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. 2021 Jun 28;62(6):959–970. doi: 10.1093/pcp/pcab063

Phosphate Suppression of Arbuscular Mycorrhizal Symbiosis Involves Gibberellic Acid Signaling

Eva Nouri 1, Rohini Surve 2, Laure Bapaume 3, Michael Stumpe 4, Min Chen 5, Yunmeng Zhang 6, Carolien Ruyter-Spira 7,8, Harro Bouwmeester 9, Gaëtan Glauser 10, Sébastien Bruisson 11, Didier Reinhardt 12,*
PMCID: PMC8504448  PMID: 34037236

Abstract

Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase—GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.

Keywords: Symbiosis, Arbuscular mycorrhiza, Petunia hybrida, Rhizoglomus irregularis, Phosphate, Gibberellin

Introduction

Arbuscular mycorrhizal (AM) fungi provide multiple benefits to their host (Chen et al. 2018), in particular increased supply with macronutrient elements such as phosphorus (Karandashov and Bucher 2005), nitrogen (Govindarajulu et al. 2005) and sulfur (Allen and Shachar-Hill 2009) and with the microelements copper (Lehmann and Rillig 2015) and zinc (Cavagnaro 2008). In addition, AM fungi increase the resistance of their hosts against biotic and abiotic stresses (Pozo and Azcon-Aguilar 2007, Abdel-Latef and Miransari 2014), prevent soil erosion by wind and water (Chaudhary et al. 2009) and reduce nutrient leaching from the soil (Cavagnaro et al. 2015). These combined benefits promote plant productivity and biodiversity (van der Heijden et al. 1998) and are considered to be of central importance for future strategies toward more sustainable agriculture systems (Gianinazzi et al. 2010, Solaiman et al. 2014).

However, AM fungal diversity and inoculum potential are often decreased in agricultural soils—an effect that is attributed primarily to intensive farming practices, in particular plowing and fertilization (Douds and Millner 1999, Oehl et al. 2004, Verbruggen and Kiers 2010, van Geel et al. 2015). The major inhibitory component in fertilizers is inorganic phosphate (Pi) (Breuillin et al. 2010, Balzergue et al. 2011, Nouri et al. 2014). Split-root experiments have shown that Pi does not act directly on AM fungi but through the increased P-status of the host plant (Breuillin et al. 2010, Balzergue et al. 2011). Pi is known to decrease host secretion of the AM fungal stimulant strigolactone (SL) (Yoneyama et al. 2007a, 2007b, Balzergue et al. 2011); however, this effect cannot be the main reason for the inhibitory effect of Pi, since the application of exogenous SL cannot rescue AM symbiosis at high Pi levels (Breuillin et al. 2010). Alternative potential mechanisms, including the induction of defense against the fungal partner, have not received experimental support in a transcriptomic study on P. hybrida (Breuillin et al. 2010). Hence, the mechanism of action of Pi in the inhibition of AM symbiosis has remained elusive.

Here, we have tested the hypothesis that the inhibitory effect of Pi on AM symbiosis may involve hormonal pathways, which are known to regulate various aspects of AM symbiosis (Gutjahr 2014). Gibberellic acid (GA) has been identified as a negative regulator of AM symbiosis, whose application can reduce AM colonization and arbuscule abundance, although insufficient GA signaling has also been shown to inhibit AM development (El Ghachtouli et al. 1996, Floss et al. 2013, Foo et al. 2013, Yu et al. 2014, Takeda et al. 2015a, 2015b). Based on these findings, we tested whether the inhibition of AM symbiosis by Pi may involve GA signaling. Our results show that Pi fertilization tends to increase GA levels in roots and that transgenic lines with reduced GA signaling exhibit increased mycorrhizal colonization levels, both in the presence and in the absence of high Pi. In addition, we show that Pi strongly affects the expression levels of several genes involved in GA biosynthesis or signaling. Taken together, these data indicate that endogenous GA has a role in controlling AM colonization at low Pi levels, as well as under high inhibitory Pi conditions.

Results

Assessing hormonal influence in AM of petunia

In order to assess the potential of GA to interfere with AM in petunia (Petunia hybrida), plantlets were inoculated with Rhizoglomus irregulare (MUCL 43204) in the presence of various GA3 concentrations. GA3 concentrations of 1 μM or more significantly reduced AM development, whereby 3 μM GA3 resulted in 63% inhibition of root colonization (from 51.3% to 19%) (Fig. 1A). For comparison, the hormones abscisic acid (ABA), ethylene [applied as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC)] and auxin [in the form of indole-3-acetic acid (IAA)] had only minor effects and only at elevated concentrations (Supplementary Fig. S1). Notably, the stress and defense hormones jasmonic acid (JA; applied as its methyl ester) and salicylic acid (SA) and elicitors of defense responses (flg22, chitin oligosaccharides and yeast extract) had only weak effects on AM symbiosis (Supplementary Fig. S1). Hence, GA3 had the strongest negative impact on AM symbiosis among all tested hormones and elicitors, since it inhibited AM development at the lowest concentrations (1 µM), and it reduced colonization more than any other treatment (compare Fig. 1A and Supplementary Fig. S1). By comparison, even higher concentrations of the other hormones did not cause comparable negative effects (compare Fig. 1A and Supplementary Fig. S1), with the strongest inhibition observed with 100 µM ACC, which reduced colonization by only 40% (from 70% to 41%). ACC is commonly used as a reliable proxy for ethylene in symbiosis research (Okazaki et al. 2004), although ACC can also exert hormonal function on its own (Polko and Kieber 2019). In general, it should be noted that endogenous hormone concentrations may not always be proportional to exogenously applied hormone concentrations, since uptake rates at the root surface may vary between hormones.

Fig. 1.

Fig. 1

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 Effect of GA3 on AM fungal development in P. hybrida. (A) Plants were inoculated with R. irregulare and watered with various concentrations of GA3 from 0.1 μM to 10 μM. Total root colonization was determined after 5 weeks from inoculation. Bars represent the mean of six biological replicates + standard deviation. (B) Plants were inoculated with R. irregulare and the leaves were sprayed with 10 µM GA3. Roots were harvested after 5 weeks and root colonization was determined. Columns represent the mean of four biological replicates + standard deviation. Columns that do not share a letter are significantly different (P < 0.05; one-way ANOVA).

In order to test whether GA may act directly on the AM fungus or indirectly through physiological changes in the plant host, we applied GA3 (10 μM) by spraying it to the leaves of inoculated plants. This resulted in an even stronger reduction of colonization (Fig. 1B), showing that GA3 acts in the host plant and not directly on the AM fungus.

Closer microscopical inspection revealed that GA3 had a pronounced effect on the formation of arbuscules (Fig. 2), fungal structures with which AM fungi supply their hosts with Pi (Harrison 2012), and which are thought to serve for the uptake of lipids from the host (Bravo et al. 2017, Jiang et al. 2017, Keymer et al. 2017, Roth and Paszkowski 2017, Rich et al. 2017b, Brands et al. 2018). Most arbuscules were malformed and exhibited considerably fewer fine branches than in control roots (Fig. 2), suggesting that they could be impaired in their function as an interface for nutrient exchange. Besides the effects on arbuscule formation, GA3 also inhibited the formation of storage vesicles, consistent with the assumption that malformed arbuscules result in reduced carbon transfer to the fungus.

Fig. 2.

Fig. 2

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 GA3 affects arbuscule formation. (A) Quantification of AM fungal structures in mycorrhizal roots after cultivation in the absence of GA (open bars), with 1 μM GA3 (gray bars) or with 10 μM GA3 (black bars) for 5 weeks. The occurrence of fungal structures was quantified for hyphopodia (Hyphop), normal arbuscules (Arbusc), abnormal arbuscules (Abnorm) and vesicles (Vesic). Columns represent the mean of six biological replicates + SD. Columns that do not share a letter are significantly different (P < 0.05; one-way ANOVA).

Confocal microscopic analysis revealed additional subcellular detail, showing that GA3 caused progressive inhibition of arbuscule differentiation over 14 d, with a lesser effect at the infection stage (Fig. 3B, E, H). This effect resembled the inhibitory effects of Pi on AM (Breuillin et al. 2010), which was also particularly pronounced for arbuscule formation (Fig. 3C, F, G). While the effects of Pi and GA3 on AM symbiosis were qualitatively similar, the extent of inhibition of arbuscule formation tended to be more extreme with GA3, in particular at the 14 d time point (Fig. 3H). The fact that the formation of hyphopodia (Fig. 2) and infection of hypodermal cells with hyphal coils (Fig. 3A–C) were not affected indicates that Pi and GA3 act at the level of arbuscule formation rather than at the stage of root infection.

Fig. 3.

Fig. 3

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 Confocal laser scanning analysis of AM colonization in P. hybrida in response to GA3 and phosphate. Plants were inoculated for 5 weeks with R. irregulare and subsequently treated with control solution (A, D, G), 10 µM GA3 (B, E, H) or 5 mM KH2PO4 (C, F, I) for 7 d (A-F) or 14 d (G–I). Then, root samples were cleared with KOH and stained with basic fuchsin and WGA-Alexa488 for confocal laser scanning microscopic analysis. (A–C) hyphal coils in hypodermal cells after 7 d of treatment (D–F) colonization in the cortex after 7 d of treatment (G–I) colonization in the cortex after 14 d of treatment Asterisks denote arbuscules, arrows indicate infection hyphae in hypodermal cells and arrowheads indicate defective fungal structures in the root cortex. All pictures show representative examples from at least 10 assessed root fragments. Size bars, 25 µm.

Expression of GA-related genes in mycorrhizal roots and under high Pi conditions

To test whether Pi impinges on GA, we next explored how Pi affects the expression of GA-related genes, since GA biosynthesis and catabolism are regulated primarily at the transcriptional level (Hedden and Thomas 2012). First, the homologs of GA biosynthetic and metabolizing genes were identified in the genome sequence of the parent of P. hybrida, Petunia axillaris, which is 98.5% identical at the DNA level with P. hybrida (Bombarely et al. 2016). The respective amino acid sequences encoded by GA-related genes from Arabidopsis thaliana and pea (Pisum sativum) were used as queries for searches of the predicted P. axillaris transcriptome by tblastn. Most enzymes in GA biosynthesis and components in GA signaling were found to be encoded in P. axillaris by gene families with multiple members like in A. thaliana and other species (Sun 2008, Hedden and Thomas 2012).

Among a total of 47 predicted GA-related petunia genes, 11 were found to be expressed in control roots or mycorrhizal roots, or in roots treated with 5 mM KH2PO4, a Pi concentration that has been shown to result in efficient inhibition of AM (Breuillin et al. 2010, Nouri et al. 2014) (Supplementary Table S1). In order to test whether Pi influences the expression of these GA-related genes and to see whether they respond to exogenous GA, plants were inoculated and allowed to be colonized by the AM fungus for 4 weeks, followed by a treatment with 5 mM KH2PO4 or with 10 µM GA3, for 3 d or 7 d. Non-mycorrhizal plants were treated in the same way. Established mycorrhizal marker genes (RAM1, RAM2, STR and PT4; Supplementary Table S1) (Rich et al. 2017a) served as markers for AM colonization and for the influence of the treatments on AM symbiosis. This alternative experimental design was chosen because long-term treatments reduce colonization (Breuillin et al. 2010; Fig. 1); hence differences in gene expression could be due directly to the treatment or, indirectly, to the reduced colonization level. Exogenous treatment of mycorrhizal plants changes colonization levels only after >7 d of treatment (Breuillin et al. 2010); hence, in such a scenario, effects on gene expression can be assigned to the treatment with confidence.

Altogether, the expression pattern showed a conspicuous trend. Several GA biosynthetic genes were induced in mycorrhizal roots versus controls (Table 1; AM/c), indicating that GA production may be increased during symbiosis. However, even stronger was the induction of the GA-degrading enzyme GA2 oxidase (GA2ox), represented by the genes GA2ox1 and GA2ox3 (Table 1). These results suggest that GA biosynthesis is induced in mycorrhizal roots, but that at the same time, GA levels are attenuated by GA catabolism. In contrast, Pi repressed most GA-related genes, both in non-mycorrhizal as well as in mycorrhizal roots (Table 1; Pi/c and Pi-AM/AM, respectively). In addition, Pi repressed the AM marker genes in mycorrhizal roots.

Table 1.

Regulation of GA-related genes and AM marker genes by phosphate and GA3

AM/c Pi/c GA/c Pi-AM/AM GA-AM/AM
Group Function 3 d 7 d 3 d 7 d 3 d 7 d 3 d 7 d 3 d 7 d
GA biosynthesis CPS1 6.46* 0.58 0.03 0.01* 1.97 0.23* 0.9 0.4* 0.20* 0.27*
CPS3 2.83 0.66 0.28 0.37* 3.38 0.92 0.83 0.85 1.26 1.95
KS2 0.37* 2.09* 2.05 3.40* 2.00 0.97 1.19 1.85* 0.001 0.96
KS5 2.34 2.23 0.50* 2.00 0.61* 1.27 0.36* 0.30 0.31* 0.92
KO 2.51* 2.24* 0.15* 0.11* 0.43* 0.38* 0.12* 0.21* 0.28* 0.45*
KAO1 1.34 3.50* 0.18* 0.53 0.73 1.55 0.22* 0.44* 1.18 0.71
KAO2 1.64 5.38* 0.71 1.10 0.94 0.65 0.99 0.51 0.87 0.72
GA0ox1 1.20 4.46* 0.69 1.06 0.21 0.06* 0.43 0.50 2.04 0.12*
GA20ox6 3.65 9.82* 0.57 0.23* 0.07 0.18* 0.03* 0.82 0.001* 2.64
GA degradation GA20ox1 33.30* 26.48* 0.26* 3.53 4.06 6.53 0.34 0.08 0.73 1.73
GA0ox3 31.98* 21.88* 0.67* 3.45 2.33* 5.73* 0.63* 0.12 4.08* 1.06
AM-related genes RAM1 66.52* 26.26* 0.51 0.41 7.64 0.72 0.47 0.15* 3.90* 0.43
RAM2 143,086* 9,437* 1.00 2.08 505.4* 1.50 0.10* 0.04 0.42* 1.73
STR 201.3* 5,337* 0.91 1.18 8.11 1.56 0.28 0.02 16.97 1.21
PT4 83,733* 51,766* 0.72 3.31* 169.6* 4.13* 0.08* 0.02* 0.35 1.36
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Gene expression of GA-related genes was determined for ent-copalyl synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20 oxidase (GA20ox) and GA2 oxidase (GA2ox). The genes Required for Arbuscular Mycorrhiza1 (RAM1), RAM2, Stunted Arbuscule (STR) and Phosphate Transporter4 (PT4) served as AM marker genes. Plants were inoculated for 4 weeks and then treated with 5 mM KH2PO4 or 10 µm GA3 for 3 d or 7 d. qPCR was performed with actin7 and GAPDH as reference genes. Relative gene expression values were calculated by the ΔΔCt method (Pfaffl 2001). Data are expressed as fold changes relative to gene expression in the relative control (treatment/control). Gene IDs are available in Supplementary Table S1.

*

Significant differences (P-value < 0.05) are indicated by asterisks.

GA had a similar inhibitory effect as Pi on GA biosynthetic genes, in particular in mycorrhizal roots at the earlier time point (GA-AM/AM; 3 d), consistent with negative feedback regulation (Hedden and Thomas 2012) (Table 1; compare GA-AM/AM and Pi-AM/AM). In contrast, the GA-metabolizing genes GA2ox1 and GA2ox3 tended to be induced by GA, indicative of feed-forward regulation to stimulate removal of GA (Hedden and Thomas 2012). While the effects of Pi and GA were similar on GA biosynthetic genes, they had opposite effects on AM-related genes: GA strongly induced the AM-related genes (Table 1; GA/c), whereas Pi strongly repressed them in mycorrhizal roots (Table 1; Pi-AM/AM), as it was shown in a previous report (Breuillin et al. 2010). Comparison of gene expression in Pi- and GA-treated mycorrhizal roots versus non-treated controls (Supplementary Table S2; Pi-AM/c and GA-AM/c) showed that several GA-related and all AM-related genes were still induced in the mycorrhizal roots but to a lesser extent than in non-treated mycorrhizal roots (AM/c). The unexpected stimulatory effect of GA on AM-related marker genes was confirmed in a second independent experiment (Supplementary Table S3), although in this case the induction of RAM1 by GA was not significant. As in the first experiment, the induction was stronger after 3 d, compared to 7 d after treatment (Supplementary Table S3, GA/c), indicating that GA has a transient effect. On the other hand, a third time point for the Pi treatment (14 d) showed that repression of AM-related marker genes became increasingly strong and was not transient, consistent with earlier findings (Breuillin et al. 2010), and with the strong long-term effect of Pi treatments on AM symbiosis.

Role of GA in phosphate suppression of AM symbiosis

To test whether Pi could potentially act through GA, we measured the levels of GA in the roots of P. hybrida in mycorrhizal and control roots treated with 5 mM KH2PO4. Interestingly, the levels of both GA1 and GA3 tended to be increased upon treatment with exogenous Pi in both mycorrhizal roots (inoculated with R. irregulare) as well as non-mycorrhizal controls (Fig. 4A, B), but in the case of the mycorrhizal roots, the differences were not statistically significant. Overall, two-way analysis of variance (ANOVA) revealed a significant interaction of Pi treatment with time. However, AM had no significant effect on GA levels (Supplementary Table S6), although in general mycorrhizal roots tended to contain lower levels of GA than non-mycorrhizal controls (Fig. 4A, B). To test whether Pi may also act by enhancing the general defense status, we measured the levels of the defense marker SA (Fu and Dong 2013). All samples contained between 40 µg/g and 60 µg/g of SA (Fig. 4C), irrespective of the mycorrhizal status or Pi levels. This is in the range of non-challenged tissues in various plant species (Gao et al. 2015) and therefore indicates that Pi does not act by increasing SA-dependent defense mechanisms in petunia roots. In order to test for other stress- and defense-related pathways, we also quantified the levels of the stress hormones ABA and JA after Pi treatments (Supplementary Fig. S2). In both cases, the levels were very low (approximately 1 ng g−1 fresh weight) and were not affected by Pi treatment in a consistent fashion (Supplementary Fig. S2). Finally, the potential of Pi to induce the stress and defense hormone ethylene was assessed; however, as in a previous study on petunia roots (Chen et al. 2021), ethylene levels were consistently around the detection limit for all treatments. Furthermore, ethylene induction caused by the experimental procedure (root excision and ethylene accumulation in gas-tight glass vials) dominated the very low natural ethylene production rates, hence precluding consistent analyses (data not shown).

Fig. 4.

Fig. 4

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 GA levels in P. hybrida roots in response to AM and phosphate. Plants were inoculated for 5 weeks with R. irregulare and subsequently treated with 5 mM KH2PO4 for 3 d or 14 d. Levels of GA1 (A), GA3 (B) and SA (C) were determined in non-mycorrhizal control plants (open bars), or inoculated plants (filled bars). A significant effect of Pi on GA accumulation was revealed by two-way ANOVA with P = 0.0006 for GA1 (A) and P = 0.0023 for GA3 (B). No effect was observed on the levels of SA. Columns represent the mean of six biological replicates + SD. Columns that do not share a letter are significantly different (P < 0.05; one-way ANOVA).

Based on the finding that Pi tended to induce GA levels, we next wanted to test whether GA may be causally related to the inhibitory effect of Pi on AM. To this end, we used transgenic lines that overexpress a GA-metabolizing enzyme, GA methyltransferase (GAMT) from A. thaliana. GAMT esterifies the carboxyl group of GA, thereby inactivating it (Varbanova et al. 2007). We used two lines, GAMT1 and GAMT2 that had previously been characterized at the molecular level (Varbanova et al. 2007). Transgenic petunia plants expressing these constructs were infertile and did not survive (Varbanova et al. 2007); hence, transgenic tobacco (Nicotiana tabacum) expressing GAMT1 and GAMT2 (Varbanova et al. 2007) were used in our experiments. Firstly, the effect of Pi and GA3 on AM development in tobacco was investigated to see whether this species can serve as an experimental model system to investigate the relationship between Pi and GA3 in AM symbiosis. We quantified AM colonization in response to Pi and GA3 in tobacco and found that, as in petunia, overall colonization was reduced by both 5 mM Pi as well as 10 μM GA3 (Supplementary Fig. S3, compare with Figs. 1–3). Ethanol treatment (0.04%) served as a control for GA3, because this hormone can only be applied from a stock solution in ethanol. Arbuscule formation was particularly sensitive to GA3 and Pi, and the latter had the stronger effect, by almost completely suppressing AM, while GA3 caused a reduction to approximately half the AM levels in controls (compare with Fig. 1A). Taken together, these results show that AM colonization in tobacco is affected by Pi and GA in a similar fashion as in petunia.

Next, we performed a time course experiment to investigate the dynamics of AM development in GAMT lines. Both GAMT1 and GAMT2 plants were colonized more quickly than the wild type (Fig. 5), suggesting that colonization of the wild type is attenuated by endogenous GA. Hence, GA appears to be involved in the regulation of fungal proliferation under conditions that favor AM (low Pi). Next, we explored whether the action of Pi on AM symbiosis is influenced by GAMT activity; hence, plants were treated with 5 mM KH2PO4. In the wild type, this treatment resulted in a 7.5-fold reduction of AM colonization relative to the controls (Fig. 6, left; compare with Supplementary Fig. S3). In contrast, both transgenic lines were less affected by Pi. GAMT1 showed a reduction of only 2.1-fold (Fig. 6, middle) and reached levels of AM colonization in the range of wild-type plants grown at low Pi levels. In GAMT2, AM colonization was reduced 3.9-fold, significantly less than in the wild type (Fig. 6, right). Hence, both GAMT lines exhibited a weaker reaction to Pi than the wild type. The weaker effect of GAMT2 on Pi-dependent inhibition of AM (relative to GAMT1) correlates with higher residual GA levels in GAMT2 over-expressor lines compared to the respective GAMT1 lines (Varbanova et al. 2007). Taken together, these results suggest that at least partially the inhibition of AM by high Pi levels involves GA signaling.

Fig. 5.

Fig. 5

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 GAMT lines exhibit accelerated AM colonization. Wild-type tobacco (N. tabacum) (open bars), transgenic GAMT1 plants (gray bars) and GAMT2 plants (black bars) were inoculated with R. irregulare and grown with basic nutrient solution (low Pi) for the indicated times (3–5 weeks). Columns represent the mean of six biological replicates + SD. Columns that do not share a letter are significantly different (P < 0.05; one-way ANOVA).

Fig. 6.

Fig. 6

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 GAMT lines exhibit reduced inhibition of AM symbiosis by phosphate. Wild-type tobacco (N. tabacum) and transgenic lines (GAMT1 and GAMT2) were inoculated with R. irregulare and grown with and without 5 mM KH2PO4 for 5 weeks. Columns represent the mean of six biological replicates + SD. Columns that do not share a letter are significantly different (P < 0.05; one-way ANOVA). Reduction is indicated as fold change for Pi-treated versus the corresponding control (e.g. 7.5× for the wild type).

Discussion

Exogenous nutrients have a tremendous impact on the development of the root system in plants (Shahzad and Amtmann 2017) and they influence the interaction with AM fungi (Carbonnel and Gutjahr 2014). AM symbiosis is particularly sensitive to Pi, which is the central nutrient provided by AM fungi (Breuillin et al. 2010, Balzergue et al. 2011, Nouri et al. 2014). This provides a strong negative feedback mechanism that allows plants to reduce the costs of AM fungal colonization in case of optimal nutrient supply. Under these conditions, the plant can reach its maximal growth potential without the fungus; thus, fungal colonization would not confer any benefit and could even reduce plant growth (Smith et al. 2009), since mycorrhizal roots consume 4–20% more photosynthates than control roots (Bago et al. 2000). Therefore, AM colonization can result in growth suppression, also known as negative mycorrhizal growth response under conditions that allow non-symbiotic plants to reach their full growth potential. This effect creates a strong selection pressure toward reduction of AM fungal colonization under conditions that are not beneficial for the host.

Pi supply has long been known to be negatively correlated with SL secretion (Yoneyama et al. 2007a, 2007b, Balzergue et al. 2011), such that high phosphate can potentially interfere with early signaling in AM symbiosis. However, this effect cannot explain the strong inhibitory effect of Pi on AM, since SL application cannot restore AM at high Pi levels (Breuillin et al. 2010, Balzergue et al. 2011). Although it has been shown that Pi acts not directly on the fungus, but indirectly by increasing the P-status of the plant (Breuillin et al. 2010, Balzergue et al. 2011), it has long remained unclear how the P-status interferes with AM fungal colonization. GA has been identified as a strong negative regulator of AM in species such as pea (P. sativum), Medicago truncatula, rice (Oryza sativa) and Lotus japonicus (El Ghachtouli et al. 1996, Floss et al. 2013, Foo et al. 2013, Yu et al. 2014, Takeda et al. 2015b). This raises the question whether Pi may act through induction of GA levels or GA signaling. In P. hybrida, GA is the strongest inhibitor of AM among a range of hormones, including the stress and defense hormones ethylene, SA and JA, and established defense signals such as chitin oligosaccharides, flagellin peptide (flg22) and yeast extract, all potent elicitors of a robust defense response (Fig. 1; Supplementary Fig. S1). In addition, exogenous Pi induced GA levels (Fig. 4), consistent with a potential role of GA in the repression of AM by Pi. However, quantitative real-time reverse transcriptase (RT)-PCR indicated that the role of GA in AM may be more complex. Exogenous Pi and GA3 both tended to repress GA biosynthetic genes (Table 1), which is not in line with the induction of GA levels by Pi, but with negative feedback regulation of GA homeostasis by GA (Sun 2008). However, GA levels are not only regulated by GA biosynthesis, but also by GA catabolism, initiated mainly by GA2ox (Sun 2008, Hedden and Thomas 2012). GA2ox was induced by GA and in mycorrhizal roots, while it was repressed by Pi (Table 1).

A more striking difference between Pi and GA was the effect on AM-related genes such as RAM1, RAM2, STR and PT4. These genes that are normally expressed only in mycorrhizal roots were strongly and rapidly induced by exogenous GA3 (at 3 d), whereas they were repressed in mycorrhizal roots by Pi (Table 1, Supplementary Tables S2 and S3 see also Breuillin et al. 2010). Taken together, gene expression analysis reveals that GA3 treatments have overlapping effects with both Pi and AM, respectively, and suggests that GA plays a complex role in AM. The fact that GAMT lines exhibited elevated levels of colonization at low Pi levels (Fig. 5) indicates that GA attenuates colonization even under AM-promoting conditions. In addition, the observation that GAMT lines tolerated higher levels of AM colonization at high Pi levels (relative to the wild type) (Fig. 6) suggests that GA signaling may contribute to the inhibitory effect of Pi on AM.

How can the promotive effect of GA on AM-related genes and the induction of GA biosynthetic genes in mycorrhizal roots be reconciled with the well-documented inhibitory role of GA in AM? GA is required for proper AM development (Takeda et al. 2015a, 2015b), and the fact that gain-of-function mutants in the AM-related signaling component CCaMK induced GA biosynthetic genes (Takeda et al. 2015b) indicates that GA is an essential component of AM development. In light of the strong inhibitory effects of GA on AM, it follows that GA homeostasis (biosynthesis and degradation) and signaling must be under tight control to allow for a balance between promotion and repression of AM. Interestingly, GA has been proposed to be involved in a feedback mechanism by which mycorrhizal plants attenuate AM colonization in response to the increased Pi levels resulting from symbiosis (Floss et al. 2013).

Taken together, these results suggest a mechanism in which mycorrhizal roots exhibit an increased flux through the GA biosynthetic pathway, while steady state GA levels remain relatively low due to high activity of the GA-degrading enzyme GA2ox (Fig. 7). Feedback regulation is known to regulate GA biosynthesis at multiple levels, thereby keeping GA levels in the tissues in narrow limits (Hedden and Thomas 2012). Due to GA inactivation by GA2ox, DELLA proteins in mycorrhizal roots are protected from degradation, allowing for the induction of RAM1 and other AM-related marker genes (Rich et al. 2015, Pimprikar et al. 2016) and resulting in AM development (Floss et al. 2013).

Fig. 7.

Fig. 7

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 Model for the role of the GA pathway in regulation of AM symbiosis by phosphate. Schematic representation of GA function in AM symbiosis based on gene expression patterns and effects of GA3 and Pi on AM symbiosis. Promoting (arrows) and inhibiting (blocks) actions of AM fungi, phosphate (Pi) and GA3 are indicated on GA biosynthesis, GA inactivation by GA2ox; on the induction of AM-related genes (RAM1, RAM2, STR and PT4); and ultimately on functional symbiosis (represented by an arbuscule). GA acts by negative feedback onto GA biosynthesis, feed-forward activation of GA2ox, and by transient induction of AM-related genes (arrow with asterisk). Inhibitory action of GA onto AM symbiosis through the canonical DELLA pathway ultimately counteracts the transient induction of AM-related genes and leads to inhibition of AM symbiosis. Thick arrows exerted by AM fungi indicate the strong induction of GA2ox genes and AM-related genes in the functional symbiosis (see Table 1). Dashed arrows represent multistep pathways.

At elevated Pi levels, GA biosynthetic genes exhibited low expression, but GA degradation was repressed as well, potentially resulting in elevated GA levels (Fig. 4), degradation of DELLA and hence a lack of induction of AM-related genes—therefore inhibition of AM (Fig. 7). The similar expression pattern of GA2ox1 and GA2ox3 relative to the AM-related marker genes (RAM1, RAM2, STR and PT4) (Table 1) indicates that they may be under control of the same regulatory mechanism. Taken together, we suggest that Pi suppression of AM symbiosis is at least partly regulated by GA signaling. However, we cannot exclude that Pi and GA act in parallel independent pathways that converge on AM symbiosis. Furthermore, the role of GA in AM symbiosis is complex (Floss et al. 2013, Takeda et al. 2015a, 2015b) and requires further investigation, in particular the transient induction of AM-related genes by GA3 (Table 1, Supplementary Table S3, Fig. 7).

GA alone cannot account for the entire effect of phosphate on AM, since the overall effect of Pi on AM is stronger than that of GA and the GAMT lines still showed an effect of Pi, although the extent of inhibition was weaker, and higher levels of colonization were tolerated in these lines after Pi addition. These results are compatible with the view that Pi triggers a multi-factorial syndrome that results in the robust inhibition of AM (Breuillin et al. 2010), of which GA is one component.

Hence, future breeding programs should seek to generate crops that exhibit weaker Pi-related inhibition of AM, in order to reconcile the multiple benefits of AM symbiosis with the advantages of fertilization. Such crops may be characterized by attenuated GA signaling in the roots, analogous to the green-revolution crops that exhibit weaker GA signaling in the shoots (Hedden 2003).

Materials and Methods

Plant materials, growth conditions and hormone treatments

Seeds of petunia (P. hybrida, line W115) or of tobacco (N. tabacum) transgenic for GAMT1 or GAMT2 (Varbanova et al. 2007), as well as wild-type tobacco (Xanthi) were germinated on seedling substrate (Klasmann, http://www.klasmann-deilmann.com). After 4 weeks, plantlets were transferred to small pots (volume: 20 ml) with a sterilized mixture of 75% sand with 25% unfertilized soil (vol/vol; further referred to as ‘substrate’) for another 2 weeks. Subsequently, plantlets were transferred to larger pots (volume: 150 ml) with the same substrate and inoculated with one teaspoon (ca. 10 g) of mycorrhizal inoculum per plant directly to the root system. Plants were further cultured as described (Nouri et al. 2014). Plants were weekly fertilized with a nutrient solution containing the following mineral nutrients (Strullu and Romand 1987): 3 mM MgSO4, 0.75 mM KNO3, 0.87 mM KCl, 0.2 mM KH2PO4, 1.52 mM Ca(NO3)2, 20 µM NaFeEDTA, 11 µM MnSO4, 1 µM ZnSO4, 30 µM H3BO3, 0.96 µM CuSO4, 0.03 µM (NH4)6Mo7O24 and 0.01 µM Na2MoO4. For hormonal treatments and Pi or elicitor treatments (Figs. 1–3, 5, 6, Supplementary Figs. S1–S3), plants were watered twice per week with 25 ml of nutrient solution containing the indicated concentrations of GA3 (Sigma-Aldrich; product No. G7645), IAA (Sigma-Aldrich; product No. I2886), ABA (Sigma-Aldrich; product No. 862169), methyl-JA (Sigma-Aldrich; product No. 392707), SA (Sigma-Aldrich; product No. S5922), ACC (Sigma-Aldrich; product No. A3903), flg22 (Meindl et al. 2000), chitin oligosaccharides (Thuerig et al. 2005) or yeast extract (Alfa Aesar, product No. J23547), at indicated concentrations. For ethylene treatments, the precursor ACC was employed, because prolonged treatments during AM development with the gaseous hormone in closed air-tight vessels would cause multiple pleiotropic effects not (or only indirectly) related to ethylene. Foliar GA treatment was performed by spraying the leaves twice per week with 10 µm GA3, while the soil was covered by aluminum foil to avoid contact with the root system. For gene expression and metabolite analysis, plants were first inoculated and grown for 4 weeks in order to reach a colonization rate of approximately 50% and then plants were watered twice per week with 25 ml of 5 mM KH2PO4 or with GA3 as indicated for 3 d, 7 d or 14 d. For all GA3 treatments, the respective control plants were treated with 0.04% ethanol.

AM fungal inoculation

Inoculum of R. irregulare (MUCL 43204) was produced in chive pot cultures. The inoculum consisted of a mixture of soil and roots and was tested for the presence of spores before use. Root staining and quantification of mycorrhizal colonization were carried out as described (Sekhara Reddy et al. 2007).

Microscopy and quantification of root colonization

Roots were harvested, washed and stored overnight in 10% KOH (w/v) in glass tubes. Then, they were cleared for 30 min at 95°C, washed twice with water, stained for 10 min with Trypan Blue (TB) staining solution at 95°C and rinsed with 10% lactic acid (vol/vol). TB staining solution consisted of 10% glycerol (wt/vol), 10% lactic acid (vol/vol) and 0.01% Trypan Blue (wt/vol). Microscopic inspection and quantification of root colonization was carried out as described with a modified grid intersection method (Sekhara Reddy et al. 2007).

Mycorrhizal roots were stained with wheat germ agglutinin coupled to Alexa488 (WGA-Alexa488; Invitrogen, catalog no. W11261) in Soerensen’s phosphate buffer (0.133 M, pH = 7.2), followed by counterstaining with 0.2% basic fuchsin (Sigma, 857343). For microscopy, the roots were immersed in a modified version of ClearSee (Kurihara et al. 2015) containing 10% (w/v) xylitol, 25% (w/v) urea and 2% (w/v) sodium dodecyl sulfate. Images were acquired on a Leica SP5 confocal microscope.

Identification of GA-related genes

GA-related genes were identified by searching the P. axillaris predicted transcriptome at the SolGenomics database (https://solgenomics.net) using established GA-related genes from A. thaliana (Hedden and Thomas 2012) by tblastx. Primers for quantitative real-time RT-PCR were designed by the primer3 tool (https://bioinfo.ut.ee/primer3-0.4.0/) (see Supplementary Table S1). Preliminary analysis of gene expression was performed in mycorrhizal and non-mycorrhizal roots to identify genes that are expressed in any of the tested conditions, and these were used for further qRT-PCR analysis.

RNA extraction and quantitative real-time RT-PCR

Frozen petunia roots were placed in a 2-ml Eppendorf tube containing a glass bead and were ground using a ball mill. Total RNA was extracted from the powdered roots according to the protocol of the Direct-zol RNA miniprep kit from Zymo Research, using trizol solution for lysis (38% vol/vol), saturated phenol (pH 8), 0.8 M guanidine thiocynate, 0.4 M ammonium thiocynate, 0.1 M Na-acetate at pH 5, and 5% (vol/vol) glycerol. The Direct-zol RNA kit involves a DNAse step to remove genomic DNA during RNA extraction. RNA was used for reverse transcription according to the protocol of the SensiFAST™ cDNA synthesis kit from Bioline. PCR reactions were carried out with 5 μl of 100× diluted cDNA solution, 1 μl of 10 μM forward and reverse primers, 12.5 μl of SensiMix SYBR Hi-ROX (BIO-RAD) and DNAse/RNAse free water up to 15 μl. The reaction cycle was 95°C for 10 min followed by 45 amplification cycles (95°C for 20 s, 64°C for 20 s and 72°C for 20 s). All samples were analyzed in technical duplicates from seven independent replicate plants. Actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as reference genes. Relative expression values were calculated using the delta-delta-Ct (ΔΔCt) method (Pfaffl 2001). Gene expression data are shown in Table 1, Supplementary Tables S2 and S3 as fold changes relative to gene expression in the respective control treatment (treatment/control). Averaged expression values of individual treatments, normalized to the two reference genes (actin and GAPDH) are shown in Supplementary Tables S4 and S5.

Extraction and quantification of GAs from roots

GAs were collected from 5-week-old petunia plants in two replicates with each replicate consisting of six plants. For root extracts, 200–250 mg fresh weight of ground root tissue was extracted by 1 ml of methanol: Milli-Q®-water (80:20) containing 2.5 mM diethyldithiocarbamic acid (as antioxidant) and 1 ml of a mixture of internal standards (ISs) at a concentration of 0.02 nmol/ml. After vortexing, samples were shaken at 4°C overnight. Then, the extraction was repeated with 2 ml of methanol:Milli-Q® water (80:20) without IS. The two solvent fractions were pooled in the same 4-ml glass vials for each sample after centrifugation (10 min at 600 g) and then dried under nitrogen flow. A third step of extraction was performed with 2 ml of ethyl acetate (EtOAc) at 4°C for 30 min. Meanwhile, the pooled fractions from methanol extraction were evaporated to near dryness and subsequently combined with the EtOAc fraction in the same 4-ml vials for each sample. Afterward, each vial was evaporated to a volume of approximately 200 µl, taken up in 1 ml of Milli-Q® water, loaded on pre-equilibrated (with 3 ml of methanol and 6 ml of Milli-Q® water) Grace Pure 500 mg C18 columns for purification. The columns were washed with 1 ml of Milli-Q® water, eluted with 4 ml of acetone and evaporated, and the residue was re-dissolved in 200 μl of 25% (vol/vol) acetonitrile in water and filtered through RC4 Minisart 0.2 μm filters before further analysis.

Gibberellins were detected and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) using a Waters Xevo TQ mass spectrometer equipped with an electrospray ionization source and coupled to a Waters Acquity ultraperformance LC system. A water/acetonitrile gradient was applied to an Acquity ultra-high performance liquid chromatography (UPLC) BEH C18 column (100 mm, 2.1 mm, 1.7 mm; Waters), starting from 5% (vol/vol) of acetonitrile for 1 min and rising to 50% (vol/vol) of acetonitrile in 6.67 min, followed by a 0.66-min gradient to 90% (vol/vol) acetonitrile, which was maintained for 0.67 min before going back to 5% (vol/vol) acetonitrile using a 0.13-min gradient, before the next run. Between two measurements, the column was equilibrated for 1.87 min with 5% (vol/vol) acetonitrile. The total run length was 10 min. The column was operated at 50°C with a flow rate of 0.5 ml· min−1. Sample injection volume was 20 μl, and the sample temperature was 10°C. The mass spectrometer was operated in positive electrospray ionization mode for GA1, 4, 5, 8, 9, 19 and 20, and in negative mode for GA3 and 7. Cone and desolvation gas flows were set to 50 and 1,000 l· h−1, respectively. The capillary voltage was set at 3 kV, the source temperature at 150°C and the desolvation temperature at 550°C. Argon was used for fragmentation by collision-induced dissociation. Multiple reaction monitoring (MRM) was used for GA identification and quantification. Parent–daughter transitions for the standards, GA1, 4, 5, 8, 9, 19, 20, 3, 7 and 53 and D2-GA1, 4, 20, 3 and 7 (used as ISs), were set by using the IntelliStart MS Console. MRM transitions selected for identification of GAs in petunia were as follows

In positive mode: for GA9, cone voltage (CV) was set to 14 eV, mass-to-charge ratio (m/z) 317.22 > 225.24 at a collision energy of 30 eV and 317.22 > 271.24 at 16 eV; for GA5, CV 12 eV, m/z 331.22 > 267.22 at 20 eV and 331.22 > 285.18 at 10 eV; for GA20, CV 18 eV, m/z 333.22 > 269.22 at 18 eV and 333.22 > 287.24 at 12 eV; for GA4 CV 12 eV, m/z 333.29 > 269.16 at 18 eV and 333.29 > 315.16 at 8 eV; for D2-GA4 CV 16 eV, m/z 335.22 > 271.23 at 14 eV and 335.22 > 317.23 at 10 eV; for GA44 CV 22 eV, m/z 347.29 > 255.25 at 22 eV and 347.29 > 301.25 at 14 eV; for GA1 CV 20 eV, m/z 349.16 > 285.17 CV at 16 eV and 349.16 > 331.16 at 10 eV; for D2-GA1 CV 10 eV, m/z 351.23 > 287.24 at 16 eV; for GA19 CV 14 eV, m/z 363.22 > 299.26 at 12 eV and 363.22 > 317.27 at 12 eV; for GA8 CV 10 eV, m/z 365.22 > 301.18 at 14 eV and 365.22 > 347.17 at 8 eV.

In the negative mode: for GA7 CV 26 eV, m/z 329.16 > 211.13 at 30 eV and 329.16 > 223.18 at 14 eV; for D2-GA7 CV 26 eV, m/z 331.22 > 213.15 at 26 eV and 331.22 > 225.19 at 24 eV; for GA3 m/z CV 28 eV, 345.16 > 143.04 at 22 eV and 345.16 > 239.21 at 14 eV; for D2-GA3 CV 30 eV, m/z 347.22 > 143.12 at 24 eV and 347.22 > 241.20 at 14 eV; for GA53 CV 48 eV, m/z 347.22 > 303.20 at 28 eV and 347.22 > 329.16 at 22 eV.

GAs were quantified by using a calibration curve with known amount of standards and based on the ratio of the peak areas of the MRM chromatogram for GA standards to the MRM chromatogram for each D2-GA as ISs. GA5, 8, 9, 19 and 53 were quantified by using a calibration curve with known amount of standards (no ISs were available). Data acquisition and analysis were performed by using MassLynx 4.1 (TargetLynx) software (Waters).

Extraction and quantification of SA, JA and ABA

SA was quantified as described (Fragniere et al. 2011) with minor modifications. Root samples of approximately 1 g were extracted with 2 ml of 70% ethanol and 200 μl of the IS ortho-anisic acid (1 ng μl−1) by blending for 30 s with a polytron (Kinematica, NY, USA). After centrifugation (10 min at 1,200 g), the supernatant was transferred to a fresh tube. The pellet was extracted with 2 ml of methanol (90% vol/vol) and centrifuged as before and the two supernatants were combined. After evaporation of the solvents under vacuum at 30°C during 40 min, ca. 400 μl of aqueous solution was left. 500 μl of trichloroacetate (5% vol/vol) were added and the mixture was centrifuged at 5,500 g for 10 min at room temperature. The supernatant was combined with 500 μl of 1:1 ethyl acetate:cyclohexane and mixed thoroughly by vortexing at maximum speed. After centrifugation (2 min at 11,000 g), the upper phase was transferred to a fresh Eppendorf tube and the lower phase was re-extracted with 500 μl of 1:1 ethylacetate:cyclohexane. The combined extracts were dried in a speedvac and dissolved in 200 μl of methanol for analysis with a reverse phase HPLC column (25 cm × 4.6 mm, 5 μm Supelco discovery® C18, Bellefonte, PA, USA), and SA was quantified relative to the IS. JA and ABA were determined by UPLC–MS/MS according to Glauser et al. (2014).

Statistical analysis

All results were tested by one-way ANOVA or two-way ANOVA as indicated. All P-values are listed in Supplementary Table S6.

Supplementary Material

pcab063_Supp
Click here for additional data file. (2.6MB, zip)

Acknowledgements

The authors thank Tatsiana Charnikhova for developing the GA extraction and quantification protocol and Eran Pichersky for providing GAMT1- and GAMT2-expressing tobacco lines. Chitin oligosaccharides were kindly provided by Georg Felix, and flg22 peptide was a gift from Thomas Boller.

Contributor Information

Eva Nouri, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Rohini Surve, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Laure Bapaume, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Michael Stumpe, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Min Chen, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Yunmeng Zhang, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 4, 6708 PB Wageningen, The Netherlands.

Carolien Ruyter-Spira, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 4, 6708 PB Wageningen, The Netherlands; Bioscience, Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 4, 6708 PB Wageningen, The Netherlands.

Harro Bouwmeester, Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 4, 6708 PB Wageningen, The Netherlands.

Gaëtan Glauser, Neuchâtel Platform of Analytical Chemistry, University of Neuchâtel, Neuchâtel 2000, Switzerland.

Sébastien Bruisson, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Didier Reinhardt, Department of Biology, University of Fribourg, Rte Albert Gockel 3, 1700 Fribourg, Switzerland.

Supplementary Data

Supplementary data are available at PCP online.

Funding

Swiss National Center of Competence in Research ‘Plant Survival’ and a grant from the Swiss National Science Foundation (31003A_135778).

Disclosures

The authors declare they have no competing financial interest.

Data Availability

All gene sequences are available at the SolGenomics database (https://solgenomics.net) and can be retrieved using the gene identifiers (ID) listed in Supplementary Table S1.

Author Contribution

E.N. and R.S. carried out most experiments; E.N., L.B., M.S., S.B. and M.C. performed gene expression analysis; E.N. carried out GA and SA measurements with the assistance of Y.Z., C.R.-S. and H.B. G.G. performed JA and ABA analytics. E.N. and D.R. designed the experiments and coordinated the project. All authors contributed to the writing of the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pcab063_Supp
Click here for additional data file. (2.6MB, zip)

Data Availability Statement

All gene sequences are available at the SolGenomics database (https://solgenomics.net) and can be retrieved using the gene identifiers (ID) listed in Supplementary Table S1.

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