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Soil organic nitrogen availability predicts ectomycorrhizal fungal protein degradation ability

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In temperate and boreal forest ecosystems, nitrogen (N) limitation on tree metabolism is alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in soil switches from inorganic (NH4 (+), NO3 (-)) to organic (mostly proteins). It has been hypothesized that ECM adapt to the most common N source in their environment, which implies that fungi growing in older forests would have higher protein degradation abilities. Moreover, recent results on a model ECM fungal species suggest that organic N uptake requires glucose supply. To test the generality of these hypotheses, we screened 55 strains of 13 Suillus species with different ecological preferences for their in vitro protein degradation abilities. Suillus species preferentially occurring in mature forests, where soil contains more organic matter, had significantly higher protease activity than those from young forests with low organic matter soils or species indifferent to forest age. Within species, the protease activities of ecotypes from soils with high or low soil organic N content did not differ significantly, suggesting resource partitioning between mineral and organic soil layers. The secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition had variable effects on secreted protease activity; in some species it triggered activity but in others it was repressed at high concentrations. Collectively, our results indicate that protease activity, a key ectomycorrhizal functional trait, is positively related to environmental N source availability, but also influenced by additional factors such as carbon availability.
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Soil organic nitrogen availability predicts ectomycorrhizal fungal protein degradation ability 1
2
Francois Rineau
a
#, Jelle Stas
a*
, Nhu H. Nguyen
b
, Thomas W. Kuyper
c
, Robert Carleer
a
, Jaco 3
Vangronsveld
a
, Jan V. Colpaert
a
, Peter G. Kennedy
b
4
Centre for Environmental Sciences, Environmental Biology group, Hasselt University, 5
Hasselt, Belgium
a
; Department of Plant Biology, University of Minnesota, St. Paul, MN, 6
USA
b
;Department of Soil Quality, Wageningen University, Wageningen, The Netherlands
c
7
8
Running head: Degradation of proteins by ECM fungi 9
#Address correspondence to Francois Rineau, francois.rineau@uhasselt.be 10
*Present address: Jelle Stas, Jaico RDP, Opglabbeek, Belgium 11
12
13
14
15
16
AEM Accepted Manuscript Posted Online 18 December 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.03191-15
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Abstract 17
In temperate and boreal forest ecosystems, nitrogen (N) limitation on tree metabolism is 18
alleviated by ectomycorrhizal (ECM) fungi. As forest soils age, the primary source of N in 19
soil switches from inorganic (NH
4
+
, NO
3
-
) to organic (mostly proteins). It has been 20
hypothesized that ECM adapt to the most common N source in their environment, which 21
implies that fungi growing in older forests would have higher protein degradation abilities. 22
Moreover, recent results on a model ECM fungal species suggest that organic N uptake 23
requires glucose supply. To test the generality of these hypotheses, we screened 55 strains of 24
13 Suillus species with different ecological preferences for their in vitro protein degradation 25
abilities. Suillus species preferentially occurring in mature forests, where soil contains more 26
organic matter, had significantly higher protease activity than those from young forests with 27
low organic matter soils or species indifferent to forest age. Within species, the protease 28
activities of ecotypes from soils with high or low soil organic N content did not differ 29
significantly, suggesting resource partitioning between mineral and organic soil layers. The 30
secreted protease mixtures were strongly dominated by aspartic peptidases. Glucose addition 31
had variable effects on secreted protease activity; in some species it triggered activity but in 32
others it was repressed at high concentrations. Collectively, our results indicate that protease 33
activity, a key ectomycorrhizal functional trait, is positively related to environmental N source 34
availability, but also influenced by additional factors such as carbon availability. 35
36
37
Introduction 38
In temperate and boreal forests, nitrogen (N) is the element that usually limits tree nutrition 39
[1]. To acquire sufficient N, trees form symbioses with microorganisms, including 40
ectomycorrhizal (ECM) fungi [2] as well as shoot endophytic bacteria [3]. In soils, ECM 41
fungi can take up N from both mineral and organic sources. Mineral N can be found as NH
4
+
42
or NO
3
-
[4], while organic N can be present as part of several different organic oligomers or 43
polymers: peptides, chitin, nucleic acids and heterocyclic N compounds [3]. Peptides are 44
considered the dominant organic N source in forest soils (representing as much as 80 % of 45
organic N [5]), with ECM fungi typically retrieving N from this source through the use of 46
proteases [3]. Despite generally broad enzymatic capacities [6], not all ECM fungi have the 47
ability to access peptide N, which has resulted in the classification of ECM fungi into 48
“protein” and “non-protein” species [7]. Multiple authors have suggested that natural selection 49
should favour traits allowing mycorrhizal fungi to utilize the most abundantly available N 50
source in their environment [8]. This would suggest that “protein” ECM fungal species have 51
their ecological niche in organic N-rich soils. Empirical support for this hypothesis has been 52
shown by Lilleskov et al. [9], who found that ECM fungal species growing in a soil rich in 53
mineral N had a lower ability to grow on proteins than ones from poorer mineral N soils. 54
Similarly, Tibbett et al. [10] demonstrated that strains of the ECM fungal genus Hebeloma 55
from the arctic region (where 99 % of the N is in organic form) had the ability to use seed 56
protein as a N source, which was not the case for Hebeloma strains from temperate soils. 57
The ratio between organic and mineral N in forest soils is affected by many factors, with 58
forest succession being the most prominent [11]. Organic N forms become increasingly 59
dominant as forest ages due to the accumulation of organic matter. Hence the 60
organic/inorganic N ratio increases through succession, suggesting that organic forms are 61
increasingly important N sources in older forest soils [12]. Along with shifts in N source, 62
changes in ECM fungal community composition have also been well documented during 63
forest succession, though some species can be found at almost all stages [13]. The stage-64
specificity of ECM fungal community composition is more likely linked to the age of organic 65
horizons (i.e., the litter, fragmentation and humus layers that develop consecutively) than to 66
the age of the tree, as experiments have shown that seedlings establishing near mature trees 67
are generally colonized by ECM fungi typical of older forests [14]. Also experiments with 68
litter removal and litter addition to conifer stands of various ages provided support for the 69
larger importance of the age of the soil (including the organic layers) than of the tree [15]. 70
Taken together, it appears that the later an ECM fungal species occurs in forest soil 71
succession, the more it is likely to be in contact with organic matter, therefore making protein 72
its dominant N source. If the aforementioned hypothesis about N availability is correct, one 73
would predict ECM fungal protein degradation ability to correlate positively with forest soil 74
age. 75
Although the general functioning of ECM fungi in forest N cycling has been recognized for 76
many years, the properties of the ECM fungal enzymes involved in protein degradation are 77
relatively poorly characterized. Research on a limited number of species showed that these 78
enzymes are secreted proteases, which most of the time belong either to the aspartic or to the 79
serine peptidase class [3,16]. While their secretion appears to be induced by the presence of 80
protein [16], recent results on a model ECM species (Paxillus involutus; Boletales, 81
Basidiomycota) also showed that uptake of N from organic matter is additionally dependent 82
on the availability of a simple carbon (C) source [17]. This latter result supports the 83
hypothesis that the mining for organic N by ECM species does not occur without an energy 84
supply [18, 19]. However, since the two latter studies compared only presence and absence of 85
glucose, it is yet to be determined how different levels of C affect organic N degradation. 86
Species from the ECM genus Suillus (Boletales, Basidiomycota) offer a good model to test 87
the relationships among protein degradation ability, forest (soil) age, and carbon availability 88
because: 1) they are widely distributed in temperate and boreal forests and form a well-89
defined monophyletic group [20, 21], 2) different Suillus species show clear preferences for 90
soils with different organic matter contents and for trees of different age [22] (Table 1), 3) 91
they associate exclusively with trees in the Pinaceae [20], resulting in similar environmental 92
gradients during succession, 4) they are easily isolated into pure culture, and 5) they have fast 93
growth and high biomass production in vitro [22]. Here, we used in vitro assays to test four 94
hypotheses: i) Suillus species preferentially occurring in mature forests, where soils have high 95
organic N content, have a higher protein degradation ability than Suillus species characteristic 96
of young forests, where soil organic N content is low; ii) Suillus species that occur in both 97
young and mature forests harbour protein-degrading strains (ecotypes) in organic-rich soils 98
and non-protein-degrading strains (ecotypes) in mineral soils; iii) protein degradation occurs 99
through the secretion of a mixture of aspartic and serine proteases; and iv) protease activity, 100
when present, increases with glucose concentration. 101
102
Material & methods 103
Strains 104
We investigated protein degradation ability of 55 ECM fungal strains, isolated from 105
sporocarps, belonging to 13 species: Suillus americanus, S. brevipes, S. bovinus, S. cavipes, S. 106
caerulescens, S. granulatus, S. grisellus, S. lakei, S. laricinus, S. luteus, S. pungens, S. 107
tomentosus, S. variegatus. All species were represented by at least two strains. Thirty-two 108
strains were isolated in North America (USA), from 9 sites (Cloquet: 46.704397, -92.510528; 109
Yosemite: 37.817027 -119.712591; Mendocino1: 38.787030, -123.514499; Mendocino2: 110
39.311419, -123.760378; Cedar Creek: 45.407066, -93.199801; Ocean Shores: 47.032500, -111
124.164167; Rock Creek: 29.670780, -82.371932; Point Reyes: 38.084533, -122.870891 and 112
Berkeley Marina: 37.859584, -122.315999), and 23 in Europe (Belgium), from 2 sites (Paal: 113
51.058887, 5.175981; Zolder: 50.995371, 5.272788). All isolations were made from 114
sporocarp tissue. In cases where morphological identification was no possible, species were 115
identified by ITS sequencing. Habitat preference of each species (e.g. forest age, soil type) 116
was gathered from the primary literature, the website of the Dutch mycological society 117
(http://www.verspreidingsatlas.nl/paddenstoelen), as well as from direct field observations by 118
the authors (Table 1). 119
120
Growth 121
Fungi were maintained on solid MMN medium for 10 days, then transplanted to fresh solid 122
MMN medium, and grown again for 7 days. At this time they were considered sufficiently 123
active to perform the experiment. A 3 × 3 mm plug of active mycelium was then placed in a 124
glass bead system. This system consisted of a monolayer of sterile 4 mm diameter glass beads 125
sitting on a 9 cm Petri dish, filled with 11 ml of liquid medium, which is the volume needed to 126
cover the glass beads [16]. The liquid medium consisted of standard liquid MMN medium 127
where the N source (ammonium chloride) was replaced by a soluble protein (BSA: Bovine 128
Serum Albumin), and elemental N concentration was kept the same (53 mg l
-1
, which is 342 129
mg l
-1
of BSA). We chose BSA as a model protein since earlier results, on the same glass bead 130
system but with P. involutus, showed that the trends observed with this protein are the same as 131
with organic matter [16] and are therefore ecologically relevant. The pH was adjusted to 4.5. 132
Fungi were grown in static condition, in the dark, at 21 C and 80 % humidity for 17 days. We 133
monitored the protease activity and the protein content in the growth medium at 0, 5, 7, 11, 12 134
and 17 days after inoculation. Incubation was stopped at 17 days because further sampling 135
significantly increased the risk of contamination; the mycelium was harvested and weighed 136
after freeze-drying. 137
138
Protease assays 139
Protease assays were run using two complementary measurement procedures. The first was 140
run on culture supernatant and therefore measured secreted protease activity. This assay was 141
an adaptation of the FITC-BSA assay [23] for microplates. First, for each sample, 100µl of 142
medium supernatant was transferred to a 96-well microplate (flat bottom, Sarstedt). Then we 143
added 100 µl of 50 mM citrate buffer (pH 4.2) and 5 µl of freshly prepared 1 mg.ml
-1
FITC-144
BSA solution (Sigma-Aldrich). A preliminary experiment with a reduced number of strains 145
showed that the protease activity measured was highest at acidic pH values (we tested pH 3, 4, 146
5, 6; data not shown): we chose pH 4 as a compromise between high values and ecological 147
relevance (soil pH is most often between pH 4 and 5 in the soils of these conifer forests). The 148
plate was sealed with aluminium foil to prevent evaporation and incubated overnight (24 h) at 149
40 °C. Then, proteins were precipitated by adding 100 µl of 10% TCA (Tri Chloroacetic 150
Acid, Sigma-Aldrich), incubated for 1 h at room temperature, centrifuged for 30 min at 25 °C 151
and 2000 rpm, and 40 µl of the supernatant was transferred to a new microplate containing 152
200 µl of 1 M Tris buffer (pH 9.7). The fluorescence was read with a FLUOStar Omega 153
microplate reader at 485 nm excitation and 520 nm emission. One thousand units corresponds 154
to the fluorescence produced by 27 mg l
-1
of trypsin during 24 h at pH 8. 155
The second assay measured the protein left in the culture supernatant, which was the result of 156
the activity of both secreted and cell-wall bound proteases. This measurement was done in 157
microplates using the Bradford assay. First, 30 µl of sample were diluted with 70 µl of 158
distilled water in a transparent 96-well microplate (flat-bottom). Then, 50 µl of this solution 159
were transferred to a new transparent microplate containing 100 µl of distilled water and 100 160
µl of Bradford Quick reagent. After one hour of incubation, the absorbance of each well was 161
read at 595 nm on a FLUOStar Omega microplate reader. In order to estimate the 162
concentration of protein from the absorbance, we also measured the absorbance of 4 standard 163
solutions (342 mg l
-1
, 224 mg l
-1
, 112 mg l
-1
, 0 mg l
-1
). 164
165
Determination of the protease class 166
In order to determine the relative proportion of each protease class of the cocktail produced by 167
a given strain, we measured protease activity as previously, but added 10 µl of specific 168
protease inhibitors to the FITC-BSA mix (as in [16]): E64 (trans-epoxysuccinyl-L-169
leucylamido(4-guanidino)butane, which inhibits cysteine proteases, final concentration 10 170
µM, stock solution prepared in water), pepstatin A (which inhibits aspartic proteases, final 171
concentration 10 µM, in ethanol), EDTA (Ethylene Diamine Tetra Acetic acid, which inhibits 172
metalloproteases, final concentration 5 mM, stock solution prepared in water) and PMSF 173
(Phenyl Methane Sulfonyl Fluoride, which inhibits serine proteases, final concentration 1 174
mM, stock solution prepared in isopropanol). All chemicals were ordered from Sigma-175
Aldrich. Measurements were carried out on 17-day post-incubation samples, since it contained 176
the highest protease values for all strains. 177
178
Influence of glucose concentration on protease activity 179
We measured the effect of glucose on protease activity by growing Suillus strains in BSA 180
medium as before, but using four different glucose concentrations: 0, 1, 2.5 and 5 g l
-1
. We 181
chose to use multiple strains of 3 Suillus species with contrasting ecologies: S. luteus, which 182
grows preferentially in early forests in organic-N-poor soils; S. variegatus, which grows 183
preferentially in mature forests with high organic-N-rich soils; and S. bovinus, which occurs 184
in both young and mature forests in similar frequency. Protease activity was measured at 1, 4, 185
6, 8 and 11 days after inoculation. 186
187
Soil analyses 188
To more clearly assess the link between in vitro protein degradation ability and environmental 189
N source availability, we measured soil organic and mineral N content in two sites from 190
which several Suillus strains used in this study were isolated: a young forest (Paal) and a 191
mature forest (Zolder) in Belgium. In each site, 15 soil samples were taken with a soil corer 192
(15 cm deep, 8 cm diameter) on 11 October 2014. Core locations were arranged on a 3 × 5 m 193
grid with each node separated by 4 m in Paal. Due to the topology of the Zolder site (a 500 m 194
long, 8 m wide dyke), we harvested 5 groups of 3 cores, with each group separated by at least 195
20 m along the dyke. The soil samples were then pooled by groups of three into five 196
composite samples, which were individually passed through a 2 mm sieve. Two hundred 197
grams (200 g) of fresh composite sample were dried overnight at 60 °C. Total N was 198
measured by Kjeldahl method and inorganic N (NO
3
and NH
4
-N) by titrimetry after reduction 199
by Devarda’s alloy. Organic N was then deduced by calculating the difference between total 200
N and inorganic N. 201
202
Statistics 203
We tested the effect of four factors: species (13 species), forest age (young, multi, mature), 204
soil type (low-organic, high-organic nitrogen) and host genus (Larix, Pseudotsuga and Pinus) 205
on two variables: secreted protease activity and protein remaining in the media. To account 206
for differences in growth rates among strains as well as the positive relationship between 207
biomass and protease activity and protein degradation, we used specific protease activity (total 208
protease activity / dry mass) and specific protein remaining (% / dry mass) in the final 209
analyses. The effects of the four factors were tested using a nested ANOVA (with strain 210
nested within species to account for possible non-independence) on log-transformed data (for 211
specific protease activity) and x-transformed data (for specific protein remaining). We used 212
Tukey HSD tests to determine significant post hoc differences among factor means. To 213
compare the protease activity of different strains of the same species collected from different 214
sites (ecotypes), we used the two species that had at least two strains from both a young and 215
mature forest site (S. bovinus and S. variegatus). Significant differences between species and 216
ecotypes were assessed using an ANOVA on 1/x normalized data (secreted protease activity) 217
and on untransformed data (protein remaining) followed by a Duncan post-hoc test. To 218
determine the relative contribution of different protease classes, we compared protease 219
activity with four inhibitors with a one-way ANOVA. For that test, data were log(x+1) 220
transformed to improve variance homogeneity and a Tukey HSD test was used to determine 221
significant differences among assay means. The effect of different glucose concentrations on 222
protease activity has been estimated using an ANOVA followed by a Tukey HSD test on the 223
protease activities measured at the end of the experiment. Correlation between protease 224
activity at the end of the experiment and percentage of protein left were evaluated by a 225
Kendall correlation analysis. Statistics were run using R [24]. 226
227
Results 228
Relations between protease activity and ecological traits 229
All 55 Suillus strains displayed significant (i.e., higher than the control) secreted protease 230
activity, and the protein content decreased by at least 28 % in all assays (Table 2). The 231
protease activity and the amount of protein left at the end of the experiment were significantly 232
and negatively correlated (Kendall’s tau < 0.001, Figure S1). However, there were 8 strains 233
for which both variables were low, suggesting that in these strains a major part of protease 234
activity was through cell-wall bound proteases (< 35 fluorescence units and < 25 % protein 235
left in the medium, respectively): they belonged to S. bovinus (SboP3, SboP6, SboP7, SboZ2, 236
SboZ3, SboZ4) and S. brevipes (13, 17). For these strains, there was no correlation between 237
the amount of protein left in the medium and their biomass at the end of the experiment. 238
Differences in both specific protease activity and protein remaining in the medium were 239
significant among species (nested ANOVA, p < 0.001 and 0.019, respectively); there were 240
also significant effects of soil type (p < 0.001) and forest age (p < 0.001) in specific protease 241
activity (Table 3). Species from mature forests had a significantly higher secreted protease 242
activity and lower specific protein remaining than those from young forests and from multi-243
stage (i.e., present in both young and mature) forests (Figure 1). When grouped by soil type, 244
strains of species from high organic N soils had significantly higher secreted protease activity 245
than ones from low organic N soils; these strains also had lower amounts of remaining protein 246
in the medium but the difference was not significant (p = 0.146, Table 3). The nested 247
ANOVA showed no significant effect of host tree species on protease activity and in 248
remaining protein contents; however, in pairwise comparisons, Pinus-associated species had 249
on average significantly lower protease activities than those associated with Larix-associated 250
hosts (Figure S2). 251
252
Soil organic N and protein degradation abilities 253
To investigate the extent to which there is local adaptation within species depending on N 254
source availability (i.e., the presence of ecotypes), we compared protein degradation activities 255
of 19 strains belonging to S. bovinus and S. variegatus present at both a young (Paal, low 256
organic/mineral N ratio: 28 ± 9) and mature (Zolder, high organic/mineral N ratio: 132 ± 12) 257
forest site. The organic N content in soil was, on average, 15 times higher in the mature forest 258
compared to the young forest (Zolder: 1838 mgN.kg
-1
, Paal: 119 mgN.kg
-1
, Table S1), but 259
there was also three times more NH
4
+
in the mature forest soils as well (Zolder: 13.7 mgN.kg
-
260
1
, Paal: 4.2 mgN.kg
-1
). There was no significant difference in protease activity and protein 261
degradation between ecotypes of the same species (Table 4). Strains of S. bovinus had low 262
specific protease activity and protein degradation in both groups (Figure 3a,c), while strains of 263
S. variegatus had significantly higher protease activity and degraded significantly more 264
protein (Figure 3b,d). 265
266
Identification of the secreted protease class 267
Among the four protease classes (aspartic, serine, cysteine and metalloproteases), there were 268
no significant differences in protease activity in the presence of serine, cysteine, and 269
metalloprotease inhibitors relative to assay with no inhibitors present (Figure 4). In contrast, 270
there was a significant ~10 fold reduction in average secreted protease activity in the presence 271
of pepstatin A, which inhibits aspartic proteases. This inhibition was observed in all Suillus 272
strains where we measured a significant secreted protease activity (Table 2). 273
274
Influence of glucose concentration on protease activity 275
Glucose addition had a significant effect on secreted protease activity (expressed as 276
fluorescence units) only for one fungal species (Table 5). Strains of S. variegatus were 277
significantly affected by the levels of glucose addition. The protease activity at 1 g l
-1
and 2.5 278
g l
-1
of glucose were significantly higher than the protease activity without glucose (Figure 5). 279
However at 5g l
-1
of glucose addition, the protease activity was low again, without a 280
significant difference from the no-glucose addition. For S. luteus and S. bovinus, the addition 281
of glucose had no significant effect, with all activities being low (Figure 5). 282
283
Discussion 284
Relations between soil N sources and protease activity 285
We found that protease activity differed significantly among strains of Suillus species based 286
on forest age and soil type. Consistent with the hypothesis about N source and protein 287
degradation ability, strains of species restricted to mature forests and high organic N soils had 288
significantly higher protease activity than those present in younger forests and low organic N 289
soils. While these results indicate that protein degradation is linked to changes in forest age 290
and soil type, these two factors are clearly not independent: mature forests are usually 291
associated with a thick organic soil layer [13]. Since many ecological factors also change 292
significantly with forest age (e.g. host tree species composition [11], soil pH [11], litter phenol 293
concentration [29]), it is also possible that other factors besides soil organic N content 294
contribute to the observed patterns. Measuring additional soil variables at each collection site 295
was beyond the scope of this study, but future experimental work (e.g. adding organic N to 296
young soils N or removing the organic layer in mature forests, as in [15]) will be helpful in 297
differentiating the relative importance of changes in organic N availability from these 298
additional environmental factors. 299
The strains of S. bovinus and S. variegatus from the Paal site (a young pine forest with low 300
organic/mineral N ratio) and the Zolder site (a mature pine forest with high organic/mineral N 301
ratio) showed no significant differences in their protease activity. Given that previous results 302
suggested that locally adapted strains of multi-forest age species may have a higher protease 303
activity in high organic N soils [8, 9], we were surprised to find no support for this kind of 304
variability. The overall higher protease activity of S. variegatus strains compared to those of 305
S. bovinus, however, is consistent with closer observations of the ecology of these two 306
species. Despite being a multi-forest age species, S. variegatus preferentially inhabits mature 307
forests, while S. bovinus grows there only as satellite populations [22]. Hence, the presence of 308
S. variegatus may depend on the development of an organic layer in the forest soil, where its 309
protein degradation ability would give a competitive advantage for N uptake. Moreover, S. 310
bovinus was also stimulated by litter removal in pine stands that exposed mineral soil [30]. 311
Interestingly, Suillus luteus, which is classified as a species characteristic for young trees on 312
mineral soils, does occur in older stands as well, but its root tips and mycelium are located in 313
the mineral not organic layer [31, 32]. Taken together, these results suggest that the presence 314
of S. variegatus in young forests may be attributable to local organic niches in young forest 315
soil, and conversely local mineral N patches may facilitate the persistence of S. bovinus in 316
mature forests. 317
For 8 of the 55 strains, protein content in the medium significantly decreased while protease 318
activity was low, meaning that the protease activity was very likely cell-wall bound. 319
Alternative mechanisms could involve as well adsorption of BSA to the mycelium [33], but 320
this hypothesis can be partially ruled out by the fact that there was no correlation between 321
protein left in the medium and the mycelial biomass for these strains. Therefore, we conclude 322
that most of the protease activity of the aforementioned strains of S. bovinus and S. brevipes 323
were cell-wall bound. Moreover, the strains preferentially inhabiting mature forests or high 324
organic soils were always characterized by high secreted protease activity. In the range of 325
Suillus species tested here, secreted proteases could therefore be an adaptation to organic N 326
rich environment. 327
While our results suggest that ecological filtering or natural selection favours physiological 328
capacities in ECM fungi that allow them to utilize the dominant N source in their 329
environment, they do not imply that protein degradation is necessarily the rate-limiting step of 330
N uptake, or that ECM protein degradation only controls N availability. Litter breakdown and 331
N mineralization depends on its lignin and polyphenolic content [29]. Lignin degradation 332
mechanisms (e.g. lignin peroxidases, Mn peroxidases, laccases, Fenton reaction) hence may 333
play a role as important as protein degradation in ECM-mediated plant N uptake. Moreover, 334
proteins are not the only source of organic N in forest soils: simple amino acids, chitin (fungal 335
or arthropod necromass) or heterocyclic N (chlorophyll, nucleic acids) can also contribute to 336
N assimilation, and all the associated enzyme activities may contribute significantly to N 337
mineralization. 338
339
Classes of proteases 340
All protease cocktails of the strains that had a significant activity were strongly repressed by 341
pepstatin A, but the other inhibitors did not significantly decrease protease activity in the 342
experimental assays. From this, we conclude that the protease activity was dominated by 343
aspartic proteases. Shah et al. [16] also showed that the cocktail of proteases secreted by P. 344
involutus was also dominated by aspartic proteases, and, as a consequence, had an acidic 345
optimum. Moreover, the authors also showed that this class of proteases accounted for most of 346
the protease activity when the fungus was growing on BSA, but also on other N sources such 347
as gliadin, pollen and dissolved soil organic matter. These findings are consistent with the 348
ecology of these systems; where organic N accumulates, soils are acidic, as observed by 349
Chalot & Brun [3]. However, partly in contrast to our study, these authors reported that ECM 350
fungal proteases belonged to aspartic and serine protease classes. We therefore suggest that 351
secreted aspartic proteases are key agents in organic N acquisition for the ECM species, at 352
least in the order Boletales. 353
354
Effects of glucose on protease activity 355
Because glucose has been previously found to trigger organic matter oxidation and N 356
acquisition from that organic matter by the ECM fungus P. involutus [17], we measured 357
protease activity of strains of three species of contrasting ecologies: S. luteus (pioneer), S. 358
bovinus (preference for young forest stages) and S. variegatus (preference for old forest 359
stages), at different glucose concentrations. S. luteus strains did not respond to glucose input, 360
possibly because of inherently low protease activities. For S. bovinus, we observed protease 361
activity only in one of the strains coming from the high organic N site, and only at the highest 362
glucose load (5 g l
-1
). For S. variegatus, the protease activity was influenced by glucose 363
concentration, but not in a linear manner. Protease activity reached peak values at 1 and 2.5 g 364
l
-1
, and was relatively low at 5 g l
-1
. Repression of protease activity by high glucose 365
concentration was reported by Colpaert & Van Laere [34] and is consistent with the use of 366
BSA as a carbon source. Indeed, high glucose input represses genes involved in C metabolism 367
pathways through catabolite repression (gluconeogenesis, Krebs/TCA cycle, and genes 368
involved in metabolization of C from other sources [35]). Moreover, it is known that some 369
ECM fungi can use the deaminated skeletons of the amino acids as a C source for TCA cycle 370
or as a template for synthesis of new aminoacids [3,17]. Therefore, we suggest that the BSA 371
in our experiment may have also been used by Suillus species as an alternative C source, with 372
C catabolites repressing protease activity at high glucose concentrations. This hypothesis is 373
consistent with the fact that protease activity is not immediately induced in our assays. 374
However, this does not explain why the tested Suillus species were not able to degrade protein 375
without glucose, which shows that an easily available C source is needed to trigger protease 376
activity, as already observed with P. involutus [17]. One explanation could be the following: 377
fungal protease activity is triggered by low to average host plant C supply; while high mineral 378
N availability in soil would result in faster uptake by the plant, higher photosynthesis rate, and 379
higher C flux. High C supply rates would then be an indication of plant N sufficiency and 380
therefore that fungal protease activity is not necessary. Alternatively, repression of protease 381
activity by high glucose supply could be related to the distance between the glucose 382
concentration and the place where organic N exploitation takes place: Suillus species are long-383
distance exploration types and therefore the hyphae proliferating close to a protein-containing 384
patch would be far away from glucose supply, in the Hartig net of the root tip. To better 385
understand the role of host carbon in protein degradation, greater experimental work is needed 386
in this area; for example, through the use of
13
C labeling of organic N. 387
388
Conclusions and future directions 389
In summary, we found that the protein degradation ability of Suillus strains 1) was highest in 390
species adapted to high organic soils; 2) showed little intraspecific variability; 3) was due 391
primarily to aspartic peptidases and 4) was controlled to some extent by glucose levels. 392
Though these were all obtained using an in vitro experimental system, we assert they are still 393
ecologically informative, as previous studies using pure culture approaches have yielded 394
results that correlate well with those observed in field settings [10, 25, 26]. The results of our 395
study imply that ability to forage for organic N is a crucial functional trait that may have an 396
important role in shaping ECM fungal communities, with protein-degrading species being 397
more common as the soil organic matter content increases. This doesn’t rule out, however, 398
that other important mechanisms related to N acquisition may play an important role as well, 399
such as chitinase activity, or N storage capacity. An important next step will be to test the 400
validity of these results in soil microcosms or field settings, particularly the role of host tree 401
and protein carbon in vivo. Given the contrasting protein degradation ability of co-occurring 402
species such as S. bovinus and S. variegatus, determining how competition for access to 403
different N sources may mediate species interactions and vertical niche differentiation would 404
provide a more mechanistic understanding the drivers of ECM fungal community structure. 405
This knowledge is particularly important in light of the strong effect of human-induced 406
gradients in nitrogen availability in Europe and North America [9]. Finally, examining the 407
protein degradation ability of additional Suillus species associated with these host genera will 408
be key to determining the strength of host phylogenetic signal versus other environmental 409
conditions. 410
411
Acknowledgments 412
The authors thank S. Branco and T. Bruns for assistance with collection of some of the North 413
American Suillus strains. Members of the Kennedy lab provided constructive comments on a 414
previous version of this manuscript. We also acknowledge constructive comments by three 415
reviewers on an earlier version of the manuscript. Jelle Stas and Francois Rineau are thankful 416
of the BOF (Special Research Fund) from UHasselt for financing his research. 417
418
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511
512
Tables and Figure legends 513
514
Table 1. Ecological characteristics of the 55 Suillus strains investigated. 515
516
Table 2. Protease activity (Fluorescence Units), remaining protein content (% of the initial 517
BSA concentration), dry biomass (mg) and ecological characteristics of all 55 Suillus strains 518
at the end of the experiment (17 days). The four rightmost columns represent the protease 519
activity in the presence of four protease inhibitors for all 55 strains. Cysteine = E64 inhibitor, 520
Aspartic = Pepstatin A, Metallo = EDTA, Serine = PSMF. 521
522
Table 3. ANOVA results of factors affecting specific protease activity and protein remaining 523
in the media. Significant effects are highlighted in grey. DF = Degrees of freedom. 524
525
Table 4. ANOVA results of differences in specific protease activity and protein remaining in 526
the media between ecotypes of S. bovinus and S. variegatus at two sites, one with low (Paal) 527
and the other with high organic matter (Zolder). DF = Degrees of freedom. 528
529
Table 5. ANOVA results on factors affecting protease activity when the fungal strains were 530
provided with different levels of glucose. 531
532
Figure 1. Box-and-whiskers representation of log-transformed values of specific protease 533
activity and specific protein degradation of the 55 Suillus strains categorized by forest age. 534
Different letters indicate significant differences among treatment forest age category means, 535
as determined by post-hoc Tukey HSD tests. The box represents the 2
nd
-3
rd
interquartile 536
range; the bold line in the box represents the median; the upper and lower bars outside the box 537
represent the 1
st
and the 4
th
quartiles, respectively; and the dots outside of the bars represent 538
the outliers (defined as values outside of the 1.5 times the interquartile range below Q1 and 539
above Q3). 540
541
Figure 2. Box-and-whiskers representation of log-transformed values of specific protease 542
activity and specific protein degradation of the 55 Suillus strains categorized by soil type. 543
Different letters indicate significant differences among treatment soil type means as 544
determined by post-hoc Tukey HSD tests. The box represents the 2
nd
-3
rd
interquartile range; 545
the bold line in the box represents the median; the upper and lower bars outside the box 546
represent the 1
st
and the 4
th
quartiles, respectively; and the dots outside of the bars represent 547
the outliers (defined as values outside of the 1.5 times the interquartile range below Q1 and 548
above Q3). 549
550
Figure 3. Box-and-whiskers representation of log-transformed values of specific protease 551
activity and specific protein degradation of strains of S. bovinus and S. variegatus isolated 552
from two forest sites: a young forest with soil of low organic/mineral N ratio (Paal) and a 553
mature forest with soil of high organic/mineral N ratio (Zolder). Boxplots represent the 554
variation of each parameters between species (S. bovinus: 6 strains in Paal, 7 in Zolder; S. 555
variegatus: 2 strains in Paal, 3 in Zolder). The box represents the 2
nd
-3
rd
interquartile range; 556
the bold line in the box represents the median; the upper and lower bars outside the box 557
represent the 1
st
and the 4
th
quartiles, respectively; and the dots outside of the bars represent 558
the outliers (defined as values outside of the 1.5 times the interquartile range below Q1 and 559
above Q3). 560
561
Figure 4. Box-and-whiskers representation of log-transformed values of protease activity of 562
the 55 Suillus strains treated by four different protease inhibitors. Different letters indicate 563
significant differences among treatment soil type means as determined by post-hoc Tukey 564
HSD tests. The box represents the 2
nd
-3
rd
interquartile range; the bold line in the box 565
represents the median; the upper and lower bars outside the box represent the 1
st
and the 4
th
566
quartiles, respectively; and the dots outside of the bars represent the outliers (defined as values 567
outside of the 1.5 times the interquartile range below Q1 and above Q3). 568
569
Figure 5. Box-and-whiskers representation of protease activity of the Suillus strains growing 570
in a BSA medium (expressed as fluorescence units) with different glucose concentrations (0, 571
1, 2.5 and 5 g l-1). Three species have been investigated: S. luteus (strains P1, P3, P4, P8, 572
P13), S. bovinus (strains P1, P2, P4, P10, Z1, Z2, Z3, Z4) and S. variegatus (strains Z1, 573
ZJW3, ZJW4, ZW6, ZJW13). For S. bovinus, strains from both Zolder and Paal sites were 574
investigated. Different letters indicate significant differences among treatment soil type means 575
as determined by post-hoc Tukey HSD tests. The box represents the 2nd-3rd interquartile 576
range; the bold line in the box represents the median; the upper and lower bars outside the box 577
represent the 1st and the 4th quartiles, respectively; and the dots outside of the bars represent 578
the outliers (defined as values outside of the 1.5 times the interquartile range below Q1 and 579
above Q3). 580
581
582
Species Strain Site Ecotype Host Genus
Forest age
preference
Soil organic
matter
preference
Reference
Type of fungal tissue in
the reference
51 CedarCreek (USA)
52 Cloquet (USA)
SboP1 Paal (Belgium) young
SboP3 Paal (Belgium) young
SboP4 Paal (Belgium) young
SboP5 Paal (Belgium) young
SboP6 Paal (Belgium) young
SboP7 Paal (Belgium) young
SboZ1 Zolder (Belgium) mature
SboZ2 Zolder (Belgium) mature
SboZ3 Zolder (Belgium) mature
SboZ4 Zolder (Belgium) mature
SboZ5 Zolder (Belgium) mature
SboZ6 Zolder (Belgium) mature
SboZ7 Zolder (Belgium) mature
13 Yosemite (USA)
14 Yosemite (USA)
15 Yosemite (USA)
17 Mendocino1 (USA)
18 Mendocino1 (USA)
19 OceanShores (USA)
20 OceanShores (USA)
47 RockCreek (USA)
38 Mendocino2 (USA)
39 Mendocino2 (USA)
53 Cloquet (USA)
54 Cloquet (USA)
55 Cloquet (USA)
56 CedarCreek (USA)
57 CedarCreek (USA)
60 CedarCreek (USA)
61 CedarCreek (USA)
43 PointReyes (USA)
75 PointReyes (USA)
63 CedarCreek (USA)
64 CedarCreek (USA)
65 CedarCreek (USA)
SluP1 Paal (Belgium)
SluP2 Paal (Belgium)
SluP3 Paal (Belgium)
SluP4 Paal (Belgium)
SluP8 Paal (Belgium)
27 BerkeleyMarina (USA)
28 BerkeleyMarina (USA)
30 BerkeleyMarina (USA)
Suillus brevipes 32 Mendocino1 (USA) NA Pinus multi NA Visser, 1995 Sporocarps, mycorrhizas
33 Montana (USA)
34 Montana (USA)
35 Montana (USA)
36 Montana (USA)
SvaP1 Paal young
SvaP3 Paal young
SvaZJW6 Zolder (Belgium) mature
SvaZJW8 Zolder (Belgium) mature
SvaZJW13 Zolder (Belgium) mature
Suillus variegatus
Suillus tomentosus
Suillus grisellus
Suillus lakei
Suillus viscidus
Suillus luteus
Suillus pungens
1
Visser S. 1995. Ectomycorrhizal fungal succession in jack pine stands following wildfire. New Phytol, 129: 389-401.
2
Twieg BD, Durall DM, Simard SW. 2007. Ectomycorrhizal fungal succession in mixed temperate forests. New Phytol, 176: 437-447.
3
Peay K, Bruns TD, Kennedy PG, Bergemann SE, Garbelotto M. 2007. A strong species-area relationship for eukaryotic soil microbes: island size matters for ectomycorrhizal
fungi. Ecology Letters, 10: 470-480.
4
Dahlberg A. 1997. Population ecology of Suillus variegatus in old Swedish Scots pine forests. Mycological research, 101: 47-54.
Sporocarps
Sporocarps
Sporocarps, mycorrhizas
Sporocarps, mycorrhizas
Sporocarps
Sporocarps
Mycorrhizas
Sporocarps
Sporocarps
Sporocarps
Sporocarps
Sporocarps, mycorrhizas
Sporocarps
Suillus caerulescens Pseudotsuga
Pinus
Pinus
NA
Pinus
http://www.verspreidingsatlas.nl/
Visser, 1995
1
Suillus americanus
Suillus bovinus
Suillus brevipes
Suillus cavipes http://www.verspreidingsatlas.nl/
http://www.verspreidingsatlas.nl/Suillus granulatus
Personal observations
Larix
mature
young
mature
Dahlberg, 1997
4
Personal observations
Pinus
Pinus
Pinus
Pinus
Larix
Pseudotsuga
Larix
Pinus
Peay et al., 2007
3
Personal observations
http://www.verspreidingsatlas.nl/
http://www.verspreidingsatlas.nl/
Visser, 1995
Twieg et al., 2007
2
multi
multi
multi
young
mature
young
multi
multi low organic
low organic
NA
high organic
NA
NA
NA
NA
NA
NA
NA NA
high organic
low organic
high organic
high organic
low organic
multi
multi
NA
NA
NA
NA
high organic
low organic
Strain Species Site
Successional
stage
Soil type Host plant
Activity
(Protease units,
day 17)
Protein (in % of
the initial content)
Mass (DW) cysteine aspartic metallo serine
55
cavipes
mature
high organic
Larix
448
0
13.7
334
9
357
302
53
cavipes
mature
high organic
Larix
218
2
7.9
296
10
297
201
54
cavipes
mature
high organic
Larix
105
9
10.2
110
8
116
79
60
grisellus
CedarCreek
mature
high organic
Larix
98
6
1.6
60
10
77
66
63
viscidus
CedarCreek
mature
high organic
Larix
97
9
8.9
86
12
75
58
61
grisellus
CedarCreek
mature
high organic
Larix
58
10
4.1
36
11
32
24
64
viscidus
CedarCreek
mature
high organic
Larix
17
44
6.2
18
9
18
14
SvaZJW6
variegatus
Zolder
multi
high organic
Pinus
460
10
14.3
353
8
286
278
SvaZJW13
variegatus
Zolder
multi
high organic
Pinus
306
9
13.1
329
9
320
226
SvaZJW8
variegatus
Zolder
multi
high organic
Pinus
292
7
12.8
226
10
243
174
27
pungens
BerkeleyMarina
multi
high organic
Pinus
103
12
3.1
66
9
62
46
30
pungens
BerkeleyMarina
multi
high organic
Pinus
94
11
5.7
53
9
60
49
28
pungens
BerkeleyMarina
multi
high organic
Pinus
69
9
6.4
47
5
36
42
SboZ1
bovinus
Zolder
multi
high organic
Pinus
59
4
9.8
55
6
46
32
SboZ2
bovinus
Zolder
multi
high organic
Pinus
16
16
7.4
13
8
16
10
SboZ4
bovinus
Zolder
multi
high organic
Pinus
16
17
5.4
8
9
13
8
SboZ7
bovinus
Zolder
multi
high organic
Pinus
13
50
7.2
8
8
6
10
SboZ5
bovinus
Zolder
multi
high organic
Pinus
12
65
3.6
8
10
10
8
SboZ6
bovinus
Zolder
multi
high organic
Pinus
10
61
5.8
12
6
6
8
SboZ3
bovinus
Zolder
multi
high organic
Pinus
9
23
6.1
9
6
9
8
SvaP1
variegatus
Paal
multi
low organic
Pinus
319
2
15.4
297
10
262
170
34
tomentosus
Montana
multi
low organic
Pinus
208
6
13.2
140
11
117
87
SvaP3
variegatus
Paal
multi
low organic
Pinus
186
4
11.6
253
9
240
188
33
tomentosus
Montana
multi
low organic
Pinus
110
3
12.7
170
11
164
114
20
brevipes
OceanShores
multi
low organic
Pinus
45
16
5.9
39
11
43
31
36
tomentosus
Montana
multi
low organic
Pinus
36
6
9.6
116
8
108
83
35
tomentosus
Montana
multi
low organic
Pinus
31
8
8
38
11
29
17
19
brevipes
OceanShores
multi
low organic
Pinus
23
34
4.7
15
9
15
12
SboP5
bovinus
Paal
multi
low organic
Pinus
17
52
4.9
14
6
8
14
SboP3
bovinus
Paal
multi
low organic
Pinus
16
6
6.7
14
10
10
12
51
americanus
CedarCreek
multi
low organic
Pinus
15
56
7
16
10
12
10
SboP1
bovinus
Paal
multi
low organic
Pinus
14
42
5.8
7
6
10
7
SboP7
bovinus
Paal
multi
low organic
Pinus
14
12
6.5
16
9
10
12
SboP4
bovinus
Paal
multi
low organic
Pinus
12
42
7
8
7
8
8
SboP6
bovinus
Paal
multi
low organic
Pinus
10
16
5
7
8
9
8
47
brevipes
RockCreek
multi
Pinus
77
12
4.2
57
10
63
43
32
brevipes
Mendocino1
multi
Pinus
28
25
8.2
18
13
23
11
18
brevipes
Mendocino1
multi
Pinus
24
39
3.5
33
12
29
16
17
brevipes
Mendocino1
multi
Pinus
20
3
9.8
83
4
70
64
13
brevipes
Yosemite
multi
Pinus
16
21
13
30
13
37
30
14
brevipes
Yosemite
multi
Pinus
15
64
4.3
12
10
10
7
15
brevipes
Yosemite
multi
Pinus
14
45
5.7
11
10
14
12
52
americanus
multi
Pinus
10
42
4.6
14
6
11
8
39
caerulescens
Mendocino2
multi
Pseudotsuga
35
13
2.4
27
8
14
16
38
caerulescens
Mendocino2
multi
Pseudotsuga
16
39
2.8
10
8
10
8
SluP1
luteus
Paal
young
low organic
Pinus
54
18
6.2
34
14
31
24
SluP8
luteus
Paal
young
low organic
Pinus
49
16
5.3
88
22
96
68
65
luteus
CedarCreek
young
low organic
Pinus
38
16
11.3
49
42
50
32
SluP2
luteus
Paal
young
low organic
Pinus
38
22
12.9
78
18
72
54
57
granulatus
CedarCreek
young
low organic
Pinus
11
46
6.9
12
7
10
6
SluP4
luteus
Paal
young
low organic
Pinus
11
49
5.6
9
7
10
10
56
granulatus
CedarCreek
young
low organic
Pinus
10
57
4.3
13
8
10
9
SluP3
luteus
Paal
young
low organic
Pinus
10
72
7.3
13
11
13
11
43
lakei
PointReyes
young
Pseudotsuga
182
7
7.7
216
9
204
155
75
lakei
PointReyes
young
Pseudotsuga
126
13
7.9
124
10
108
83
Soil Type
Forest Age
Host Genus
Species
Specific protease activity
Nested ANOVA
log(x)
5.00E-04
9.00E-04
5.83E-02
5.02E-06
Specific protein remaining
Nested ANOVA
sqrt(x)
0.1464
0.0428
0.2876
0.0189
DF
2
3
1
10
Factors
Variable Test Transformation
Variable Test Species x Site Species Site
Protease activity
ANOVA
0.89
1.35E-05
0.79
Protein degradation
ANOVA
0.80
3.70E-03
0.65
DF
1
1
1
Factors
Variable
Species Test
Glucose
concentration
Protease activity
Suillus luteus
ANOVA
0.21
Suillus bovinus
ANOVA
0.14
Suillus variegatus
ANOVA
2.70E-04
DF
3
Factors
... Ad question 3: Positive correlation among AMF abundance, GRSP, and soil organic C levels (Singh et al., 2016;Zhang et al., 2017) generally agrees with the evidence that high fungal-to-bacterial biomass ratio is coupled with increased N availability and litter decomposition and is accompanied by higher C storage potential of the soils (Koranda et al., 2014;Malik et al., 2016). Enhanced N availability increases fungal degradation of cellulose from plant cell walls (Koranda et al., 2014) and subsequent increased glucose availability enhances activity of exoenzymes (including proteases) secreted by saprotrophic fungi (Rineau et al., 2016). Although bacteria in such soils are considered to play a subordinate role as decomposers of smaller molecules that are primarily produced by soil fungi, the hyphal network of AMF offers a specific niche for heterotrophic soil bacteria (Nazir et al., 2010). ...
Article
The term “Glomalin” was originally used to describe a hypothetical gene product of arbuscular mycorrhizal fungi (AMF) that was assumed to be a nearly ubiquitous, thermostable and highly recalcitrant glycoprotein, deposited in soils in large amounts, and deemed to indicate soil health and quality. It was defined operationally as the fraction of soil organic matter (SOM) extractable by a hot citrate buffer and assessed either by Bradford assay or by cross-reactivity with monoclonal antibody MAb32B11. Later, it was recognized that the extracts contained a variety of compounds, including some of non-AMF origin, cross-reactive with both Bradford assay and the monoclonal antibody. This led to re-describing the pertinent (and still only operationally defined) SOM as “glomalin-related soil proteins (GRSP)”, albeit without any substantial change in the underlying concepts. Consequently, a great deal of confusion in this area arose among researchers in soil, plant, and environmental sciences. Glomalin or GRSP (often used interchangeably) has previously been linked to various soil features, including stability of soil aggregates, size of soil C and N pools, sequestration of heavy metals, and alleviation of various plant stresses. GRSP concentrations in soil often, but not always, have been correlated with AMF biomass measured by alternative (mainly microscopic) approaches. GRSP formation, deposition, and/or decomposition in soils seem to be largely dependent on a multitude of interactions among plants, AMF, and other soil microorganisms, including prokaryotes. The chemical structure of GRSP extracted from soil remains unclear and generally complex. That is due to the unspecific mode of its extraction and purification, as well as the great variety of analytical approaches that have been used heretofore to assess it. Future research needs to elucidate the exact composition of this operationally defined SOM fraction, the controls over its production and accumulation in soils, and its exact role in soil ecology generally and soil food webs in particular. Furthermore, novel and independent tools should be established to more specifically (as compared to current glomalin assays) assess AMF biomass and functioning in roots and soil and its involvement in soil processes.
... 21 Aspartic proteases are also key enzymes for protein decomposition by ectomycorrhizal fungi, which are abundant in boreal forest ecosystems. 22 BSA was chosen as a model protein because of its solubility in water and strong affinity to mineral surfaces. 10,23 It is also a wellcharacterized protein facilitating the interpretations of our experimental data. ...
Article
Full-text available
Proteins are a substantial nitrogen source in soils provided that they can be hydrolyzed into bioavailable small peptides or amino acids. However, the strong associations between proteins and soil minerals restrict such proteolytic reactions. This study focused on how an extracellular fungal protease (Rhizopus sp.) hydrolyzed iron oxide-associated BSA and the factors that affected the proteolysis. We combined batch experiments with size-exclusion and reversed-phase liquid chromatography and in situ infrared spectroscopic measurements to monitor the generation of proteolytic products in solution as well as the real-time changes of the adsorbed BSA during 24 h. Results showed that protease hydrolyzed the iron oxide-associated BSA directly at the surface without an initial desorption of BSA. Concurrently the protease was adsorbed to vacant surface sites at the iron oxides, which significantly slowed down the rate of proteolysis. This inhibiting effect was counteracted by the presence of pre-adsorbed phosphate or by increasing the BSA coverage, which prevented protease adsorption. Fast initial rates of iron oxide-associated BSA proteolysis, comparable to proteolysis of BSA in solution, and very slow rates at prolonged proteolysis suggest a large variability in mineral-associated proteins as a nitrogen source in soils and that only a fraction of the protein is bioavailable.
... Ectomycorrhizal fungi also secrete an assortment of peptidases to utilize proteins within the soil (Nehls et al., 2001;Müller et al., 2007;Shah et al., 2013;Rineau et al., 2016) and encode a number of amino acid, oligopeptide, and dipeptide transporters to take up the resulting small peptide products (Nehls et al., 1999;Benjdia et al., 2006;Lucic et al., 2008;Casieri et al., 2013). Expression of these organic N transporters and secretion of peptidase are usually reduced in the presence of ammonium, indicating the fungal preference for uptake of the more easily accessible ammonium from soil (Avolio et al., 2012;Bödeker et al., 2014). ...
Article
Full-text available
Symbiosis with ectomycorrhizal (ECM) fungi is an advantageous partnership for trees in nutrient-limited environments. Ectomycorrhizal fungi colonize the roots of their hosts and improve their access to nutrients, usually nitrogen (N) and, in exchange, trees deliver a significant portion of their photosynthetic carbon (C) to the fungi. This nutrient exchange affects key soil processes and nutrient cycling, as well as plant health, and is therefore central to forest ecosystem functioning. Due to their ecological importance, there is a need to more accurately understand ECM fungal mediated C and N movement within forest ecosystems such that we can better model and predict their role in soil processes both now and under future climate scenarios. There are a number of hurdles that we must overcome, however, before this is achievable such as understanding how the evolutionary history of ECM fungi and their inter- and intra- species variability affect their function. Further, there is currently no generally accepted universal mechanism that appears to govern the flux of nutrients between fungal and plant partners. Here, we consider the current state of knowledge on N acquisition and transport by ECM fungi and how C and N exchange may be related or affected by environmental conditions such as N availability. We emphasize the role that modern genomic analysis, molecular biology techniques and more comprehensive and standardized experimental designs may have in bringing cohesion to the numerous ecological studies in this area and assist us in better understanding this important symbiosis. These approaches will help to build unified models of nutrient exchange and develop diagnostic tools to study these fungi at various scales and environments.
... For instance, N availability plays a key role in CO 2 uptake by plants (Terrer et al., 2016). In the case of ectomycorrhizal fungi, the ability to access N org through secreted proteases has been related to their ecological niche differentiation as part of the natural forest succession (Rineau et al., 2016). For example, in temperate and boreal forest, N is the element limiting tree nutrition (Rees et al., 2001). ...
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In this study we investigated how the source of organic carbon (Corg) and nitrogen (Norg) affects the interactions between fungi of the genus Morchella and bacteria dispersing along their hyphae (fungal highways; FH). We demonstrated that bacteria using FH increase the hydrolysis of an organic nitrogen source that only the fungus can degrade. Using purified fungal exudates, we found that this increased hydrolysis was due to bacteria enhancing the activity of proteolytic enzymes produced by the fungus. The same effect was shown for various fungal and bacterial strains. The effect of this enhanced proteolytic activity on bacterial and fungal biomass production varied accordingly to the source of Corg and Norg provided. An increase in biomass for both partners 5 days post-inoculation was only attained with a Norg source that the bacterium could not degrade and when additional Corg was present in the medium. In contrast, all other combinations yielded a decrease on biomass production in the co-cultures compared to individual growth. The coupled cycling of Corg and Norg is rarely considered when investigating the role of microbial activity on soil functioning. Our results show that cycling of these two elements can be related through cross-chemical reactions in independent, albeit interacting microbes. In this way, the composition of organic material could greatly alter nutrient turnover due to its effect on the outcome of interactions between fungi and bacteria that disperse on their mycelia.
... All rights reserved. Martin et al., 2016) are used by ECM fungi to decay SOM (Rineau et al., 2015), whereas others, suggest that class II fungal peroxidases are of greater relative importance for ECM decay since they can degrade more 'recalcitrant' compounds present within SOM pools (Kyaschenko et al., 2017;Baskaran et al., 2017). Recent models that include both hydrolysable and oxidizable 'fractions' of SOM (Baskaran et al., 2017) incorporate a more mechanistic view of ECM decay that can inform experimentation and articulation of the relative importance of different enzymatic or radical based decay pathways. ...
Article
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The extent to which ectomycorrhizal (ECM) fungi enable plants to access organic nitrogen (N) bound in soil organic matter (SOM) and transfer this growth‐limiting nutrient to their plant host, has important implications for our understanding of plant–fungal interactions, and the cycling and storage of carbon (C) and N in terrestrial ecosystems. Empirical evidence currently supports a range of perspectives, suggesting that ECM vary in their ability to provide their host with N bound in SOM, and that this capacity can both positively and negatively influence soil C storage. To help resolve the multiplicity of observations, we gathered a group of researchers to explore the role of ECM fungi in soil C dynamics, and propose new directions that hold promise to resolve competing hypotheses and contrasting observations. In this Viewpoint, we summarize these deliberations and identify areas of inquiry that hold promise for increasing our understanding of these fundamental and widespread plant symbionts and their role in ecosystem‐level biogeochemistry.
Chapter
The global market of superabsorbent materials (SAMs) has been estimated at 7.5 billion € in 2017 and is expected to keep growing annually at a rate of 6.2%. Most SAMs used nowadays are based on acrylic derivatives, but their toxicity, low biodegradability and petrochemical nature, together with their high price and incompatibility with agricultural lands and underground water, have arisen the interest in novel superabsorbent biopolymers (SABs). These alternative SABs can be obtained from different natural resources (e.g., cellulose, starch, blood plasma, gelatine, chitosan, among others) that are by-products or biowastes from different food and agricultural industries. Therefore, the development of this new generation of SABs enhances the biocompatibility and biodegradability of the pursued products, as well as the sustainability of several industrial segments. This new generation of biopolymers also involves changes in material processing since nowadays most fabrication procedures are based on graft copolymerisation of vinylic monomers (e.g., acrylonitrile, acrylic acid, acrylamide) onto natural polymers (starch, cellulose and their derivatives), but there are emerging studies focusing on protein-based SABs. This chapter presents an overall diagnosis of the current state of SABs.
Chapter
Proteases play essential role in diverse physiological, metabolic and regulatory processes in all living organisms. Proteolytic enzymes are also the most dominant sector of the industrial enzyme market. Filamentous fungi have found applications in many industries and represent an important source of proteases. Diverse fungal proteases are widely applied in food and pharmaceutical industries. They are also of great interest for their applications in leather and fabric processing, waste management, and detergent industry. This article will review the current knowledge on proteases from filamentous fungi, their classification and potential industrial applications.
Article
Mutualisms are ubiquitous in natural systems, but less is known about how these positive interactions influence species distributions compared with antagonistic interactions, such as competition and predation. The niche concept is one useful approach for thinking about factors that shape species ranges, which we apply here towards understanding how the nature of plant-mycorrhizal symbioses change across large environmental gradients. We used a continuous niche mapping approach to examine how two ectomycorrhizal fungi (Thelephora terrestris, Suillus pungens) impact pine seedling growth across a two-dimensional soil nitrogen (N) and phosphorus (P) gradient. We found that ectomycorrhizal fungi improved plant growth most in nutrient addition treatments with highly imbalanced N:P ratios, demonstrating that mycorrhizal benefits depend on interactions between niche axes. However, T. terrestris (highN:lowP) and S. pungens (lowN:highP) benefited plants most at opposite ends of the resource ratio spectrum, consistent with niche partitioning and functional specialization. While ectomycorrhizal fungi are often thought of as being most beneficial for nitrogen uptake, our results suggest that members of the Thelephoraceae may specialize in improving plant P uptake. Ectomycorrhizal colonization by a single fungus increased plant niche volume (calculated as convex hull volumes of plant growth response surfaces across N and P gradients) compared to non-mycorrhizal control plants and shows the overall positive effects of mutualisms on plant niche volume. Despite plant host benefits in S. pungens and T. terrestris single species treatments, the presence of both fungi together decreased plant niche volume. The lack of functional complementarity, despite functional specialization, indicates that in some environments, either fungal competition or the cost of maintaining a suboptimal mycorrhizal partner can limit the benefits of a higher quality partner. The niche mapping approach we present has the potential to answer fundamental questions about the dimensions of functional diversity in ectomycorrhizal fungi and the distributions of mycorrhizal symbioses.
Article
Soil organic nitrogen is largely composed of proteinaceous material, hence, the extracellular peptidases that are widely produced by microorganisms play a critical role in the recycling of soil organic nitrogen. But why do microbes produce such a variety of functionally different peptidases? In theory, this could be an adaptation to substrate heterogeneity, but it may also be an adaptation to variable soil conditions. Here we characterized the contribution of different catalytic types, or classes, of peptidases present in soil with the intent to determine if their relative contributions would vary as a function of soil properties. We screened specific peptidase inhibitors and optimized their concentrations to work effectively in soil. Total potential proteolytic activity was partitioned among several peptidase classes by adding class-specific inhibitors to the peptidase assay. Using Pepstatin A, EDTA (ethylenediaminetetraacetic acid), PMSF (phenylmethylsulfonyl fluoride), and E64 (epoxysuccinyl-L-leucylamido (4-guanidino) butane), we were able to discriminate among aspartic, metallo-, serine, and cysteine peptidases, respectively. We found that diverse peptidases were active and contributed to the proteolytic activity in soil. Extracellular peptidase profiles varied among different soils and were associated with soil chemical and microbial properties. Metallopeptidases contributed 30–50% of the soil proteolytic activity in all soils. A higher relative contribution of metallopeptidase activity was found in less acidic soils, reflecting its neutral pH optimum. Serine peptidases were only detected in soil from Douglas-fir (Pseudotsuga menziesii) stands (10–20% of total proteolytic activity) but not in soils under red alder (Alnus rubra). The relative activity of aspartic peptidase correlated positively with the fungal:bacterial ratios of the soils. Our results lend support to the view that microbial communities might modify their peptidase profile to optimize protein utilization in response to soil and other environmental factors.
Thesis
En Europe, le bois est la première source d’énergie renouvelable. La transition énergétique se traduit par une intensification de l’exploitation des forêts. L’effet de ces pratiques sylvicoles sur les communautés microbiennes du sol est encore peu étudié. Au cours de ma thèse, j’ai évalué les conséquences d’une manipulation artificielle de matière organique en forêt tempérée sur la diversité fonctionnelle et taxonomique des communautés bactériennes et fongiques telluriques dans six sites expérimentaux (réseau expérimental MOS). Parallèlement, une caractérisation fonctionnelle des communautés microbiennes a également été réalisée dans un contexte proche des réalités de l’intensification des pratiques sylvicoles sous climat tropical en plantation d’Eucalyptus. Si certains descripteurs fonctionnels de la dégradation de la matière organique sont particulièrement informatifs, les activités microbiennes de dégradation de la chitine, polymère azoté des arthropodes et champignons, sont apparues très sensibles au retrait de matière organique. C’est pourquoi, par des approches de génomiques comparatives, nous avons cherché à estimer le potentiel chitinolytique des différentes guildes fongiques des sols. En conditions contrôlées, nous avons ensuite quantifié les capacités potentielles de mobilisation et de transfert du carbone et de l’azote, à partir d’une matière organique microbienne riche en chitine, par un champignon ectomycorhizien en symbiose avec son hôte. Enfin, la généricité des fonctions chitinolytiques d’un plus large spectre d’espèces fongiques ectomycorhiziennes a été évaluée par le couplage d’approches enzymatiques et isotopiques. L’ensemble de nos résultats met en lumière le rôle significatif des champignons ectomycorhiziens dans la mobilisation du carbone et de l’azote à partir de certaines formes de matière organique, et la nécessité de prendre en compte le compartiment microbien dans les études d’impact des pratiques sylvicoles
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In Pinaceae, the chloroplast, mitochondrial, and nuclear genomes are paternally, maternally, and biparentally inherited, respectively. Examining congruence and incongruence of gene phylogenies among the three genomes should provide insights into phylogenetic relationships within the family. Here we studied intergeneric relationships of Pinaceae using sequences of the chloroplast matK gene, the mitochondrial nad5 gene, and the low-copy nuclear gene 4CL. The 4CL gene may exist as a single copy in some species of Pinaceae, but it constitutes a small gene family with two or three members in others. Duplication and deletion of the 4CL gene occurred at a tempo such that paralogous loci are maintained within but not between genera. Exons of the 4CL gene have diverged approximately twice as fast as the matK gene and five times more rapidly than the nad5 gene. The partition-homogeneity test indicates that the three data sets are homogeneous. A combined analysis of the three gene sequences generated a well-resolved and strongly supported phylogeny. The combined phylogeny, which is topologically congruent with the three individual gene trees based on the Templeton test, is likely to represent the organismal phylogeny of Pinaceae. This phylogeny agrees to a certain extent with previous phylogenetic hypotheses based on morphological, anatomical, and immunological data. Disagreement between the previous hypotheses and the three-genome phylogeny suggests that morphology of both vegetative and reproductive organs has undergone convergent evolution within the pine family. The strongly supported monophyly of Nothotsuga longibracteata, Tsuga mertensiana, and Tsuga canadensis on all three gene phylogenies provides evidence against previous hypotheses of intergeneric hybrid origins of N. longibracteata and T. mertensiana. Divergence times of the genera were estimated based on sequence divergence of the matK gene, and they correspond well with the fossil record.
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I. II. III. IV. V. References SUMMARY: Although hypothesized for many years, the involvement of ectomycorrhizal fungi in decomposition of soil organic matter remains controversial and has not yet been fully acknowledged as an important factor in the regulation of soil carbon (C) storage. Here, we review recent findings, which support the view that some ectomycorrhizal fungi have the capacity to oxidize organic matter, either by 'brown-rot' Fenton chemistry or using 'white-rot' peroxidases. We propose that ectomycorrhizal fungi benefit from organic matter decomposition primarily through increased nitrogen mobilization rather than through release of metabolic C and question the view that ectomycorrhizal fungi may act as facultative saprotrophs. Finally, we discuss how mycorrhizal decomposition may influence organic matter storage in soils and mediate responses of ecosystem C sequestration to environmental changes. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.
Article
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Conifers predominantly occur on soils or in climates that are suboptimal for plant growth. This is generally attributed to symbioses with mycorrhizal fungi and to conifer adaptations, but recent experiments suggest that aboveground endophytic bacteria in conifers fix nitrogen (N) and affect host shoot tissue growth. Because most bacteria cannot be grown in the laboratory very little is known about conifer–endophyte associations in the wild. Pinus flexilis (limber pine) and Picea engelmannii (Engelmann spruce) growing in a subalpine, nutrient-limited environment are potential candidates for hosting endophytes with roles in N 2 fixation and abiotic stress tolerance. We used 16S rRNA pyrosequencing to ask whether these conifers host a core of bacterial species that are consistently associated with conifer individuals and therefore potential mutualists. We found that while overall the endophyte communities clustered according to host species, both conifers were consistently dominated by the same phylotype, which made up 19–53% and 14–39% of the sequences in P. flexilis and P. engelmannii, respectively. This phylotype is related to Gluconacetobacter diazotrophicus and other N 2 fixing acetic acid bacterial endophytes. The pattern observed for the P. flexilis and P. engelmannii needle microbiota—a small number of major species that are consistently associated with the host across individuals and species—is unprecedented for an endophyte community, and suggests a specialized beneficial endophyte function. One possibility is endophytic N fixation, which could help explain how conifers can grow in severely nitrogen-limited soil, and why some forest ecosystems accumulate more N than can be accounted for by known nitrogen input pathways.
Article
Ectomycorrhizal fungi are symbiotically associated microorganisms which ecological importance has been repeatedly demonstrated. There has been a considerable amount of research aimed at assessing the ability of ectomycorrhizal fungi and ectomycorrhizas to utilize organic nitrogen sources. The fate of soil proteins, peptides and amino acids has been studied from a number of perspectives. Exocellular hydrolytic enzymes have been detected and characterized in a number of ectomycorrhizal and ericoid fungi. Studies on amino acid transport through the plasma membrane have demonstrated the ability of ectomycorrhizal fungi to take up the products of proteolytic activities. Investigations on intracellular metabolism of amino acids have allowed the identification of the metabolic pathways involved. Possible intracellular compartmentation of amino acids will be examined by immunocytochemistry. Further translocation of amino acids in symbiotic tissues has been established by experiments using isotopic tracers, although the exact nature of the nitrogenous compounds transferred at the symbiotic interface remained unclear. One of the main future challenges in the physiology of organic nitrogen acquisition is to determine the nature, the regulation and the location of N-compound transporters at the soil-fungus and fungus-plant interfaces. The molecular approach which is just emerging in this particular research area will greatly improve our knowledge. Future research should also address the extent of competition between different ectomycorrhizal species and between different microbial populations for organic nitrogen. (C) 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.
Article
Studies of nutrient cycling in forests span more than 100 yr. In earlier years, most attention was given to the measurement of the pools of nutrients in plants and soil and of the return of nutrients from plant to soil in litterfall. The past 20 yr or so have seen a major concentration on the processes of nutrient cycling, with particular emphasis on those processes by which the supply of nutrients to the growing forest is sustained. In the more highly productive forests, up to 10 tonnes of litter of low nutritional quality is deposited annually on the forest floor. The decomposition of this litter, the mineralization of the nutrients it holds, and the uptake of nutrients by tree roots in the carbon-rich environment which results are the themes of this review.
Article
The ectomycorrhizal (ECM) fungal communities associated with the host genus Alnus have been widely noted for their low richness and high proportion of host-specific species, but the processes underlying their atypical structure remain poorly understood. In this study, we investigated whether the high acidity and nitrate concentrations characteristic of Alnus soils may act as important environmental filters that limit the membership in Alnus ECM fungal communities. Using a pure culture approach, we grew four species from two host groups (Alnus and non-Alnus) in liquid media containing different acidity and nitrate concentrations. We found that the growth of the Alnus-associated ECM fungi was not, on average, affected by high acidity, while the non-Alnus-associated ECM fungi had a significantly negative growth response under the same conditions. Similarly, when grown at high nitrate, the non-Alnus-associated ECM fungi also generally performed more poorly. Growth responses of the Alnus-associated ECM fungi in both the high acidity and high nitrate treatments indicated tolerance rather than preference for those chemical conditions. The mechanism underlying the differential acidity tolerance may involve active hyphal buffering of local acidity environments. Taken together, our results suggest that soil chemical conditions likely do act as significant environmental filters that, along with other ecological and evolutionary factors, drive the atypical specificity of Alnus ECM interactions.