon allocated by the plant to the root (Smith and Read 1997). In exchange, the fungus improves the mineral supply to the plant (mainly phosphate) through the external mycelium, extending through and beyond the nutrient depletion area that surrounds the root (Jakobsen 1995). In fact, mineral nutrients such as phosphorus have very limited mobility in soil, and depletion zones – where the entire available nutrient has been scavenged – quickly appear around roots (Marschner 1995). To obtain phosphorus, plants have to extend their root surface area, and they are helped in this task by mycorrhizal fungal hyphae, which are thinner and more extensive than the root hairs themselves.

The biological meaning of the symbiotic association between the plant roots and some soil fungi, defined for the first time as “mycorrhiza” by Frank 1885), has become more and more apparent in recent years, especially since the corresponding research covers important aspects of ecology, evolutionary biology, genetics, and developmental biology.

Despite their enormous ecological relevance, knowledge of the cellular and molecular mechanisms that control the success of plant–fungal symbiotic associations is still limited, as highlighted by recent reviews and books (Martin et al. (2007; Gianinazzi-Pearson et al. 2007; Pühler and Strack 2007). However, the development of technological platforms in plant genomics is greatly facilitating the comprehensive identification of genes that are activated during mycorrhizal symbiosis. For these reasons, research on mycorrhizas has entered the mainstream of biology, since new tools to uncover symbiont communication and associated developmental mechanisms, diversity, and contributions of symbiotic partners to functioning of mycorrhizal associations are now available. All of these new aspects have been documented in the above-mentioned reviews, as well as in Paszkowski 2006), Bucher (2007), and Genre and Bonfante (2007).

The aim of this chapter is to investigate the importance of root hairs in the establishment of mycorrhizal interactions, and to verify whether plant (root hairs) and fungal (extraradical hyphae) structures work synergistically to provide efficient mineral uptake. For these reasons, a short summary of the main mycorrhizal typologies and morphological features is provided to address the question “what is the involvement of root hairs in mycorrhizal associations?”

Appendices

Appendix: How to Study the Interactions Between Root Hairs and Arbuscular Mycorrhizal Fungi: Technical Aspects

The achievement of mycorrhizas under controlled conditions is mandatory to investigate plant–fungal interactions according to a defined time scale. Two major techniques can be followed, one suitable for quantitative and morphological analysis on fixed material, the other for in vivo observation.

Morphological and Quantitative Analysis on Fixed Material

2.1 Mycorrhizal Synthesis

Ten-day-old seedlings were inoculated with Gigaspora margarita Becker and Hall (strain deposited in the Bank of European Glomales as BEG 34) using the Millipore (Bedford, MA) sandwich method (Giovannetti et al. (1993). Two seedlings were placed between two membranes (Millipore, Bedford; pore diameter of 0.45 μm), with 10–15 fungal spores (or without any spores for controls). Membranes containing the seedlings were planted in sterile acid-washed quartz sand in Magenta GA-7 vessels (Sigma, St Louis, MO) and grown in climate chamber at 22°C, 60% humidity, with 14 h of light per day. After 3 weeks, samples from roots were cut after observation under a stereo microscope and processed for quantification and cytological analyses.

2.2 Mycorrhizal Intensity Evaluation

Mycorrhized root segments (1 cm long) were incubated overnight at room temperature in 0.1% Cotton blue (w/v) in lactic acid. Segments were then washed in lactic acid, and observed under microscope for quantification assays.

According to the method by Trouvelot et al. 1986), segments were classified into four categories depending on the percentage of segment length occupied by mycelium and by arbuscules. Five parameters were considered:

• (1) F%, reporting the percentage of segments showing internal colonization (frequency of mycorrhization)

• (2) M%, indicating the average percent colonization of root segments (intensity of mycorrhization)

• (3) a%, quantifying the average presence of arbuscules within the infected areas (percentage of arbuscules)

• (4) A%, quantifying the presence of arbuscules in the whole root system (percentage of arbuscules in the root system)

2.3 Morphological Observation

Root segments were fixed in 2.5% (v/v) glutaraldehyde in 10 mM sodium phosphate buffer (PBS, pH 7.2) for 2 h at 4°C. After rinsing with the same buffer, samples were postfixed in 1% (w/v) osmium tetroxide in double-distilled water for 1 h, washed three times with double-distilled water, and dehydrated in an ethanol series (30, 50, 70, 90, and 100%; each step for 10 min) at room temperature. The root segments were infiltrated in 2:1 (v/v) ethanol–LR White resin (Polysciences, Warrington, PA) for 1 h at room temperature, in 1:2 (v/v) ethanol–LR White for 1 h at room temperature and in 100% LR White overnight at 4°C.

Semithin sections (1 μm) cut from root-embedded samples were stained with 1% toluidine blue for morphological observations.

Thin sections (70 nm) were counterstained with uranyl acetate and lead citrate and observed under a transmission electron microscope.

2.4 Cytochemical Localization of Polysaccharides (Thiéry et al. 1967)

Thin sections were treated with periodic acid (1%) for 30 min, washed three times in distilled water, and incubated overnight at 4°C in thiocarbohydrazide (0.2%) in 20% acetic acid. They were then washed in descending concentration of acetic acid and incubated for 30 min in silver proteinate (1%) in the dark. Silver-stained sections were viewed without additional staining under a transmission electron microscope.

In Vivo Analysis

3.1 In Vivo Microscopic Observation of Mycorrhizal Infection

The targeted AM inoculation technique developed by Chabaud et al. (2002) is currently the most suitable method for studying early stages of the symbiotic association between Gigaspora species and A.-rhizogenes-transformed root cultures of M. truncatula. Axenic spores of G. rosea or G. gigantea are gently inserted into the M medium containing 0.5% Phytagel in square Petri dishes, and cultured at 32°C at a slope of ~70° in a 2% CO₂ atmosphere for optimal germination according to Bécard et al. (1992). Under these conditions, spores germinate within 3–6 days. Germinated spores are then transferred within a gel plug to the Petri dish containing the transformed root culture, and positioned underneath a growing secondary root, in such a way that the germination hyphae (with negative geotropism) quickly reach the roots, thereby facilitating the identification of potential infection sites. Hyphal growth and root contacts can be recorded daily on the underside of the dish. For confocal microscopy observations, the root and fungus are covered with 1 mL of sterile water, on top of which is laid a 25-μm gas-permeable plastic film (bioFOLIE 25, Sartorius AG, Vivascience Support Center, Göttingen, Germany). The refractive index of the film is compatible with the use of long-distance water-immersion confocal objectives, thus allowing continuous prolonged microscopic observation, convenient transfer of the dish between the growth chamber and the microscope stage, as well as minimizing potential contamination of the coculture. Initial hyphal contact with the root can be monitored using a stereomicroscope. The potential infection points are then observed and followed in detail under a confocal microscope, using a long distance 40× water-immersion objective (Genre et al. 2005).