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Plants grow in the company of myriads of other organisms: many are direct or indirect competitors, or even pathogenic, others can be beneficial. To differentiate between friend and foe is therefore essential for a plant’s survival. Chemical signalling often precedes and enables consideration as to whether a foreign organism is a friend. Plant cells detect extracellular chemical cues often before physical contact, which when perceived, trigger the generation of secondary signals and downstream events. Decoding this complexity subsequently leads to the plant’s response enabling a friendly relationship. In the arbuscular mycorrhizal symbiosis (AMS) of most terrestrial plants with soil fungi of the Glomeromycota, presymbiosis (i.e. the chemical phase before physical interaction) is prerequisite for reprogramming plant cells towards accommodation of the microbial symbiont. In this issue of New Phytologist, Kuhn et al. (pp. 716–733) report new results suggesting that steroids may act as early arbuscular mycorrhizal (AM) signals.
‘…steroids could indeed play an important role in many aspects of AMS including hyphal penetration and arbuscule formation. Steroids would then join the club of lipids as mycorrhizal signals …’
Epidermal responses and cellular accomodation
Presymbiosis is a prerequisite for hyphopodium (or appressorium) formation, which is the first morphological element in the AMS. The formation of an AM fungal hyphopodium on host roots is probably induced upon contact through thigmotropic signals specifically from the host rhizodermal cell wall (Giovannetti et al., 1993; Nagahashi & Douds, 1997); it cannot be induced on artificial surfaces or on nonhost roots. How do AM fungi subsequently penetrate root cells? There are two options. Penetration of the epidermal layer has been shown to occur via opening of a rhizodermal cleft between two cells through which the fungus enters (Parniske, 2004). Alternatively, the AM fungus can cross epidermal cells directly through a tunnel-like intracellular structure that forms subsequent to hyphopodium formation and before fungal penetration. The tunnel, named the prepenetration apparatus (PPA) (Genre et al., 2005), is formed as a result of massive membrane restructuring and biosynthesis throughout hyphal colonization and arbuscule formation in roots (Genre et al., 2008). Kuhn et al. used an elegant approach to identify marker genes for the early developmental phase in the AMS of Medicago truncatula, a widely used model species for root symbiosis research. An in vitro system allowed harvesting of root segments containing contact points between fungus and host plants or even developing hyphopodia. Gene expression profiling using RNA extracted from these segments yielded, among others, a candidate gene encoding the membrane-localized steroid-binding protein 1 (MtMSBP1). The results further indicated that MtMSBP1 gene expression was stimulated by a diffusible fungal signal(s). This work agrees with that of other groups indicating that perception of diffusible fungal signals precedes the transcriptional cellular reprogramming of host epidermal cells.
The enigmatic ‘Myc factor’
These attempts, destined to identify soluble compounds involved in early signal perception mechanisms during AMS establishment, postulate the existence of a ‘Myc factor’, an AM fungal signal analogous to the rhizobial Nod factor that induces molecular responses in the host root. After many years, its chemical nature is still enigmatic. Kosuta et al. (2003) demonstrated that the symbiosis-specific M. truncatula early nodulin ENOD 11 (MtENOD11) gene is induced in the roots in absence of physical contact between both symbiotic partners. Overall, evidence was provided for a diffusible AM fungus-specific signal of < 3500 Da (Chabaud et al., 2002). The Bonfante laboratory later showed that spores of Gigaspora margarita, as well as of two Glomus isolates, released diffusible molecules into the culture medium, which were perceived by plant roots via Ca2+-mediated signalling (Navazio et al., 2007). The fungal molecules were found to be thermostable, to have a molecular mass of > 3000 Da and to be amphiphilic. Moreover, a diffusible AM fungal factor from Gigaspora and Glomus species was found to stimulate lateral root formation in M. truncatula (Olah et al., 2005). It is unknown whether the MtENOD11 promoter-activating factor, the described fungal spore signal, the diffusible AM fungal factor and the MtMSBP1 gene-inducing factor are the same, or different, ‘Myc factors’. It is certainly of outstanding interest to characterize the chemical nature of these factors and underlying processes.
Biosynthesis and transport of plant steroids
Steroids consist of a sterane core (saturated tetracyclic hydrocarbon 1,2-cyclopentanoperhydrophenanthrene), which is a carbon structure of four fused rings, three cyclohexane rings and one cyclopentane ring that are partially or completely hydrogenated. This core is generally substituted by additional functional groups at distinct C positions (Fig. 1). Natural steroids are derived from squalene and may thus be considered as modified triterpenes.
Knowledge of steroid biosynthesis is based upon groundbreaking discoveries made by the three Nobel laureates, Otto Wallach (1910), Leopold Ruzicka (1939) and Feodor Lynen (1964). Steroid biosynthesis is an anabolic metabolic pathway that produces steroids from isopentenylpyrophosphate, which is generated in plants via two different isopreonid (or terpenoid) biosynthetic pathways: (1) with two molecules of acetyl-CoA as the original substrate in the cytosol or (2) with pyruvate and glycerinaldehyde-3-phosphate in the plastids. Steroids are made from two molecules of farnesyl pyrophosphate forming the triterpene molecule squalene (C30). In plants, steroids are basic products for membranes and defense compounds; they are also recognized as essential hormones in plants as well as in animals. Among numerous plant steroids, brassinolide (BL) is the most active form of the known growth-regulating brassinosteroids (BRs). The structure of BL was determined by Grove et al. (1979) and showed similarity to animal steroid hormones. As a result of its low concentration, the identification of BL took 10 yr of dedicated work (Mandava, 1988). MSBP1 from Arabidopsis is involved in the inhibition of cell elongation (Yang et al., 2005) possibly through affecting vesicle trafficking and auxin distribution (Yang et al., 2008). MSBP1 was shown to bind to multiple steroid molecules, including BR, with different affinities (Yang et al., 2005). Sterols (i.e. steroid alcohols) of AM fungal origin are used as biochemical markers to determine the abundance of AM fungi and AM fungal biomass (see Olsson et al., 2003). Only recently, the first gene involved in the steroid biosynthetic pathway, so far described from an AM fungus has been described (Oger et al., 2009). Overall, the knowledge on steroid biosynthetic pathways and the complexity of steroid chemical diversity, in mycorrhizal roots, is rather poor (Campagnac et al., 2008; Oger et al., 2009).
It is uncertain whether steroids can act as diffusible ‘Myc factors’. Steroids are relatively nonpolar molecules. Thus, whilst short-distance intracellular or intercellular transport of steroids appears possible, it seems unlikely that it occurs through the simple passive diffusion of free bioactive steroids within and between cells. As a consequence, it is likely that short-distance steroid transport involves carrier mechanisms, which allows these molecules to move through the diverse cellular environments of a mycorrhiza. Kuhn et al. subcellularly localized the MtMSBP1 protein to the plant endoplasmic reticulum (ER) where steroids may be produced on membranes then enter the cytosol and move to the plasma membrane, cross the plasma membrane and enter the extracellular environment, where they are perceived at the surface of surrounding cells. Along this scenario, steroids, instead of being an extracellular fungal signal, could be of plant origin and act as secondary signals. Indeed, membrane-bound, ATP-binding cassette (ABC) transporters facilitate the movement of steroids out of animal cells, and it is possible that homologous mechanisms may exist in plants (Young & Fielding, 1999).
Provided that the function of MtMSBP1 follows sequence homology, a striking mycorrhizal phenotype, demonstrated in Kuhn et al. as a result of an MtMSBP1 gene knockdown in M. truncatula, indicated that steroids could indeed play an important role in many aspects of the AMS, including hyphal penetration and arbuscule formation. Steroids would then join the club of lipids as mycorrhizal signals, namely the lysolipid lysophosphatidylcholine (LPC) originating from phosphatidylcholine, a common lipid in eukaryotic membranes. Lysophosphatidylcholine was shown to trigger phosphate transporter gene expression in a mycorrhiza-specific manner (Drissner et al., 2007).
Life requires membranes, and AMS may require lipid signals. These signals could be generated whilst membrane invaginations during PPA formation early, and arbuscule development late, occur in AM development. They would thus pave the way towards symbiotic exchange of goods in this friendly partnership. Overall, the broadening horizon of lipid research in AMS is opening up exciting vistas for the future.