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Negative effects of potassium supplies and calcium signal inhibitors on oxalate efflux by ectomycorrhizal fungi
South African Journal of Botany 106 (2016) 232–237
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Negative effects of potassium supplies and calcium signal inhibitors on oxalate efflux by ectomycorrhizal fungi H. Yang a, W. Xiang b, J. Huang a, L. Yuan a,⁎, H. Wang c a b c
College of Resources and Environment, Southwest University, Chongqing 400715, China College of Biotechnology, Southwest University, Chongqing 400715, China School of Resources and Environment, University of Jinan, Jinan 250022, China
a r t i c l e
i n f o
Article history: Received 18 October 2015 Received in revised form 8 June 2016 Accepted 21 July 2016 Available online xxxx Edited by R Bottini Keywords: Ectomycorrhizal fungi Potassium Calcium signals Oxalate
a b s t r a c t The main role of ectomycorrhizal fungi is to extract phosphorus (P) for trees. Organic acids, particularly oxalate, are also beneficial to mobilize nutrients such as potassium (K), calcium (Ca) and magnesium (Mg) from minerals and rocks in soils. In the paper, a pure liquid culture experiment was carried out to elucidate the influence of K supplies and inhibitors related to Ca signals and anion channels on the efflux of oxalate and protons by the isolates of Pisolithus tinctorius, Cenococcum geophilum, Lactarius deliciosus and Boletus badius. The fungal isolates varied greatly in the biomass and the absorption of N, P and K. Oxalate, acetate, malate, citrate and succinate were detected in the culture solutions. All the studied fungal isolates could effuse oxalate and the faster exudation was observed at low K supply than high K. The fungal K accumulation correlated negatively with the efflux rates of oxalate (r = −0.359, n = 60) and protons (r = −0.630, n = 60). The stimulation of oxalate and proton efflux by external hyphae in soil with low K could be beneficial to K mobilization from minerals. However, the inhibitors of calmodulin (trifluoperazine and ruthenium red), Ca2+ (verapamil) and anion channels (niflumic acid) decreased the fungal oxalate efflux at low K supply. Therefore, both Ca signals and anion channels involved in the process of the fungal oxalate exudation in low K conditions. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Potassium (K), an element required slightly lower than nitrogen (N), is one of the essential major nutrients for trees in forests. K deficiency retards tree growth and limits forest productivities (Oosterhuis and Berkowitz, 1996; William and Norman, 2004; Tripler et al., 2006). Soils deficient in K are commonly found in the tropical and subtropical areas because of a deep weathering and intensive leaching. Therefore, they usually lack available K, and ectomycorrhizal trees such as pine and eucalyptus in the soil have to extract K from minerals and rocks in soils to satisfy their nutrient requirements. Many forest trees have evolved mutualistic symbiosis with ectomycorrhizal fungi that contribute to their nutrition. In the fungus–tree associations, the fungi obtain carbohydrates from host trees and, in turn, provide the plants with mineral nutrients such as phosphorus (P) and K. Most of K in soils could not be utilized directly by plants because the available K for plants is usually less than 2% of the total soil K (Mengel and Uhlenbecker, 1993). Numerous studies have shown that mycorrhizal plants can extract nutrients, including calcium (Ca), Mg (magnesium), P and K from soil minerals (Landeweert et al., 2001; Zahoor and Zaffar, 2013). The ⁎ Corresponding author. Tel./fax: +86 23 68250426. E-mail address: [email protected] (L. Yuan).
http://dx.doi.org/10.1016/j.sajb.2016.07.021 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
growth of Pinus sylvestris seedlings was significantly stimulated by Paxillus involutus when microcline was used as the sole K source (Wallander and Wickman, 1999). Ectomycorrhizal inoculation might improve the growth and K nutrition of the trees in artificial plantation in nutrient-deficient soils. Yuan et al. (2004) have reported the weathering of minerals and utilization of HCl-extractable K by eucalyptus seedlings inoculated with Pisolithus microcarpus and the concomitant acceleration of the tree's growth rate. Organic acids including oxalate, malate, citrate, succinate and acetate were found in the culture solutions for growing ectomycorrhizal fungal isolates. In European forest soils, the organic acids reached 1 mmol kg− 1 in the soil and numerous 10–50 μm hollow vessels could be observed on the surface of the rocks. They were filled usually with the external hyphae of ectomycorrhizas, and high concentrations of organic acids, especially oxalate, which was detected at the hyphal ends (Landeweert et al., 2001). These organic acids could dissolve the rocks and release nutrient elements such as Ca, Mg and K. It is necessary to point out that both [Fe(C2O4)3]3 − and [Al(C2O4)3]3 − have very high chelation constants of 2.0 × 10 16 and 3.9 × 10 16 (Lapeyrie, 1988; Adeleke et al., 2012). Therefore, oxalate may chelate Al3+ and Fe3+ in the crystal lattice of minerals containing K, resulting in the weathering of these minerals and K release (Fox et al., 1990; Gadd, 1999). Lapeyrie et al. (1987) reported that the biological weathering of minerals was accelerated by the oxalate efflux from
H. Yang et al. / South African Journal of Botany 106 (2016) 232–237
ectomycorrhizal fungus hyphae, resulting in the release of otherwise unavailable K from minerals. Wallander and Wickman (1999) found high concentrations of citrate and oxalate in culture media containing biotite that were used to grow pine seedlings colonized by Suillus variegates. In addition, a large amount of protons were detected in culture media for culturing ectomycorrhizal fungal isolates (Rosling, 2009). Protons can replace interlayer K in 2:1 clay minerals and even destroy their lattice structures, whereby making K available for plants (Wallander, 2000). Thus, the release of protons and organic acids, particularly oxalate, may be a mechanism for ectomycorrhizal fungi to mobilize and utilize unavailable K from minerals and rocks in soils. Moreover, the growth stimulation and concomitant increment of nutrient uptake by mycorrhizal plants is usually observed in poor soils (Huang and Lapeyrie, 1996; Pradeep and Vrinda, 2010). Simultaneous depletion of K and Mg in culture media leads to the weathering of phlogopite by ectomycorrhizal fungi in pure culture, which might be related to the oxalate efflux of ectomycorrhizal fungi (Yuan et al., 2004). Plant roots release organic anions through their channels on plasma membranes and Ca2+ signals participate in this process. Many factors such as channel phosphorylation and allostery, hormones, and cytosolic cations and anions could influence Ca2 + signal network and organic anion efflux (Roberts, 2006). For example, aluminum and copper stimulated significantly the efflux of citrate by Arabidopsis thaliana roots and malate by wheat roots. The stimulation by the metal ions and concomitant reduction in Ca2+ flow across plasma membranes of roots were inhibited by Ca2 + signal and anion channel inhibitors (Yang et al., 2003). K+, as a companying ion of organic acids, activates voltagegated channel of anions through membrane polarization in efflux of organic acids by guard cells in stemma behaviors (Hedrich and Jeromin, 1992). K+ may thus act as a signal factor whereby regulating the efflux of organic acids by plant cells. In our previous studies, we also found that external K supplies could influence the efflux of acetate by the isolates of Pisolithus tinctorius and Lactarius deliciosus grown in culture media, which was also related to Ca2+ signal network (Zhang et al., 2014). It is interesting to understand the efflux of organic acids, particularly oxalate by ectomycorrhizal fungi in response to K supply and Ca2+ signal network. In general, the role of organic acids in mineral weathering on the soil scale remains controversial and the evidence on the influence of external K supply and Ca2+ signals on the efflux of oxalate and protons by ectomycorrhizal fungi is limited. Therefore, the objectives of this study are (i) to study efflux of oxalate and protons by four ectomycorrhizal isolates under different K level supplies in liquid medium; (ii) to explore how Ca2+ signals and anion channels to control oxalate and proton efflux by fungi. 2. Materials and methods 2.1. Fungal strains Four ectomycorrhizal fungal strains used in the experiment, namely P. tinctorius, Cenococcum geophilum, L. deliciosus and Boletus badius, were kept in the microbiology laboratory of the College of Resources and Environment, Southwestern University, Chongqing, China. L. deliciosus and B. badius were originally isolated from subtropical acidic forest soils (pH 4.00–4.52) in Chongqing. C. geophilum from a temperate neutral forest soil (pH 6.47) in Inner Mongolia (all from mycorrhizal Pinus spp.) and P. tinctorius was isolated from a tropical eucalyptus soil (pH 5.77) in Puer, Yunnan, China. Mycelia for inoculation were grown on Pachlewski agar medium for 2 weeks at 25 ± 1 °C in the dark. The medium contained (g L−1) ammonium tartrate 0.5, KH2PO4 1.0, MgSO4 0.5, glucose 20, maltose 5.0, vitamin B1 0.1, agar 20 and 1 ml L−1 microelement solution. (1 L microelement solution contained (mg) H3BO3 8.45, MnSO4 5.0, FeSO4 6.0, CuSO4 0.625, ZnCl2 2.27 and (NH4)2MoO4 0.27).
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2.2. Culture of fungal isolates The KH2PO4 in Pachlewski liquid medium was replaced by NaH2PO4 to give equivalent P and the K treatments were established by adding K2SO4 to the liquid medium at the following concentrations (mg K L−1): 8, 40 and 200. Forest soils in tropic areas are usually considered as K-deficient if available K is less than 40 mg kg−1 (Yuan et al., 2005). Therefore, these K concentrations in the culture media are referred to as low K (K1), normal K (K2) and high K (K3), respectively. A total of 20 mL of Pachlewski liquid medium was transferred into a 100 mL Erlenmeyer flask and steam-sterilized at 121 °C for 30 min. Each flask was inoculated with a mycelial plug (6 mm in diameter) and incubated without agitation for two weeks at 25 ± 1 °C in the dark. 2.3. Experimental treatments Thereafter, the culture solutions were poured out and the mycelia were washed with the sterilized distillation water to remove culture solution on their surfaces. Concerning the absence of Ca2+ in Pachlewski medium and the importance of Ca2 + in plasma membrane integrity, anion channel activation and cytosolic Ca2 + concentrations, 5 mL of CaCl2 sterilized solution (0.1 mol L−1) was then added into each Erlenmeyer flask to incubate fungal mycelia for 24 h. Fungal mycelia were washed with sterilized water and CaCl2 solution was replaced by Pachlewski liquid medium (20 mL) with the same K concentrations as previously described. Simultaneously, the inhibitors related to calmodulin (trifluoperazine, TFP), Ca2 + (verapamil, VP; ruthenium red, RR) and anion channels (niflumic acid, NIF) were added into the culture solutions, respectively. Their concentrations in the mediums were 150.0 μmol L− 1 TFP, 8.0 μmol L− 1 RR, 150.0 μmol L− 1 VP and 15.0 μmol L− 1 NIF. The control was established exactly in the same way except that no inhibitors were added. The fungal mycelia were cultured statically for 48 h at 25 ± 1 °C in the dark with 6 replicate flasks in full randomized design. 2.4. Harvest and analysis Fungal mycelia were harvested by filtration and washed with deionized water to remove the liquid culture medium from the surfaces. They were then oven-dried at (80 ± 2 °C) for 24 h, weighed and digested with H2SO4–H2O2. Nitrogen (N) in digests was analyzed by Kjeldahl procedure, phosphorus (P) by molybdenum blue colorization (Murphy and Riley, 1962) and potassium (K) by flame photometry (Ohyama et al., 1991). Filtrate pH was detected using a PHS-3C pH meter (Shanghai Analysis Instrument Company, China). The proton concentration in solution was obtained according to pH = − log10 [H+]. Thereafter, the culture solutions were acidified by 0.1 mol L− 1 HCl and then analyzed for organic acids by high-performance liquid chromatography (HPLC; HITACHI, Japan). Samples (20 μL) were injected into an Ion300 organic acid analysis column (Phenomenex, Torrance, CA, USA) with 2.5 mmol L− 1 H2 SO 4 as mobile phase at 0.5 mL min − 1 and 450 psi. The retention time was 9.57 min for oxalate, 11.52 min for citrate, 13.31 min for malate, 14.53 min for lactate, 15.95 min for succinate, 17.47 min for formate and 20.72 min for acetate. Standards of organic acids were prepared and analyzed before and after the sample solutions. 2.5. Statistical analysis All data were subjected to analysis of variance using SPSS 20.0 model (ANOVA, Duncan's multiple range test and Pearson's correlation coefficient). All parameters were checked for normality (Shapiro–Wilk) and homogeneity of variance (Levene's test), which showed that all variables fit normality assumption. Differences obtained at levels of P b 0.01 were considered significant.
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H. Yang et al. / South African Journal of Botany 106 (2016) 232–237
Biomass (mg flask-1)
45
isolates (963.23 μg flask−1) and the other three fungal isolates had similar N accumulation (range, 620.21–703.61 μg flask−1). The mean P concentration in mycelia of B. badius was 3.62 mg g−1 dw, significantly higher than L. deliciosus, P. tinctorius and C. geophilum that showed similar mean P concentrations (range, 1.73–2.19 mg g−1 dw). B. badius isolate also had the highest P accumulation (72.74 μg flask−1), followed by L. deliciosus (53.86 μg flask− 1), P. tinctorius (46.24 μg flask−1) and C. geophilum (38.03 μg flask−1). There was no significant difference in mean K concentration between B. badius and C. geophilum (range, 16.08–17.34 mg g− 1 dw), but there is a much higher than those in L. deliciosus and P. tinctorius (range, 5.10–8.43 mg g−1 dw). The mean K accumulation in the fungal isolates had a similar trend to the mean K concentrations.
a
36 b
27
bc
c 18 9 0
B. badius
L. deliciosus
P. tinctorius
C. geophilum
Fig. 1. Fungi biomass (different letters on the bars are significantly different at P b 0.01 and are the same below).
3. Results and analysis 3.3. Efflux of organic acids and protons by ectomycorrhizal fungal isolates 3.1. Fungal growth As shown in Table 1, fungal isolates varied greatly in the efflux of organic acids. Oxalate and acetate were detectable in all culture solutions, malate only in the liquid culture media with L. deliciosus, citrate with P. tinctorius and L. deliciosus, and succinate with L. deliciosus and B. badius. The concentrations of oxalate also varied remarkably in the fungal culture solutions, with the highest oxalate concentration in the culture solution of P. tinctorius isolates but lowest with C. geophilum. In addition, there was no significant difference in acetate concentration in fungal culture solution (range, 30.44–34.26 mg L−1). The total concentration of tested organic acids ranged from 44.79 mg L− 1 (C. geophilum) to 115.82 mg L−1 (P. tinctorius) in culture solutions. The concentrations of total proton ions ranged from 0.04 mmol L−1 (C. geophilum) to 0.50 mmol L−1 (P. tinctorius) in the liquid culture mediums. However, the concentrations of protons ionized from organic acids, calculated by their concentrations and ionization coefficients,
After incubation for two weeks, the four fungal isolates varied significantly in their growth rates, with L. deliciosus showing the highest mean biomass of about 38.54 mg flask− 1, followed by C. geophilum (23.45 mg flask−1), P. tinctorius (19.74 mg flask−1) and B. badius with 16.75 mg flask−1 (Fig. 1). 3.2. Fungal nutrient uptake Ectomycorrizhal fungus isolates also differed greatly in nutrient concentration and accumulation (Fig. 2). After being cultured for 14 days, the mean N concentration in fungal hyphae ranged from 18.26 mg g−1 dw (dw refers to dry weight, and same below) (L. deliciosus) to 41.07 mg g−1 dw (C. geophilum). The mean N accumulation, calculated by multiplying biomass by N concentration, was highest in C. geophilum 50
a
-1
-1
a
30 b
20 10 0
B. badius
L. deliciosus
P. tinctorius
a
1000 N accumulation (µg flask )
N concentration (mg g dw)
a 40
800
b
P. tinctorius
200 0
C. geophilum
B. badius
80
a
-1
b b
b
2 1 B. badius
L. deliciosus
P. tinctorius
bc c
40 20 0
C. geophilum
b
60
B. badius
P concentration
L. deliciosus
P. tinctorius
15 b
10 b 5 0
B. badius
L. deliciosus
P. tinctorius
K concentration
C. geophilum
a
-1
a
K accumulation (µg flask )
K concentration (mg g-1 dw)
a
C. geophilum
P accumulation
500 20
C. geophilum
N accumulation
P accumulation (µg flask )
P concentration (mg g-1 dw)
L . deliciosus
400
a
3
0
b
600
N concentration
4
b
400 a 300 b
b
200 100 0
B. badius
L. deliciosus
P. tinctorius
K accumulation
Fig. 2. Nutrient concentration and accumulation in ectomycorrhizal fungi.
C. geophilum
H. Yang et al. / South African Journal of Botany 106 (2016) 232–237
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Table 1 The concentrations of organic acids in the fungal culture solutions (mg L−1). Fungal strains
Oxalate
Malate
Citrate
Succinate
Acetate
∑X
B. badius L. deliciosus P. tinctorius C. geophilum LSD0.01
22.60 ± 2.30ab 15.59 ± 1.69bc 29.23 ± 4.33a 10.58 ± 1.56c 10.32
ND 22.95 ± 2.35 ND ND
ND 20.11 ± 2.01b 55.71 ± 5.56a ND 19.11
28.61 ± 4.28a 19.39 ± 1.95b ND ND 8.16
30.44 ± 4.10a 34.26 ± 4.25a 30.88 ± 3.98a 34.21 ± 4.20a 7.53
81.64 ± 7.58b 112.30 ± 11.20a 115.82 ± 12.15a 44.79 ± 4.48c 24.57
ND means not detected. In each column, data followed by different small letters are significantly different at P b 0.01 (Duncan's multiple range test) and same below.
changed from 1.14 × 10−3 mmol L−1 to 4.33 × 10−2 mmol L−1 in culture solution, less than 8.49% of total proton ions (Table 2).
through the interlayer of clay minerals such as mica and phlogopite and replace the K+ present. Likely, fast proton efflux by external fungal hyphae at low K occurs in ectomycorrhizas, which could be beneficial to extract K in interlayer of clay minerals. The weathering of minerals containing K caused by oxalate and the replacement of the interlayer K by protons have been demonstrated, albeit not unequivocally, by some scientists (Banfield et al., 1999; Arocena and Glowa, 2000) and might be important in K mobilization and nutrition for trees in forests (Lapeyrie, 1988; Adeleke et al., 2012). The release of organic acids and protons could thus be the mechanism for ectomycorrhizal fungi to obtain K element from the soils lack of available K to satisfy their nutrient demands. It is worth to point out that the four fungal isolates in the present experiment varied greatly in the efflux of organic acids and protons, indicating various abilities of ectomycorrhizal fungus strains to mobilize K from soils. C. geophilum, originally isolated from a neutral and K-rich soil in Inner Mongolia, had less ability to effuse both organic acids and protons than P. tinctorius, L. deliciosus and B. badius from acidic and K-deficient soils in South China. The latter three ectomycorrhizal fungi in K-deficient soils for a long time might evolve the abilities to adapt poor K environments. Efflux of oxalate and protons by ectomycorrhizal fungal isolates varied in response to the K supplies in our present experiment. Higher efflux rates were observed at low K supply (K1) than high K (K2 and K3), and fungal K accumulation showed negative correlations with oxalate (r = − 0.359, n = 60) and proton efflux rates (r = − 0.630, n = 60). Our results are consistent with those of Lapeyrie et al. (1987) and Paris et al. (1995a, 1996) who studied the fungal organic acid efflux and found that simultaneous depletion of K+ and Mg2+ in culture medium in vitro leads to enhanced weathering of phlogopite, and this biological weathering is related to the efflux of protons and oxalate by fungal hyphae (Lapeyrie et al., 1987; Paris et al., 1995b, 1996). Many scientists also found that mineral nutrient deficiencies, particularly P, stimulated greatly the efflux of organic acids and protons from both ectomycorrhizal fungi in pure culture and ectomycorrhizal trees in pot experiments (Paris et al., 1996; Van Hees et al., 2003; Van Schöll et al., 2006a, 2006b; Ouahmane et al., 2009; Turpault et al., 2009). Taking into account of the high solubility of minerals in acidic solutions (Drever, 1994), the organic acids and protons excreted from plant roots and mycorrhizal fungi could be beneficial to decompose mineral grains and otherwise to release nutrients such as Mg, Ca, K and P (Wallander, 2000; Landeweert et al., 2001). Therefore, oxalate and proton exudation by ectomycorrhizal trees in forests could be ecologically and physiologically important for tree nutrition and soil nutrient availabilities. K present in soils might act as a signal to control oxalate efflux by mycorrhizal roots and the increment of oxalate efflux in K-deficient soils could be beneficial to mobilize K from minerals to satisfy the
3.4. Effect of K and signal inhibitor on fungal oxalate efflux As shown in Table 3, ectomycorrhizal fungal isolates released more oxalate into culture solutions with K1 than with K2 and K3. The mean rate of oxalate efflux by four fungal isolates with K1 was doubled at least compared to those with K2 and K3. Inhibitors of Ca2+ signals and anion channels decreased significantly the oxalate efflux rate of the fungal isolates with K1 supply. However, there was no influence of the inhibitors on the oxalate efflux of the fungal isolates with K2 and K3 except that by P. tinctorius, which was decreased as TFP added into culture solution with K2. 3.5. Relations between fungal K and oxalate efflux by ectomycorrhizal fungal isolates The fungal K accumulation by ectomycorrhizal fungal isolates showed significant negative correlations with the efflux rates of oxalate (r = −0.359, n = 60) and protons (r = −0.630, n = 60) (Table 4), and the fungal K concentrations also showed significant negative correlations with the efflux rates of oxalate (r = −0.270, n = 60) and protons (r = −0.256, n = 60). A significant negative correlation between oxalate efflux rate and fungal biomass was also observed (r = − 0.473, n = 60). However, fungal K accumulation was correlated positively with K concentrations in the hyphae (r = 0.468, n = 60) and the Oxalate efflux rate showed significant positive correlations with protons (r = 0.512, n = 60). 4. Discussion Oxalate is one of strongest organic acids, which can ionize a large amount of protons into solutions, because of conjugation in the molecular structure and the high ionization coefficient (Wang, 2009). However, our results showed that the protons ionized from organic acids accounted for small amount of total proton ions in fungal culture solutions, indicating most of protons directly effused from the fungal isolates. The result is coincident with previous reports of Marschner (1995), Fomina et al. (2004, 2006), Gadd (2007); studies by Neumann and Romheld (1999) and Balogh-Brunstad et al. (2008) found that some ectomycorrhizal fungi and trees under nutrient deficiencies, notably including P, Ca, Mg and K, released much more protons than organic acids into culture media. Both protons and K are monovalent. The diameter of protons (0.32 × 10− 10 m) is much smaller than that of K (2.03 × 10− 10 m). Protons may thus have a strong capacity to move
Table 2 The concentrations of protons in culture solutions (mmol L−1). Fungal strains B. badius L. deliciosus P. tinctorius C. geophilum LSD0.01
∑H+ 0.24 ± 0.03b 0.42 ± 0.10ab 0.51 ± 0.12a 0.04 ± 0.002c 0.19
H+ ionized from organic acids −3
3.28 × 10 ± 0.0002c 1.78 × 10−2 ± 0.002b 4.33 × 10−2 ± 0.003a 1.14 × 10−3 ± 0.0001d 1.24 × 103
∑H+ minus H+ ionized from organic acids 0.23 ± 0.02b 0.40 ± 0.09ab 0.47 ± 0.11a 0.04 ± 0.002c 0.19
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Table 3 The effects of potassium and signal inhibitors on fungal oxalate efflux rates. K levels
Signal inhibitors (μmol L−1)
K1
No inhibitor RR TFP NIF VP Averages No inhibitor RR TFP NIF VP Averages No inhibitor RR TFP NIF VP Averages
K2
K3
K effect Inhibitor effect Fungus effect
The secretion rate of oxalic acid in ectomycorrhizal fungi (mg g−1 dw d−1) B. badius
L. deliciosus
P. tinctorius
C. geophilum
1.68 ± 0.34a 0.88 ± 0.25cde 0.80 ± 0.22cde 1.29 ± 0.23b 0.89 ± 0.25cde 1.11 0.65 ± 0.02de 0.66 ± 0.14de 0.52 ± 0.13ef 0.64 ± 0.13def 0.66 ± 0.05de 0.63 0.58 ± 0.13ef 0.47 ± 0.10ef 0.65 ± 0.14de 0.52 ± 0.13ef 0.45 ± 0.08f 0.53 P = 0.0002 ** P = 0.0010 ** P = 0.0001 **
0.86 ± 0.04a 0.44 ± 0.12 cd 0.46 ± 0.12c 0.58 ± 0.16b 0.62 ± 0.03bc 0.59 0.19 ± 0.04e 0.11 ± 0.04e 0.07 ± 0.03e 0.17 ± 0.05e 0.13 ± 0.01e 0.13 0.18 ± 0.01e 0.25 ± 0.05de 0.22 ± 0.01e 0.13 ± 0.04e 0.25 ± 0.02de 0.21 P = 0.0001 ** P = 0.0384 *
1.48 ± 0.09a 0.84 ± 0.10 cd 0.74 ± 0.03cde 1.17 ± 0.30b 0.76 ± 0.10 cd 1.00 0.84 ± 0.07 cd 0.81 ± 0.01 cd 0.42 ± 0.03e 0.63 ± 0.07 cde 0.59 ± 0.10de 0.66 0.94 ± 0.01bc 0.92 ± 0.05 cd 0.90 ± 0.08 cd 0.71 ± 0.17cde 0.84 ± 0.11 cd 0.85 P = 0.0001 ** P = 0.0025 **
0.88 ± 0.13a 0.55 ± 0.16b 0.39 ± 0.10bc 0.36 ± 0.10bc 0.35 ± 0.04bc 0.51 0.34 ± 0.07bc 0.22 ± 0.05 cd 0.18 ± 0.02 cd 0.18 ± 0.02 cd 0.14 ± 0.01 cd 0.21 0.03 ± 0.002d 0.03 ± 0.002d 0.02 ± 0.001d 0.01 ± 0.001d 0.01 ± 0.001d 0.02 P = 0.0001 ** P = 0.0003 **
** means significant differences at P b 0.01; * means significant differences at P b 0.05; NS means no significant differences at P N 0.05.
nutrient requirements of mycorrhizal trees. However, the slow release of oxalate in K rich soils could prevent K from leaching. K+ uptake by Arabidopsis roots from soils involves a K+ uptake module consisting of the two K+ channel alpha-subunits, AKT1 and AtKC1 (Wang et al., 2010). These two channels are essential for root K uptake and cell K homeostasis when plants experience K limited conditions. The channel-mediated K+ uptake from K-deficient soils and K+ leakage vice versa depends on the calcium-sensing proteins CBL and their interacting kinase CIPK. Short supply of K+ is suggested to feed back on cytosolic calcium signals and then the K+ channel protein is subject to activation via a Ca2+-dependent kinase/phosphatase signal network (Li et al., 2006). At low K supply, the fungal oxalate efflux was decreased significantly by inhibitors of Ca2+ signals and ion channels in contrast to high K at which the oxalate efflux rates changed little, if any, as the various inhibitors added. As an accompanying ion of organic acids, K+ maintains charge balance inside and outside cells and activates voltagegated channel of anions through membrane polarization (Bellando et al., 1995). Protons can replace K+ as an accompanying ion of organic acids in K+ limit condition and thus K+ deficiency might act as a signal factor whereby regulating fungal organic acid efflux. In our present experiment, the signal and ion channel inhibitors added into culture solutions included TFP, VP, RR and NIF. The primary application of TFP is for schizophrenia treatment. Most studies have indicated that TFP is a potent calmodulin-specific inhibitor and that calmodulin has two high-affinity binding sites for TFP. Verapamil, a Ca2+ channel blocker, could cease Ca2+ transport across plasma membranes. RR is often used as a pharmacological tool to study specific functions of cellular proteins. This chemical is known to interact with a large number of proteins, including ion channels on plasma membranes, and inhibit intracellular Ca2+ release by the ryanodine receptors. The effects of this Ca2+ channel blocker cannot be removed by the addition of CaCl2 to the external medium in contrast to
ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), which behaved otherwise by the addition of external Ca2+. NIF is widely used to inhibit Ca2+-activated anion channels, being characterized by a very fast block that dramatically decreased current amplitude without altering open-probability. Our results suggest that both Ca2+ signal network and Ca2+-activated anion channels involved the process of the fungal oxalate exudation in low K conditions. Similar findings were also reported by Tang et al. (2008) that Ca2+ signals and anion channels controlled the efflux of malate and citrate by ryegrass and barley roots. Ca2+ signal and anion channel inhibitors decreased simultaneously the efflux of organic acids by lupine and the nutrient extraction from minerals. Therefore, it is reasonable to suggest that both Ca2+ signal network and anion channels participated in the process of fungal oxalate exudation in low K conditions. K deficiencies could be a primary signal acting on calmodulin and Ca2+ channels on the plasma membranes of fungi, which changed the Ca2+ distribution inside and outside of the fungal cells, and then activated a series of reactions responsible for oxalate efflux, probably including gene expression, oxalate synthesis and anion channel activation. In general, ectomycorrhizal fungal isolates differed in the efflux of organic acids and protons. Fungal oxalate exudation was stimulated in low K supplies but inhibitors of Ca signals and anion channels decreased the oxalate efflux by ectomycorrhizal fungal isolates at low K supply. Taking into account the difference between fungal isolates and mutualistic symbionts, further studies need to be carried out with ectomycorrhizal trees. Acknowledgements This work was funded by the 973 Program of China (Project 2013CB127405), National Natural Science Foundation of China (Projects
Table 4 Correlation analysis for K nutrient, efflux rate of oxalate and proton, growth of ectomycorrhizal fungi. Correlation coefficients
K concentration
K accumulation
Oxalate efflux rate
H+ efflux rate
Biomass
K concentration K accumulation Oxalate efflux rate H+ efflux rate Biomass
1 0.468** −0.270* −0.256* 0.123
1 −0.359** −0.630** 0.199
1 0.512** −0.473**
1 −0.026
1
** means significant differences at P b 0.01; * means significant differences at P b 0.05.
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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb
Negative effects of potassium supplies and calcium signal inhibitors on oxalate efflux by ectomycorrhizal fungi H. Yang a, W. Xiang b, J. Huang a, L. Yuan a,⁎, H. Wang c a b c
College of Resources and Environment, Southwest University, Chongqing 400715, China College of Biotechnology, Southwest University, Chongqing 400715, China School of Resources and Environment, University of Jinan, Jinan 250022, China
a r t i c l e
i n f o
Article history: Received 18 October 2015 Received in revised form 8 June 2016 Accepted 21 July 2016 Available online xxxx Edited by R Bottini Keywords: Ectomycorrhizal fungi Potassium Calcium signals Oxalate
a b s t r a c t The main role of ectomycorrhizal fungi is to extract phosphorus (P) for trees. Organic acids, particularly oxalate, are also beneficial to mobilize nutrients such as potassium (K), calcium (Ca) and magnesium (Mg) from minerals and rocks in soils. In the paper, a pure liquid culture experiment was carried out to elucidate the influence of K supplies and inhibitors related to Ca signals and anion channels on the efflux of oxalate and protons by the isolates of Pisolithus tinctorius, Cenococcum geophilum, Lactarius deliciosus and Boletus badius. The fungal isolates varied greatly in the biomass and the absorption of N, P and K. Oxalate, acetate, malate, citrate and succinate were detected in the culture solutions. All the studied fungal isolates could effuse oxalate and the faster exudation was observed at low K supply than high K. The fungal K accumulation correlated negatively with the efflux rates of oxalate (r = −0.359, n = 60) and protons (r = −0.630, n = 60). The stimulation of oxalate and proton efflux by external hyphae in soil with low K could be beneficial to K mobilization from minerals. However, the inhibitors of calmodulin (trifluoperazine and ruthenium red), Ca2+ (verapamil) and anion channels (niflumic acid) decreased the fungal oxalate efflux at low K supply. Therefore, both Ca signals and anion channels involved in the process of the fungal oxalate exudation in low K conditions. © 2016 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Potassium (K), an element required slightly lower than nitrogen (N), is one of the essential major nutrients for trees in forests. K deficiency retards tree growth and limits forest productivities (Oosterhuis and Berkowitz, 1996; William and Norman, 2004; Tripler et al., 2006). Soils deficient in K are commonly found in the tropical and subtropical areas because of a deep weathering and intensive leaching. Therefore, they usually lack available K, and ectomycorrhizal trees such as pine and eucalyptus in the soil have to extract K from minerals and rocks in soils to satisfy their nutrient requirements. Many forest trees have evolved mutualistic symbiosis with ectomycorrhizal fungi that contribute to their nutrition. In the fungus–tree associations, the fungi obtain carbohydrates from host trees and, in turn, provide the plants with mineral nutrients such as phosphorus (P) and K. Most of K in soils could not be utilized directly by plants because the available K for plants is usually less than 2% of the total soil K (Mengel and Uhlenbecker, 1993). Numerous studies have shown that mycorrhizal plants can extract nutrients, including calcium (Ca), Mg (magnesium), P and K from soil minerals (Landeweert et al., 2001; Zahoor and Zaffar, 2013). The ⁎ Corresponding author. Tel./fax: +86 23 68250426. E-mail address: [email protected] (L. Yuan).
http://dx.doi.org/10.1016/j.sajb.2016.07.021 0254-6299/© 2016 SAAB. Published by Elsevier B.V. All rights reserved.
growth of Pinus sylvestris seedlings was significantly stimulated by Paxillus involutus when microcline was used as the sole K source (Wallander and Wickman, 1999). Ectomycorrhizal inoculation might improve the growth and K nutrition of the trees in artificial plantation in nutrient-deficient soils. Yuan et al. (2004) have reported the weathering of minerals and utilization of HCl-extractable K by eucalyptus seedlings inoculated with Pisolithus microcarpus and the concomitant acceleration of the tree's growth rate. Organic acids including oxalate, malate, citrate, succinate and acetate were found in the culture solutions for growing ectomycorrhizal fungal isolates. In European forest soils, the organic acids reached 1 mmol kg− 1 in the soil and numerous 10–50 μm hollow vessels could be observed on the surface of the rocks. They were filled usually with the external hyphae of ectomycorrhizas, and high concentrations of organic acids, especially oxalate, which was detected at the hyphal ends (Landeweert et al., 2001). These organic acids could dissolve the rocks and release nutrient elements such as Ca, Mg and K. It is necessary to point out that both [Fe(C2O4)3]3 − and [Al(C2O4)3]3 − have very high chelation constants of 2.0 × 10 16 and 3.9 × 10 16 (Lapeyrie, 1988; Adeleke et al., 2012). Therefore, oxalate may chelate Al3+ and Fe3+ in the crystal lattice of minerals containing K, resulting in the weathering of these minerals and K release (Fox et al., 1990; Gadd, 1999). Lapeyrie et al. (1987) reported that the biological weathering of minerals was accelerated by the oxalate efflux from
H. Yang et al. / South African Journal of Botany 106 (2016) 232–237
ectomycorrhizal fungus hyphae, resulting in the release of otherwise unavailable K from minerals. Wallander and Wickman (1999) found high concentrations of citrate and oxalate in culture media containing biotite that were used to grow pine seedlings colonized by Suillus variegates. In addition, a large amount of protons were detected in culture media for culturing ectomycorrhizal fungal isolates (Rosling, 2009). Protons can replace interlayer K in 2:1 clay minerals and even destroy their lattice structures, whereby making K available for plants (Wallander, 2000). Thus, the release of protons and organic acids, particularly oxalate, may be a mechanism for ectomycorrhizal fungi to mobilize and utilize unavailable K from minerals and rocks in soils. Moreover, the growth stimulation and concomitant increment of nutrient uptake by mycorrhizal plants is usually observed in poor soils (Huang and Lapeyrie, 1996; Pradeep and Vrinda, 2010). Simultaneous depletion of K and Mg in culture media leads to the weathering of phlogopite by ectomycorrhizal fungi in pure culture, which might be related to the oxalate efflux of ectomycorrhizal fungi (Yuan et al., 2004). Plant roots release organic anions through their channels on plasma membranes and Ca2+ signals participate in this process. Many factors such as channel phosphorylation and allostery, hormones, and cytosolic cations and anions could influence Ca2 + signal network and organic anion efflux (Roberts, 2006). For example, aluminum and copper stimulated significantly the efflux of citrate by Arabidopsis thaliana roots and malate by wheat roots. The stimulation by the metal ions and concomitant reduction in Ca2+ flow across plasma membranes of roots were inhibited by Ca2 + signal and anion channel inhibitors (Yang et al., 2003). K+, as a companying ion of organic acids, activates voltagegated channel of anions through membrane polarization in efflux of organic acids by guard cells in stemma behaviors (Hedrich and Jeromin, 1992). K+ may thus act as a signal factor whereby regulating the efflux of organic acids by plant cells. In our previous studies, we also found that external K supplies could influence the efflux of acetate by the isolates of Pisolithus tinctorius and Lactarius deliciosus grown in culture media, which was also related to Ca2+ signal network (Zhang et al., 2014). It is interesting to understand the efflux of organic acids, particularly oxalate by ectomycorrhizal fungi in response to K supply and Ca2+ signal network. In general, the role of organic acids in mineral weathering on the soil scale remains controversial and the evidence on the influence of external K supply and Ca2+ signals on the efflux of oxalate and protons by ectomycorrhizal fungi is limited. Therefore, the objectives of this study are (i) to study efflux of oxalate and protons by four ectomycorrhizal isolates under different K level supplies in liquid medium; (ii) to explore how Ca2+ signals and anion channels to control oxalate and proton efflux by fungi. 2. Materials and methods 2.1. Fungal strains Four ectomycorrhizal fungal strains used in the experiment, namely P. tinctorius, Cenococcum geophilum, L. deliciosus and Boletus badius, were kept in the microbiology laboratory of the College of Resources and Environment, Southwestern University, Chongqing, China. L. deliciosus and B. badius were originally isolated from subtropical acidic forest soils (pH 4.00–4.52) in Chongqing. C. geophilum from a temperate neutral forest soil (pH 6.47) in Inner Mongolia (all from mycorrhizal Pinus spp.) and P. tinctorius was isolated from a tropical eucalyptus soil (pH 5.77) in Puer, Yunnan, China. Mycelia for inoculation were grown on Pachlewski agar medium for 2 weeks at 25 ± 1 °C in the dark. The medium contained (g L−1) ammonium tartrate 0.5, KH2PO4 1.0, MgSO4 0.5, glucose 20, maltose 5.0, vitamin B1 0.1, agar 20 and 1 ml L−1 microelement solution. (1 L microelement solution contained (mg) H3BO3 8.45, MnSO4 5.0, FeSO4 6.0, CuSO4 0.625, ZnCl2 2.27 and (NH4)2MoO4 0.27).
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2.2. Culture of fungal isolates The KH2PO4 in Pachlewski liquid medium was replaced by NaH2PO4 to give equivalent P and the K treatments were established by adding K2SO4 to the liquid medium at the following concentrations (mg K L−1): 8, 40 and 200. Forest soils in tropic areas are usually considered as K-deficient if available K is less than 40 mg kg−1 (Yuan et al., 2005). Therefore, these K concentrations in the culture media are referred to as low K (K1), normal K (K2) and high K (K3), respectively. A total of 20 mL of Pachlewski liquid medium was transferred into a 100 mL Erlenmeyer flask and steam-sterilized at 121 °C for 30 min. Each flask was inoculated with a mycelial plug (6 mm in diameter) and incubated without agitation for two weeks at 25 ± 1 °C in the dark. 2.3. Experimental treatments Thereafter, the culture solutions were poured out and the mycelia were washed with the sterilized distillation water to remove culture solution on their surfaces. Concerning the absence of Ca2+ in Pachlewski medium and the importance of Ca2 + in plasma membrane integrity, anion channel activation and cytosolic Ca2 + concentrations, 5 mL of CaCl2 sterilized solution (0.1 mol L−1) was then added into each Erlenmeyer flask to incubate fungal mycelia for 24 h. Fungal mycelia were washed with sterilized water and CaCl2 solution was replaced by Pachlewski liquid medium (20 mL) with the same K concentrations as previously described. Simultaneously, the inhibitors related to calmodulin (trifluoperazine, TFP), Ca2 + (verapamil, VP; ruthenium red, RR) and anion channels (niflumic acid, NIF) were added into the culture solutions, respectively. Their concentrations in the mediums were 150.0 μmol L− 1 TFP, 8.0 μmol L− 1 RR, 150.0 μmol L− 1 VP and 15.0 μmol L− 1 NIF. The control was established exactly in the same way except that no inhibitors were added. The fungal mycelia were cultured statically for 48 h at 25 ± 1 °C in the dark with 6 replicate flasks in full randomized design. 2.4. Harvest and analysis Fungal mycelia were harvested by filtration and washed with deionized water to remove the liquid culture medium from the surfaces. They were then oven-dried at (80 ± 2 °C) for 24 h, weighed and digested with H2SO4–H2O2. Nitrogen (N) in digests was analyzed by Kjeldahl procedure, phosphorus (P) by molybdenum blue colorization (Murphy and Riley, 1962) and potassium (K) by flame photometry (Ohyama et al., 1991). Filtrate pH was detected using a PHS-3C pH meter (Shanghai Analysis Instrument Company, China). The proton concentration in solution was obtained according to pH = − log10 [H+]. Thereafter, the culture solutions were acidified by 0.1 mol L− 1 HCl and then analyzed for organic acids by high-performance liquid chromatography (HPLC; HITACHI, Japan). Samples (20 μL) were injected into an Ion300 organic acid analysis column (Phenomenex, Torrance, CA, USA) with 2.5 mmol L− 1 H2 SO 4 as mobile phase at 0.5 mL min − 1 and 450 psi. The retention time was 9.57 min for oxalate, 11.52 min for citrate, 13.31 min for malate, 14.53 min for lactate, 15.95 min for succinate, 17.47 min for formate and 20.72 min for acetate. Standards of organic acids were prepared and analyzed before and after the sample solutions. 2.5. Statistical analysis All data were subjected to analysis of variance using SPSS 20.0 model (ANOVA, Duncan's multiple range test and Pearson's correlation coefficient). All parameters were checked for normality (Shapiro–Wilk) and homogeneity of variance (Levene's test), which showed that all variables fit normality assumption. Differences obtained at levels of P b 0.01 were considered significant.
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Biomass (mg flask-1)
45
isolates (963.23 μg flask−1) and the other three fungal isolates had similar N accumulation (range, 620.21–703.61 μg flask−1). The mean P concentration in mycelia of B. badius was 3.62 mg g−1 dw, significantly higher than L. deliciosus, P. tinctorius and C. geophilum that showed similar mean P concentrations (range, 1.73–2.19 mg g−1 dw). B. badius isolate also had the highest P accumulation (72.74 μg flask−1), followed by L. deliciosus (53.86 μg flask− 1), P. tinctorius (46.24 μg flask−1) and C. geophilum (38.03 μg flask−1). There was no significant difference in mean K concentration between B. badius and C. geophilum (range, 16.08–17.34 mg g− 1 dw), but there is a much higher than those in L. deliciosus and P. tinctorius (range, 5.10–8.43 mg g−1 dw). The mean K accumulation in the fungal isolates had a similar trend to the mean K concentrations.
a
36 b
27
bc
c 18 9 0
B. badius
L. deliciosus
P. tinctorius
C. geophilum
Fig. 1. Fungi biomass (different letters on the bars are significantly different at P b 0.01 and are the same below).
3. Results and analysis 3.3. Efflux of organic acids and protons by ectomycorrhizal fungal isolates 3.1. Fungal growth As shown in Table 1, fungal isolates varied greatly in the efflux of organic acids. Oxalate and acetate were detectable in all culture solutions, malate only in the liquid culture media with L. deliciosus, citrate with P. tinctorius and L. deliciosus, and succinate with L. deliciosus and B. badius. The concentrations of oxalate also varied remarkably in the fungal culture solutions, with the highest oxalate concentration in the culture solution of P. tinctorius isolates but lowest with C. geophilum. In addition, there was no significant difference in acetate concentration in fungal culture solution (range, 30.44–34.26 mg L−1). The total concentration of tested organic acids ranged from 44.79 mg L− 1 (C. geophilum) to 115.82 mg L−1 (P. tinctorius) in culture solutions. The concentrations of total proton ions ranged from 0.04 mmol L−1 (C. geophilum) to 0.50 mmol L−1 (P. tinctorius) in the liquid culture mediums. However, the concentrations of protons ionized from organic acids, calculated by their concentrations and ionization coefficients,
After incubation for two weeks, the four fungal isolates varied significantly in their growth rates, with L. deliciosus showing the highest mean biomass of about 38.54 mg flask− 1, followed by C. geophilum (23.45 mg flask−1), P. tinctorius (19.74 mg flask−1) and B. badius with 16.75 mg flask−1 (Fig. 1). 3.2. Fungal nutrient uptake Ectomycorrizhal fungus isolates also differed greatly in nutrient concentration and accumulation (Fig. 2). After being cultured for 14 days, the mean N concentration in fungal hyphae ranged from 18.26 mg g−1 dw (dw refers to dry weight, and same below) (L. deliciosus) to 41.07 mg g−1 dw (C. geophilum). The mean N accumulation, calculated by multiplying biomass by N concentration, was highest in C. geophilum 50
a
-1
-1
a
30 b
20 10 0
B. badius
L. deliciosus
P. tinctorius
a
1000 N accumulation (µg flask )
N concentration (mg g dw)
a 40
800
b
P. tinctorius
200 0
C. geophilum
B. badius
80
a
-1
b b
b
2 1 B. badius
L. deliciosus
P. tinctorius
bc c
40 20 0
C. geophilum
b
60
B. badius
P concentration
L. deliciosus
P. tinctorius
15 b
10 b 5 0
B. badius
L. deliciosus
P. tinctorius
K concentration
C. geophilum
a
-1
a
K accumulation (µg flask )
K concentration (mg g-1 dw)
a
C. geophilum
P accumulation
500 20
C. geophilum
N accumulation
P accumulation (µg flask )
P concentration (mg g-1 dw)
L . deliciosus
400
a
3
0
b
600
N concentration
4
b
400 a 300 b
b
200 100 0
B. badius
L. deliciosus
P. tinctorius
K accumulation
Fig. 2. Nutrient concentration and accumulation in ectomycorrhizal fungi.
C. geophilum
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Table 1 The concentrations of organic acids in the fungal culture solutions (mg L−1). Fungal strains
Oxalate
Malate
Citrate
Succinate
Acetate
∑X
B. badius L. deliciosus P. tinctorius C. geophilum LSD0.01
22.60 ± 2.30ab 15.59 ± 1.69bc 29.23 ± 4.33a 10.58 ± 1.56c 10.32
ND 22.95 ± 2.35 ND ND
ND 20.11 ± 2.01b 55.71 ± 5.56a ND 19.11
28.61 ± 4.28a 19.39 ± 1.95b ND ND 8.16
30.44 ± 4.10a 34.26 ± 4.25a 30.88 ± 3.98a 34.21 ± 4.20a 7.53
81.64 ± 7.58b 112.30 ± 11.20a 115.82 ± 12.15a 44.79 ± 4.48c 24.57
ND means not detected. In each column, data followed by different small letters are significantly different at P b 0.01 (Duncan's multiple range test) and same below.
changed from 1.14 × 10−3 mmol L−1 to 4.33 × 10−2 mmol L−1 in culture solution, less than 8.49% of total proton ions (Table 2).
through the interlayer of clay minerals such as mica and phlogopite and replace the K+ present. Likely, fast proton efflux by external fungal hyphae at low K occurs in ectomycorrhizas, which could be beneficial to extract K in interlayer of clay minerals. The weathering of minerals containing K caused by oxalate and the replacement of the interlayer K by protons have been demonstrated, albeit not unequivocally, by some scientists (Banfield et al., 1999; Arocena and Glowa, 2000) and might be important in K mobilization and nutrition for trees in forests (Lapeyrie, 1988; Adeleke et al., 2012). The release of organic acids and protons could thus be the mechanism for ectomycorrhizal fungi to obtain K element from the soils lack of available K to satisfy their nutrient demands. It is worth to point out that the four fungal isolates in the present experiment varied greatly in the efflux of organic acids and protons, indicating various abilities of ectomycorrhizal fungus strains to mobilize K from soils. C. geophilum, originally isolated from a neutral and K-rich soil in Inner Mongolia, had less ability to effuse both organic acids and protons than P. tinctorius, L. deliciosus and B. badius from acidic and K-deficient soils in South China. The latter three ectomycorrhizal fungi in K-deficient soils for a long time might evolve the abilities to adapt poor K environments. Efflux of oxalate and protons by ectomycorrhizal fungal isolates varied in response to the K supplies in our present experiment. Higher efflux rates were observed at low K supply (K1) than high K (K2 and K3), and fungal K accumulation showed negative correlations with oxalate (r = − 0.359, n = 60) and proton efflux rates (r = − 0.630, n = 60). Our results are consistent with those of Lapeyrie et al. (1987) and Paris et al. (1995a, 1996) who studied the fungal organic acid efflux and found that simultaneous depletion of K+ and Mg2+ in culture medium in vitro leads to enhanced weathering of phlogopite, and this biological weathering is related to the efflux of protons and oxalate by fungal hyphae (Lapeyrie et al., 1987; Paris et al., 1995b, 1996). Many scientists also found that mineral nutrient deficiencies, particularly P, stimulated greatly the efflux of organic acids and protons from both ectomycorrhizal fungi in pure culture and ectomycorrhizal trees in pot experiments (Paris et al., 1996; Van Hees et al., 2003; Van Schöll et al., 2006a, 2006b; Ouahmane et al., 2009; Turpault et al., 2009). Taking into account of the high solubility of minerals in acidic solutions (Drever, 1994), the organic acids and protons excreted from plant roots and mycorrhizal fungi could be beneficial to decompose mineral grains and otherwise to release nutrients such as Mg, Ca, K and P (Wallander, 2000; Landeweert et al., 2001). Therefore, oxalate and proton exudation by ectomycorrhizal trees in forests could be ecologically and physiologically important for tree nutrition and soil nutrient availabilities. K present in soils might act as a signal to control oxalate efflux by mycorrhizal roots and the increment of oxalate efflux in K-deficient soils could be beneficial to mobilize K from minerals to satisfy the
3.4. Effect of K and signal inhibitor on fungal oxalate efflux As shown in Table 3, ectomycorrhizal fungal isolates released more oxalate into culture solutions with K1 than with K2 and K3. The mean rate of oxalate efflux by four fungal isolates with K1 was doubled at least compared to those with K2 and K3. Inhibitors of Ca2+ signals and anion channels decreased significantly the oxalate efflux rate of the fungal isolates with K1 supply. However, there was no influence of the inhibitors on the oxalate efflux of the fungal isolates with K2 and K3 except that by P. tinctorius, which was decreased as TFP added into culture solution with K2. 3.5. Relations between fungal K and oxalate efflux by ectomycorrhizal fungal isolates The fungal K accumulation by ectomycorrhizal fungal isolates showed significant negative correlations with the efflux rates of oxalate (r = −0.359, n = 60) and protons (r = −0.630, n = 60) (Table 4), and the fungal K concentrations also showed significant negative correlations with the efflux rates of oxalate (r = −0.270, n = 60) and protons (r = −0.256, n = 60). A significant negative correlation between oxalate efflux rate and fungal biomass was also observed (r = − 0.473, n = 60). However, fungal K accumulation was correlated positively with K concentrations in the hyphae (r = 0.468, n = 60) and the Oxalate efflux rate showed significant positive correlations with protons (r = 0.512, n = 60). 4. Discussion Oxalate is one of strongest organic acids, which can ionize a large amount of protons into solutions, because of conjugation in the molecular structure and the high ionization coefficient (Wang, 2009). However, our results showed that the protons ionized from organic acids accounted for small amount of total proton ions in fungal culture solutions, indicating most of protons directly effused from the fungal isolates. The result is coincident with previous reports of Marschner (1995), Fomina et al. (2004, 2006), Gadd (2007); studies by Neumann and Romheld (1999) and Balogh-Brunstad et al. (2008) found that some ectomycorrhizal fungi and trees under nutrient deficiencies, notably including P, Ca, Mg and K, released much more protons than organic acids into culture media. Both protons and K are monovalent. The diameter of protons (0.32 × 10− 10 m) is much smaller than that of K (2.03 × 10− 10 m). Protons may thus have a strong capacity to move
Table 2 The concentrations of protons in culture solutions (mmol L−1). Fungal strains B. badius L. deliciosus P. tinctorius C. geophilum LSD0.01
∑H+ 0.24 ± 0.03b 0.42 ± 0.10ab 0.51 ± 0.12a 0.04 ± 0.002c 0.19
H+ ionized from organic acids −3
3.28 × 10 ± 0.0002c 1.78 × 10−2 ± 0.002b 4.33 × 10−2 ± 0.003a 1.14 × 10−3 ± 0.0001d 1.24 × 103
∑H+ minus H+ ionized from organic acids 0.23 ± 0.02b 0.40 ± 0.09ab 0.47 ± 0.11a 0.04 ± 0.002c 0.19
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Table 3 The effects of potassium and signal inhibitors on fungal oxalate efflux rates. K levels
Signal inhibitors (μmol L−1)
K1
No inhibitor RR TFP NIF VP Averages No inhibitor RR TFP NIF VP Averages No inhibitor RR TFP NIF VP Averages
K2
K3
K effect Inhibitor effect Fungus effect
The secretion rate of oxalic acid in ectomycorrhizal fungi (mg g−1 dw d−1) B. badius
L. deliciosus
P. tinctorius
C. geophilum
1.68 ± 0.34a 0.88 ± 0.25cde 0.80 ± 0.22cde 1.29 ± 0.23b 0.89 ± 0.25cde 1.11 0.65 ± 0.02de 0.66 ± 0.14de 0.52 ± 0.13ef 0.64 ± 0.13def 0.66 ± 0.05de 0.63 0.58 ± 0.13ef 0.47 ± 0.10ef 0.65 ± 0.14de 0.52 ± 0.13ef 0.45 ± 0.08f 0.53 P = 0.0002 ** P = 0.0010 ** P = 0.0001 **
0.86 ± 0.04a 0.44 ± 0.12 cd 0.46 ± 0.12c 0.58 ± 0.16b 0.62 ± 0.03bc 0.59 0.19 ± 0.04e 0.11 ± 0.04e 0.07 ± 0.03e 0.17 ± 0.05e 0.13 ± 0.01e 0.13 0.18 ± 0.01e 0.25 ± 0.05de 0.22 ± 0.01e 0.13 ± 0.04e 0.25 ± 0.02de 0.21 P = 0.0001 ** P = 0.0384 *
1.48 ± 0.09a 0.84 ± 0.10 cd 0.74 ± 0.03cde 1.17 ± 0.30b 0.76 ± 0.10 cd 1.00 0.84 ± 0.07 cd 0.81 ± 0.01 cd 0.42 ± 0.03e 0.63 ± 0.07 cde 0.59 ± 0.10de 0.66 0.94 ± 0.01bc 0.92 ± 0.05 cd 0.90 ± 0.08 cd 0.71 ± 0.17cde 0.84 ± 0.11 cd 0.85 P = 0.0001 ** P = 0.0025 **
0.88 ± 0.13a 0.55 ± 0.16b 0.39 ± 0.10bc 0.36 ± 0.10bc 0.35 ± 0.04bc 0.51 0.34 ± 0.07bc 0.22 ± 0.05 cd 0.18 ± 0.02 cd 0.18 ± 0.02 cd 0.14 ± 0.01 cd 0.21 0.03 ± 0.002d 0.03 ± 0.002d 0.02 ± 0.001d 0.01 ± 0.001d 0.01 ± 0.001d 0.02 P = 0.0001 ** P = 0.0003 **
** means significant differences at P b 0.01; * means significant differences at P b 0.05; NS means no significant differences at P N 0.05.
nutrient requirements of mycorrhizal trees. However, the slow release of oxalate in K rich soils could prevent K from leaching. K+ uptake by Arabidopsis roots from soils involves a K+ uptake module consisting of the two K+ channel alpha-subunits, AKT1 and AtKC1 (Wang et al., 2010). These two channels are essential for root K uptake and cell K homeostasis when plants experience K limited conditions. The channel-mediated K+ uptake from K-deficient soils and K+ leakage vice versa depends on the calcium-sensing proteins CBL and their interacting kinase CIPK. Short supply of K+ is suggested to feed back on cytosolic calcium signals and then the K+ channel protein is subject to activation via a Ca2+-dependent kinase/phosphatase signal network (Li et al., 2006). At low K supply, the fungal oxalate efflux was decreased significantly by inhibitors of Ca2+ signals and ion channels in contrast to high K at which the oxalate efflux rates changed little, if any, as the various inhibitors added. As an accompanying ion of organic acids, K+ maintains charge balance inside and outside cells and activates voltagegated channel of anions through membrane polarization (Bellando et al., 1995). Protons can replace K+ as an accompanying ion of organic acids in K+ limit condition and thus K+ deficiency might act as a signal factor whereby regulating fungal organic acid efflux. In our present experiment, the signal and ion channel inhibitors added into culture solutions included TFP, VP, RR and NIF. The primary application of TFP is for schizophrenia treatment. Most studies have indicated that TFP is a potent calmodulin-specific inhibitor and that calmodulin has two high-affinity binding sites for TFP. Verapamil, a Ca2+ channel blocker, could cease Ca2+ transport across plasma membranes. RR is often used as a pharmacological tool to study specific functions of cellular proteins. This chemical is known to interact with a large number of proteins, including ion channels on plasma membranes, and inhibit intracellular Ca2+ release by the ryanodine receptors. The effects of this Ca2+ channel blocker cannot be removed by the addition of CaCl2 to the external medium in contrast to
ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), which behaved otherwise by the addition of external Ca2+. NIF is widely used to inhibit Ca2+-activated anion channels, being characterized by a very fast block that dramatically decreased current amplitude without altering open-probability. Our results suggest that both Ca2+ signal network and Ca2+-activated anion channels involved the process of the fungal oxalate exudation in low K conditions. Similar findings were also reported by Tang et al. (2008) that Ca2+ signals and anion channels controlled the efflux of malate and citrate by ryegrass and barley roots. Ca2+ signal and anion channel inhibitors decreased simultaneously the efflux of organic acids by lupine and the nutrient extraction from minerals. Therefore, it is reasonable to suggest that both Ca2+ signal network and anion channels participated in the process of fungal oxalate exudation in low K conditions. K deficiencies could be a primary signal acting on calmodulin and Ca2+ channels on the plasma membranes of fungi, which changed the Ca2+ distribution inside and outside of the fungal cells, and then activated a series of reactions responsible for oxalate efflux, probably including gene expression, oxalate synthesis and anion channel activation. In general, ectomycorrhizal fungal isolates differed in the efflux of organic acids and protons. Fungal oxalate exudation was stimulated in low K supplies but inhibitors of Ca signals and anion channels decreased the oxalate efflux by ectomycorrhizal fungal isolates at low K supply. Taking into account the difference between fungal isolates and mutualistic symbionts, further studies need to be carried out with ectomycorrhizal trees. Acknowledgements This work was funded by the 973 Program of China (Project 2013CB127405), National Natural Science Foundation of China (Projects
Table 4 Correlation analysis for K nutrient, efflux rate of oxalate and proton, growth of ectomycorrhizal fungi. Correlation coefficients
K concentration
K accumulation
Oxalate efflux rate
H+ efflux rate
Biomass
K concentration K accumulation Oxalate efflux rate H+ efflux rate Biomass
1 0.468** −0.270* −0.256* 0.123
1 −0.359** −0.630** 0.199
1 0.512** −0.473**
1 −0.026
1
** means significant differences at P b 0.01; * means significant differences at P b 0.05.
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