pH signature for the responses of arbuscular mycorrhizal fungi to external stimuli

Abstract

Environmental and developmental signals can elicit differential activation of membrane proton (H⁺) fluxes as one of the primary responses of plant and fungal cells. In recent work,¹ we could determine that during the presymbiotic growth of arbuscular mycorrhizal (AM) fungi specific domains of H⁺ flux are activated by clover root factors, namely host root exudates or whole root system. Consequently, activation on hyphal growth and branching were observed and the role of plasma membrane H⁺-ATPase was investigated. The specific inhibitors differentially abolished most of hyphal H+ effluxes and fungal growth. As this enzyme can act in signal transduction pathways, we believe that spatial and temporal oscillations of the hyphal H⁺ fluxes could represent a pH signature for both early events of the AM symbiosis and fungal ontogeny.

Addendum to: Ramos AC, Façanha AR, Feijó JA. Proton (H⁺) flux signature for the presymbiotic development of the arbuscular mycorrhizal fungi. New Phytol 2008; 178:177-88.

The 450-million-year-old symbiosis between the majority of land plants and arbuscular mycorrhizal (AM) fungi is one of the most ancient, abundant and ecologically important symbiosis on Earth.2,3

The development of AM interaction starts before the physical contact between the host plant roots and the AM fungus. The hyphal growth and branching are induced by the root factors exudated by host plants, followed by the formation of appressorium leading to the hyphal penetration in the root system. These root factors seems to be specifically synthesized by host plants, since exudates from non-host plants are not able to promote neither hyphal differentiation nor appressorium formation.4,5 Most root exudates contain several host signals or better, active compounds including flavonoids6,19 and strigolactones,7,8 however many of them are not yet known.

Protons (H⁺) may have an important role on the fungal growth and host signal perception.1 In plant and fungal cells, H⁺ can be pumped out through two different mechanisms: (1) the activity of the P-type plasma membrane (PM) H⁺-ATPase9 and (2) PM redox reactions.10 The proportional contribution from both mechanisms is not known, but in most plant cells the PM H⁺-ATPase seems to be the major responsible by the H⁺ efflux across plasma membrane. AM Fungal cells also energize their PM using P-type H⁺-pumps quite similar to the plant ones. Indeed, some genes codifying isoforms of P-type H⁺-ATPase have been isolated of AM fungi,11–13 and AM fungal ATP hydrolysis activity was shown by cytochemistry, localized mainly in the first 70 µm from the germ tube tip.14 This structural evidence correlates with data obtained by H⁺-specific vibrating probe (Fig. 1A and B), which indicates that the H⁺ efflux in Gigaspora margarita is more intense in the subapical region of the lateral hyphae1 (Fig. 1A). Furthermore, the correlation between the cytosolic pH profile previously obtained by Jolicoeur et al.,15 with the H⁺ efflux pattern (erythrosine-dependent), seems to clearly indicate that an active PM H⁺-ATPase takes place at the subapical hyphal region. Using orthovanadate, we could show that those H⁺ effluxes are susceptible mainly in the subapical region, but no effect in the apical was found.1 Recently, a method to use fluorescent marker expression in an AM fungus driven by arbuscular mycorrhizal promoters was published.31 It could be adjusted as an alternative to measure “in vivo” PM H⁺-ATPase expression in AM fungal hyphae and their responses to root factors.31

The H⁺ electrochemical gradient generated by PM H⁺-ATPases provides not only driving force for nutrient uptake,9,16 but also can act as an intermediate in signal transduction pathways.18 The participation of these H⁺ pumps in cell polarity and tip growth of plant cells was recently reported,27 addressing their crucial role on apical growth.28 Naturally, in the absence of root factors the AM fungi have basal metabolic8,21–23 and respiratory activity.24 However when root signals are recognized and processed by AM fungal cells they might become activated.22 We thus searched for pH signatures that could reflect the alterations on fungal metabolism in response to external stimuli. In fact, preliminary analyses from our group demonstrate that AM fungal hyphae increase their H⁺ efflux in response not only to root exudates recognition, but also to other root factors (Fig. 1C). The incubation for 30 min of AM fungal hyphae with several root factors induces hyphal H⁺ efflux similar to the response to intact root system (5 days of incubation). The major increases were found with 1% CO₂ (750%) followed by root cell wall proteins (221%), root exudates (130%) and synthetic strigolactone (5%) (Fig. 1C). Those stimulations could define the transition from the state without root signals to the presymbiotic developmental stage (Fig. 1C). In the case of CO₂, the incorporation of additional carbon could represent a new source of energy, since CO₂ dark fixation takes place in Glomus intraradices germ tubes.22,25

Interestingly, after the treatment with synthetic strigolactone (10⁻⁵ M GR24), no significant stimulation was found compared to the remaining factors (Fig. 1C). It opens the question if the real effect of strigolactone is restrict to hyphal branching and does not intervene in very fast response pathways. Likewise, strigolactones need additional time to exhibit an effect, as recently discussed by Steinkellner et al.,26 However, at the moment, no comprehensive electrophysiological analyses are presently available separating the effects of strigolactone and some flavonoids in AM fungal hyphae.

The next target of our work is the study of ionic responses of single germ tubes or primary hyphae to root factors (Fig. 2). As reported by Ramos et al.,1 we have been observing that the pattern of ion fluxes at the apical zone of primary hyphae is differentiated from secondary or lateral hyphae. In the primary, two interesting responses were detected in the absence of root factors: (1) a “dormant Ca²⁺ flux” and (2) Cl⁻ or anion fluxes at the same direction of H⁺ ions, suggesting a possible presence of H⁺/Cl⁻ symporters at the apex, similarly to what occurs in root hairs (Fig. 2).30 In the presence of root factors such as root exudates the stimulated influxes of Cl⁻ (anion), H⁺, Na⁺ and effluxes of K⁺ and Ca²⁺ are activated. It can explain why the AM fungi hyphal tips are depolarized20,29 during the period without root signals—“asymbiosis”—as long as K⁺ efflux and H⁺ influx occur simultaneously. Indeed, H⁺ as well as Ca²⁺ ions may act as second messengers, where extra and intracellular transient pH changes are preconditions for a number of processes, including gravity responses and possibly in plant-microbe interactions.17,30

Clearly, further data on the mechanism of action of signaling molecules such as strigolactones over the signal transduction and ion dynamics in AM fungi will be very important to improve our understanding of the molecular bases of the mycorrhization process. Future studies are necessary in order to provide basic knowledge of the ion signaling mechanisms and their role on the response of very important molecules playing at the early events of AM symbiosis.

Figures and Tables

Figure 1 (A) H⁺ flux profile along growing secondary hyphae of G. margarita in the presence (open squares) or absence (closed squares) of erythrosin B and its correlation with cytosolic pH (pHc) data described by Jolicoeur et al.,15 (dotted line). Dotted area depicts the region with higher susceptibility to erythrosin B. (B) ion-selective electrode near to AM fungal hyphae. (C) Stimulation on hyphal H⁺ efflux after incubation with root factors or whole root system. R, roots; RE, root exudates; CO₂, carbon dioxide; CWP, cell wall proteins; GR24, synthetic strigolactone. The medium pH in all treatment was monitored and remained about 5.7, including with prior CO₂ incubation. Means followed by the same letter are statistically equal by Duncan's test at p < 5%.

Figure 2 Ion dynamics in the apex of primary hyphae of arbuscular mycorrhizal fungi. It represents the Stage 1 described in Ramos et al.1 After treatment with root factors, an activation of Ca²⁺ efflux is observed at the hyphal apex.

Addendum to: