Journal of Experimental Botany, Vol. 51, No. 345, pp. 747–754, April 2000
Alterations in the plasma membrane polypeptide pattern of tomato roots (Lycopersicon esculentum) during the development of arbuscular mycorrhiza Karim Benabdellah, Concepcio´n Azco´n-Aguilar and Nuria Ferrol1 Departamento de Microbiologı´a del Suelo y Sistemas Simbio´ticos, Estacio´n Experimental del Zaidı´n (CSIC), Profesor Albareda 1, 18008, Granada, Spain Received 20 August 1999; Accepted 12 November 1999
Abstract Changes induced by arbuscular mycorrhizal (AM) formation in the plasma membrane polypeptide pattern of tomato roots have been assessed by 2D-PAGE analysis. Plasma membrane fractions were isolated by aqueous two-phase partitioning from control and mycorrhizal tomato root microsomes. Analysis of 2D-PAGE gels revealed that AM colonization induces at the plasma membrane level two major changes in protein synthesis: down-regulation of some constitutive polypeptides and synthesis of new polypeptides or endomycorrhizins. A comparison of changes induced by two different levels of AM colonization showed that 16 polypeptides were differentially displayed at both AM colonization stages, while some others were transiently regulated. Five of the differentially displayed plasma membrane polypeptides at both AM colonization stages were selected for N-terminal amino acid sequencing. Reliable sequences were obtained for two of the selected spots. Sequence alignment search indicated that one of the sequenced polypeptides showed 75% identity to the N-terminal sequence of the 69 kDa catalytic subunit of the vacuolar type H+-ATPase of several plants. The possible significance of these findings is discussed in relation to the functioning of the AM symbiosis. Key words: Arbuscular mycorrhizas, plasma membrane, 2D-PAGE, endomycorrhizins, V-type H+-ATPase.
Introduction Arbuscular mycorrhizal (AM ) fungi belonging to the order Glomales (Zygomycota) are obligate biotrophs that
form mutualistic symbioses with most of the agriculturally important plant species (Barea and Jeffries, 1995). Plant colonization by the AM fungus and the development of an active symbiosis induces considerable morphological and physiological changes in both symbionts (Bonfante and Perotto, 1995). These changes are presumably the result of a complex sequence of interactions between the fungus and the plant root. As in other plant–microbe systems, it is likely that regulation of these interactions requires a continuous exchange of signals between both partners, which, when perceived through the corresponding receptors, induce a cascade of events leading to changes in the expression of certain genes ( Koide and Schreiner, 1992; Boller, 1995). Although the nature of those signals and receptors in AM symbiosis is yet unknown, there is increasing evidence that the AM association is controlled by the differential expression of certain genes (Harrison, 1997). The existence of plant mutants unable to form AM symbiosis confirms the hypothesis that specific plant genes are involved in the establishment of the symbiosis (Duc et al., 1989; Gianinazzi-Pearson et al., 1995; Barker et al., 1998; Wegel et al., 1998). This has been corraborated recently (Samra et al., 1997), when it was shown that the 2D-PAGE pattern of root proteins isolated from the AM-compatible (wild-type) and the AM-resistant (myc−nod−) pea genotypes were different and that the changes induced by AM colonization in both genotypes were also very different. It is well known that in AM symbiosis the plant benefits from a better nutritional status in exchange for photosynthate to the fungus. This bidirectional nutrient transport is crucial for the functional integration of the symbionts (Smith and Smith, 1996). The symbiotic
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748 Benabdellah et al. interfaces where the plasma membranes of both symbionts are associated, are considered to play a central role in regulating nutritional and signal exchanges between both partners (Smith and Smith, 1990). Therefore, it is expected that the activity, composition and structure of plasma membranes of both organisms are altered at the symbiotic interfaces. In this sense, it has been shown that one of the most dramatic alterations in the colonized host cell occurs at the interface level, where the plasma membrane invaginates and proliferates around the hyphal branches of the arbuscule, increasing 4–10-fold its surface area (Alexander et al., 1989). This peri-arbuscular plasma membrane has a high H+-ATPase activity, contrasting with the low activity of plasma membranes of uncolonized cells (Gianinazzi-Pearson et al., 1991). Furthermore, 2D-PAGE analysis of tomato root microsomal fractions has shown that AM colonization induces up-regulation and down-regulation of some constitutive membranebound polypeptides as well as induction of some new membrane polypeptides or endomycorrhizins (Benabdellah et al., 1998). Interestingly, immunological studies using monoclonal antibodies raised against membrane components of pea nodules revealed that some of the antigenic components were also expressed in the periarbuscular membrane (Perotto et al., 1994). Moreover, cloning and expression studies of genes encoding a plant plasma membrane H+-ATPase isozyme (Murphy et al., 1997), two phosphate transporters (Liu et al., 1998) and a sugar transporter (Harrison, 1996) have evidenced that AM symbiosis induces regulation of genes involved in membrane transport processes. Although the above-mentioned studies indicate that the host plasma membrane undergoes multiple changes as a consequence of AM formation, further studies using isolated plasma membrane vesicles need to be done. The aim of the present work was to study by 2D-PAGE, a useful technique for the global analysis of protein synthesis, changes induced by AM colonization in the polypeptide pattern of tomato root plasma membrane vesicles isolated by aqueous two-phase partitioning of microsomal fractions. Additionally, some of the differentially displayed plasma membrane proteins were N-terminal sequenced.
Materials and methods Plant material and growth conditions Tomato (Lycopersicon esculentum Mill cv. Earlymech) seeds were surface-sterilized and pregerminated in sterile vermiculite. Seedlings were grown in containers (1.0 l ) containing a sterile mixture of sand/vermiculite (1/1, v/v). Half of the plants were inoculated with a soil–sand-based inoculum containing fungal propagules and chopped mycorrhizal roots of the AM fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe (BEG 12). Control plants received a filtrate (
