Abstract
Arbuscular mycorrhizal symbiosis is an ancient interaction between plants and fungi of the phylum Glomeromycota. In exchange for photosynthetically fixed carbon, the fungus provides the plant host with greater access to soil nutrients via an extensive network of root-external hyphae. Here, to determine the impact of the symbiosis on the host ionome, the concentration of nineteen elements was determined in the roots and leaves of a panel of thirty maize varieties, grown under phosphorus limiting conditions, with, or without, inoculation with the fungus Funneliformis mosseae. Although the most recognized benefit of the symbiosis to the host plant is greater access to soil phosphorus, the concentration of a number of other elements responded significantly to inoculation across the panel as a whole. In addition, variety-specific effects indicated the importance of plant genotype to the response. Clusters of elements were identified that varied in a coordinated manner across genotypes, and that were maintained between non-inoculated and inoculated plants.
- NC
- non-colonized
- M
- mycorrhizal
- SDW
- shoot dry weight
- ICP-MS
- inductively coupled plasma mass spectrometry
- PC
- principal component
Introduction
Plants require 14 essential mineral elements to complete their lifecycle, namely nitrogen (N), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), copper (Cu), zinc (Zn), molybdenum (Mo), boron (B), chloride (Cl) and nickel (Ni) (Marschner, 2012). Depending on their concentration in the plant, these elements can be classified as macronutrients or micronutrients. In addition, elements can be classified into four major groups based on their requirement for 1) synthesis of biomolecules, 2) energy transfer, 3) ion balance and 4) electron transport. As a by-product of nutrient and water acquisition, plants will also take up a number of additional non-essential elements that may, in high concentrations, be toxic, such as Aluminum (Al), Arsenic (As), Cadmium (Cd), Cobalt (Co), Selenium (Se), Strontium (Sr) and Rubidium (Rb). A deficiency of mineral elements has detrimental consequences on plant fitness or, in the agronomic context, crop yield, and plants have developed a number of strategies to promote uptake in nutrient deficient soils and optimize the efficiency of internal use. Such responses include modification of the root system architecture, induction of high affinity nutrient transporters, remobilization of internal resources, growth arrest, down-regulation of photosynthesis and the induction of senescence (Lynch, 1995; Aibara and Miwa, 2014; Whitcomb et al., 2014). In addition, plants form mutualistic associations with rhizosphere organisms, such as nitrogen-fixing rhizobia or arbuscular mycorrhizal (AM) fungi (Bucher, 2007; Parniske, 2008; Vance, 2014).
AM symbiosis is a mutualistic interaction established between soil fungi belonging to the phylum Glomeromycota and the majority (70-90%) of land plant species (Schübler et al., 2001; Smith and Read, 2008). The capacity to form AM symbiosis has been retained in the major cereal crop species throughout domestication and improvement (e.g. Koide et al., 1988; Hetrick et al., 1992; Kaeppler et al., 2000; Sawers et al., 2008), as has a conserved molecular machinery required for symbiotic establishment and nutrient exchange (e.g. Paszkowski et al., 2002; Gutjahr et al., 2008; Yang et al., 2012; Willman et al., 2013; Liu et al., 2016; Nadal et al., 2017). One of the major benefits of AM symbiosis to the plant host is enhanced nutrient uptake as the result of enhanced soil foraging by an extensive network of root-external fungal hyphae (Bago et al., 2003; Finlay, 2008). An increase in P uptake in P limiting soils is well established as the primary physiological consequence of AM symbiosis on the plant host (Bucher, 2007). AM symbiosis, however, has a broad impact on host mineral nutrition, potentially increasing the uptake of additional essential nutrients such as N, Cu, Fe, Mn and Zn or limiting the uptake of potentially toxic elements such as Cd, Pb, Hg and As (Govindarajulu et al., 2005; Jin et al., 2005; Göhre and Paszkowski, 2006; Guether et al., 2009). These effects may be mediated through direct transport by the fungi, or by alterations in root system architecture and physiology. Transcriptomic and functional analyses have identified plant encoded nutrient transporters specifically expressed or up-regulated in mycorrhizal plants, including transporters of phosphate (e.g. Harrison et al., 2002; Paszkowski et al., 2002; Liu et al., 2016), ammonium (Koegel et al., 2013), sulfate (Giovannetti et al., 2014) and sodium (Porcel et al., 2016). To better understand the impact of AM symbiosis on plant nutrition, it is informative to consider the ionome - the total element composition - as a whole, investigating the relationships that exist between elements as a result of their interaction in soil chemistry, common uptake machinery, and the mechanisms of plant internal homeostasis (Baxter et al., 2008; Baxter, 2015).
Here, the concentration of nineteen elements was determined in the leaves and roots of a panel of thirty maize lines, consisting of the 26 parents of the nested association mapping (NAM) population (McMullen et al., 2009) and a number of additional varieties, grown in the greenhouse under P limiting conditions, with or without inoculation with the AM fungus Funneliformis mosseae. Maize is not only a crop of great agronomic importance, but also a model system well supported with genetic and genomic tools. Previous studies have shown commercial maize varieties to be well colonized by AM fungi and to typically present a strong growth response to AM inoculation under P limiting greenhouse conditions (e.g. Kaeppler et al., 2000; Sawers et al., 2017), while reverse genetics approaches are defining the importance of specific genes to the functioning of AM symbiosis in maize (Willman et al.,2013; Nadal et al., 2017). The study panel represented a non-biased sampling of the broader genetic diversity of maize breeding lines, allowing generalization ad to the effect on specific elements, investigation of correlated responses across lines, and evaluation genotype specific responses.
Although P was the limiting nutrient in the experiment, the host ionome responded broadly to AM symbiosis, reinforcing the idea that chemical elements behave as a coordinated system when growth conditions are altered. Furthermore, the response among certain groups of elements was correlated, even when the response itself differed among maize varieties. Given interest in the agronomic application of AM symbiosis to increase the efficiency of fertilizer use, improve nutritional value, and maintain the concentrations of toxic metals at safe levels (Sawers et al., 2008; Fester and Sawers, 2011), these results provide a valuable reference dataset for further characterization of the effect of mycorrhizal colonization on the maize ionome under field conditions.
RESULTS
The maize ionome responds to inoculation with Funneliformis mosseae
To assess the impact of mycorrhizal colonization on the host ionome, root and shoot samples collected from a previously reported maize evaluation (Sawers et al., 2017) were analyzed by inductively coupled plasma mass spectrometry (ICP-MS). In this experiment, 30 maize inbred lines, selected to maximize genetic diversity (McMullen et al., 2009), were grown with (M) or without (NC) inoculation with Funneliformis mosseae, under phosphorus (P) limiting conditions (Sawers et al., 2017). It was reported previously that plants were well colonized (about 60% of the total root length contained fungal structures, and about 30% of the total root length contained arbuscules) with an associated increase in shoot dry weight (SDW) of approximately two-fold (Sawers et al., 2017). Here, in addition to the concentration of P included in the initial report, concentrations of a further 18 elements are presented. Initially, all genotypes were considered together, to generalize as to the main effect of fungal inoculation on the maize ionome (Fig.1; Table 1). Inoculation with F mosseae was associated with a significant increase in P concentration in both roots (Wilcoxon test, p < 0.05) and leaves (p < 0.01). In addition, in roots, a significant increase of Na (p < 0.001) and S (p < 0.05) was observed, along with a decrease of Cd, Co, Mn and Ni (all, p < 0.001). In leaves, there were significant increases in the concentration of Al (p < 0.001), Fe (p < 0.001) and a decrease of Zn (p < 0.05) concentration.
Patterns in ion concentration shift following inoculation with F. mosseae
To characterize relationships among ions, pairwise correlations were calculated among all 38 ion-inoculation combinations, separately for roots and leaves, across the thirty varieties evaluated (Fig. 2, 3). Correlation in the concentration of any given ion between NC and M plants ranged from 0.05 to 0.71 in roots and 0.03 to 0.74 in leaves (Table 1). Clustering of the correlation matrix revealed covariation among ions and between M and NC treatments (Fig. 2, 3). For certain ions M and NC treatments grouped together (e.g. K in both roots and leaves), indicating that patterns of variation among lines were maintained, while for other ions, M and NC treatments were not close in the clustering (e.g. P in both roots and leaves), indicating patterns of relative accumulation among lines to be changing under colonization. Clustering revealed also relationships among ions. The largest cluster was seen in the roots, consisting of Al, As, Co, Fe, Ni and Rb (Fig 2). Interestingly, this cluster is maintained under both NC and M treatments, although the correlations of the ions between NC and M treatments are low: i.e. the impact of AM symbioses on the concentration of these ions was genotype specific, such that level in NC plants did not well predict the level in M plants; and yet, whatever response did occur in a given variety was coordinated across the clustered ions.
To further investigate patterns of covariance in ion concentration, a principal component (PC) analysis was performed. Root and leaf data were analyzed separately (Fig. 4; Table 2). In both tissues, NC and M plants were partially separated on the basis of the first two PCs (representing 48% and 40% of the total variance in roots and leaves, respectively). In roots, the ions with greatest representation in PC1 were Al, As, Co, Fe, Ni and Rb (combined contribution of 71%; Fig. 5). Al, As, Co, Fe, Ni and Rb were clustered in the covariance matrix, and, as is consistent, contributed similarly to the PCs (Fig. 4; Table 2), their concentration generally decreasing in M plants. A significant reduction in Co and Ni was observed also in the single ion analysis, while median root concentrations of Al, As and Fe were reduced in M plants (Fig. 1; Table 1). Ca, Na, Mo, S, Sr and Rb contributed most to root PC2 (79% total; Fig.5), largely increasing in concentration in M plants, again consistent with the single ion analysis (Fig. 1; Table 1). Rb contributed equally to both PC1 and PC2 (7.8% and 5.9%, respectively) and showed no clear pattern with respect to inoculation (Fig. 1, Fig. 5). In leaves, PC1 and PC2 were predominantly represented by Ca, Mg, Mn, Mo, Sr and S (70%), and Al, As, Co, Fe, Ni and P (74%), respectively (Fig. 5; Table 2). NC and M treatments were best distinguished by PC2, generalized by an increase in the leaf concentration of Al, As, Co, Fe, Ni and P in M plants that was consistent with the single ion analysis (Fig. 1; Table 1). The contribution of P to the PCs was notably low, and in a direction opposite to the ions making the greatest contributions (Fig. 4), reflecting the negative correlations observed between concentrations of P and these ions, not only between treatments, but among genotypes within a single treatment (Fig. 3, S1, S2).
Differences in the ionome response to AM inoculation among maize varieties indicates the importance of host genotype
To investigate the importance of plant genotype to the response to inoculation with F. mosseae, the change in ionomic PC scores between NC and M treatments was compared (Fig. 5). Reaction norm plots reflected the main effect of inoculation in roots and shoots (c.f. Fig.1), but revealed also evidence of genotype specific differences (indicated by non-parallel lines in Fig. 5). As observed in PC biplots (Fig. 4), the difference between NC and M treatments was best captured by PC2, in both roots and leaves. In roots, there was a clear trend towards an increased PC2 score in inoculated plants, related to increasing concentrations of Ca, Na, Mo, S, Sr and Rb; in leaves the trend was towards a lower score in PC2, related to increasing concentrations of Al, As, Co, Fe, Ni and P. A number of lines, however, did not follow these general trends: in roots CML247, Ki3, M162W, M37W, Oh7b and Pa36 showed a reduction in PC2 when inoculated; in leaves, the lines B97, CML52, CML103 and CML247 showed an increase in PC2 when inoculated. These lines were not found to be exceptional with regard to growth response in the previous analysis (Sawers et al., 2017), although it should be noted that P, the limiting nutrient, made only minor contributions to these PCs. As would be predicted by the clustering results (Fig. 2, 3), there were instances in which genotype specific effects were correlated among ions, well illustrated by the behavior of Al and Fe in the roots (Fig. 6).
DISCUSSION
Measurement of the concentration of nineteen ions in the leaves and roots of maize seedlings grown with or without inoculation with the AM fungus Funneliformis mosseae revealed coordinated changes in response to AM colonization. By using a panel of maize varieties designed to maximize genetic diversity, it was possible to both make meaningful generalizations and to examine patterns of covariation in ion concentration. Analysis of single ions and PCA indicated AM colonization to be associated with an increase in the root concentration of Ca, Na, Mo, P, Rb, S and Sr, and a decrease in Cd, Co, Cu, Mn, Ni and Zn (Fig. 1, 4, 5; Table 1). In the leaves, similar analyses revealed an increase in Al, As, Co, Fe, Na, Ni and P, and decrease of Mn and Zn in colonized plants (Fig. 1, 4, 5; Table 1). In single ion analysis, the concentrations of Mn, Na, Ni, P and Zn responded (p < 0.05) similarly to AM colonization in both roots and leaves, with Ni the only ion for which the sign of the response differed between the two tissues (Fig. 1; Table 1). Given that the analysis cannot distinguish between elements within the root and elements adhering to the root surface, the overlap between root and shoot provides an important indication that the changes observed in the root reflect differences in uptake. Although leaf P concentrations were at deficient levels (Reuter and Robinson, 1997), and variation in P concentration was shown previously to correlate well with plant growth in this experiment (Sawers et al., 2017), the P response to AM inoculation was far from the most significant change to the ionome, nor did P contribute greatly to PCs (Fig. 1, 5; Table 1).
One of the most significant (p < 0.001) changes in the ionome of AM plants was the reduction in concentration in roots of Co and the potentially toxic, non-essential heavy metal Cd (Fig. 1; Table1). Although Co and Cd concentrations were relatively low (Reuter and Robinson, 1997) in both NC and M plants, these data are consistent with the previously reported role of AM fungi in protecting the host plant from accumulation of toxic elements (Göhre and Paszkowski, 2006). Mn concentration was also reduced, although non-limiting, in M plants, in both roots and leaves (Fig. 1; Table 1). Reduced Mn accumulation in M plants has been reported previously in maize and other plants, and attributed to reduced plant production of P-mobilizing carboxylates (e.g. Kothari et al., 1991; Posta et al., 1994; Nazeri et al., 2013; Gerlach et al., 2015). Genotypes varied in Mn accumulation, and a negative correlation ( r = -0.35, p = 0.06; Fig. S1) was observed between P and Mn accumulation in the leaves of M plants. Measurement of the leaf Mn concentration has been proposed previously as a method to distinguish different strategies of P acquisition at higher taxonomic levels: concentrations of Mn will tend to be higher in species that exude carboxylate to favor direct P acquisition, due to increased Mn mobility in the rhizosphere when compared with species that tend to acquire P via mycorrhizae (Lambers et al., 2015). Here, an intraspecific correlation was observed between Mn and P concentrations in M plants that might be exploited in the evaluation and genetic mapping of P uptake strategies. Correlations were observed also between P and Mg concentrations in M plants: a negative correlation in leaves (r = -0.62, p < 0.01; Fig. S2) and a positive correlation (r = 0.47, p < 0.01; Fig. S2) in roots. Cluster analysis of pairwise correlations revealed further groups of elements that varied together across genotypes (Fig. 2, 3). A number of clusters were common to NC and M plants, although the correlations between the two treatments were not strong, indicating a coordinated but genotype specific response: i.e. responses differed, but for a given genotype, the ions in a cluster responded in a similar way, a pattern well illustrated by concentrations of Al and Fe in the roots (Fig. 6)
In contrast to controlled experimental systems, field soils present a far more complex range of physicochemical properties, promoting correlation and interaction in the availability of nutrients to plants. For example, P deficiency is often accompanied by insufficiency of other nutrients, such as Ca, Mg and Zn in acid soils, or Fe and Zn in alkaline conditions (Calderón-Vázquez et al., 2009; Hinsinger, 2001; Osaki et al., 1999; Uexküll and Mutert, 1995). Soil water content will have nutrient specific effects: uptake of K and P is strongly reduced at low soil water content; uptake of Ca and Mg is less effected by water content (Talha et al., 1979). It is clear that AM symbiosis has a broad impact on the host ionome beyond enhancement of P uptake. Although the direct fungal contribution to the uptake of each nutrient was not quantified, genotype specific responses are consistent with variation in symbiotic function, as reported previously with respect to P (Sawers et al., 2017). Strong genotype specific responses were observed also for a number of elements which showed no change in mean concentration between NC and M plants when the panel was considered as a whole (e.g. Al and Fe in roots; Fig. 6), suggesting AM symbiosis to indeed impact, directly or indirectly, uptake of such elements. An analogy can be made to so-called “hidden” P uptake, the significant substitution of AM mediated for direct P uptake that can occur in colonized plants, even if the final P concentration, or indeed plant performance, are equivalent to non-colonized levels (Smith et al., 2003).
Successful application of AM fungi in agricultural systems requires a suitable combination of plant host genotype, fungal community, soil environment and management (Fester and Sawers, 2012). It remains to be seen to what degree such a complex system can be manipulated, or, ultimately, optimized. AM symbiosis, however, represents far more than a simple exchange of carbon for P (Smith et al., 2009). Indeed, AM symbiosis can be considered as a particular environmental modification that impacts all aspects of plant growth and development. Data presented here reveal both the profound impact of AM symbiosis on plant nutrition, and the importance of host genotype to the symbiotic outcome. Evaluation of the ionome is relatively stable and inexpensive, and might be readily incorporated into the evaluation of AM response in the field. Here, plants were grown in a greenhouse system under P limiting conditions, with other nutrients supplied in excess, and, as is consistent, no clear pattern was observed between the concentration of elements other than P and plant growth. Under a specific set of field conditions, however, the broad impact of AM symbiosis on the ionome beyond P acquisition may indeed impact performance, through to final yield and the nutritional quality of the grain.
MATERIALS AND METHODS
Growth of maize diversity panel inoculated with Funneliformis mosseae
As described previously (Sawers et al., 2017), a panel of 30 diverse maize lines, comprising the 26 diverse inbred founders of the maize NAM population (McMullen et al., 2009), Pa36 (a line tolerant of low P availability; Kaeppler et al., 2000), and the broadly used reference lines B73 and W22, and W64A (a line used previously for study of AM symbiosis; Paszkowski et al., 2006), was evaluated with (M) or without (NC) inoculation with F. mosseae (isolate number 12, European Bank of Glomales, http://www.kent.ac.uk/bio/beg/), as previously described (Sawers et al., 2017). Briefly, plants were grown in 1 L pots, in sand/clay (9:1 vol/vol), fertilized three times per week with 100 ml of modified Hoagland solution (Hoagland and Broyer, 1936) containing 10% (100μM) of the standard concentration of KH2PO4, the potassium concentration being maintained by addition of KCl. A total of 1200 plants (30 genotypes x 2 treatments x 6 replicates) were grown in a complete block design, over five separate plantings, at the University of Lausanne, Switzerland. Plants were harvested after 8 weeks and shoot dry weight measured (SDW). For two plantings (corresponding to six complete blocks) roots were collected also, and stained to confirm efficacy of the fungal inoculum as previously described (Gutjahr et al., 2008).
Determination of elemental concentration by ICP-MS analysis
Root and shoot samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine the concentration of twenty metal ions. Weighed tissue samples were digested in 2.5mL concentrated nitric acid (AR Select Grade, VWR) with an added internal standard (20ppb In, BDH Aristar Plus). Sample digestion and dilution was carried out as described previously (Ziegler et al,2013). Concentration of the elements B11, Na23, Mg25, Al27, P31, S34, K39, Ca43, Mn55, Fe57, Co59, Ni60, Cu65, Zn66, As75, Se82, Rb85, Sr88, Mo98 and Cd111 was measured using an Elan 6000 DRC-e mass spectrometer (Perkin-Elmer SCIEX) connected to a PFA microflow nebulizer (Elemental Scientific) and Apex HF desolvator (Elemental Scientific). A control solution was run every tenth sample to correct for machine drift both during a single run and between runs. Measurements for B11 were not considered further as concentrations were apparently below the level of reliable detection. The ICP-MS technique does not allow determination of N concentration.
Statistical analysis
Least squares (LS) means (lsmeans::lsmeans; Lenth, 2016) for concentrations were obtained based on fixed effect model for each genotype x ion x inoculation x tissue (root or shoot) combination. Differences in measured traits between treatments were investigated by Wilcoxon test (stats::wilcox.test; R Core Team, 2016) for paired comparisons, and by ANOVA (stats::lm) and post hoc Tukey HSD test (agricolae::test.HSD, de Mendiburu, 2016) for multiple comparisons. Percentage root length colonization data was square root transformed prior to analysis. Ion concentration LS means were used to calculate separate correlation matrices for root and shoot (Hmisc::rcorr; Harrel, 2016) that were visualized as heatmaps (gplots::heatmap.2, Warnes et al., 2016), using the default hierarchical clustering. Principal component analysis (PCA) was performed separately for root and leaf samples, using all ion concentrations, with R statistics ade4::dudi.pca (Dray & Dufour, 2007) using centered and scaled data, and the results visualized with ade4::scatter. Only selected ions were included in the biplot.
FUNDING
This work was supported by the Swiss National Science Foundation [grant number PP00A-110874, PP00P3-130704 to U.P.]; the Gatsby Charitable Foundation [grant number RG60824 to U.P.]; and the Mexican National Council of Science and Technology [grant number CB2015-254012 to R.S.]
DISCLOSURES
Conflicts of interest: No conflicts of interest declared
Supplementary Figures
Fig. S1. P and Mn concentrations in plants with and without inoculation with Funneliformis mosseae. A - D concentrations (ppm) of P31 and Mn55 in the roots and leaves of non-colonized (NC) plants and plants inoculated with F. mosseae. Points represent mean values for each of thirty maize varieties.
Fig. S2. P and Mg concentrations in plants with and without inoculation with Funneliformis mosseae. A - D concentrations (ppm) of P31 and Mg25 in the roots and leaves of non-colonized (NC) plants and plants inoculated with F. mosseae. Points represent mean values for each of thirty maize varieties.
ACKNOWLEDGEMENTS
We thank Matthias Mueller for assistance with plant growth and evaluation.