Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content and fungal endophyte infection

Lolium perenne cultivars differing in their capacity to accumulate water soluble carbohydrates (WSCs) were infected with three strains of fungal Neotyphodium lolii endophytes or left uninfected. The endophyte strains differed in their alkaloid profiles. Plants were grown at two different levels of nitrogen (N) supply in a controlled environment. Metabolic profiles of blades were analysed using a variety of analytical methods. A total of 66 response variables were subjected to a principle components analysis and factor rotation. The first three rotated factors (46 % of the total variance) were subsequently analysed by ANOVA. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA) and fibres were decreased. The high sugar cultivar AberDove had reduced levels of nitrate, most minor amino acids (AAs), sulphur and fibres compared to the control cultivar Fennema, whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several AAs, and Mg were decreased; WSCs, lipids, some organic acids and CGA were increased. Re-growth of blades was stimulated at high N; and there was a significant endophyte x cultivar interaction on re-growth. Mannitol, a fungal specific sugar alcohol, was significantly correlated with fungal biomass. Our findings suggest that effects of endophytes on metabolic profiles of L. perenne can be considerable, depending on host plant characteristics and nutrient supply, and we propose that a shift in C/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses. indirect nature linked to the nutritional value of plants and/ or of a more direct nature linked to toxicity of secondary metabolites beyond fungal alkaloids. Our study also shows that metabolic traits of specific grass cultivars/ populations and nutrient availability can be critical factors in determining metabolic and physiological outcomes of the grass-endophyte association and must therefore be taken into consideration for future experiments.


INTRODUCTION
variables as supplementary material (Table S1). It should be noted, that when these variables are analysed as univariate response variables rather than as part of a principal component axis, some of them show significant interactions that are not readily apparent in the multivariate approach, e.g. LMW and HMW WSCs (Table S1; Rasmussen et al., 2007). We therefore attached the complete data set as supplementary material (Table S2) for those readers who would like to try alternative analyses of these data. The standardised univariate responses of these variables are shown in Figure 2b, 2d, and 2f as support for the interpretation of the multivariate responses and to allow a closer inspection of those variables loading heavily onto RF-1 (i.e. loadings ≥ 0.5 and ≤ -0.5). Variable standardisation allows direct comparison of the response magnitudes for variables with either very different concentrations, or variables with different units of measurement. As can be seen (Fig. 2b), the effect of high N supply was most prominent on major amino acids (L-Gln, L-Asp, L-Thr, L-Ala, L-Asn, L-Arg, L-Ser, L-Asn, and L-Glu), which represent 85% of the total free amino acid pool across all treatments (Table S1). Nitrate, total N and total protein were also considerably increased at high N supply and we note here that nitrate was increased almost 9-fold, whereas all other variables were increased less than 3.2 fold (based on untransformed data, see also Table   S2). The standard deviation for nitrate was very high, resulting in relatively smaller differences when standardised. Minor amino acids (L-His, L-Gly, L-Ile, L-Leu, L-Tyr, L-Val, L-Pro, L-Lys, and L-Phe) were much less affected by increased N supply. L-Met was the only AA analysed here that was not affected by N supply and did not load strongly onto any of the three rotated factors. All variables loading negatively onto RF-1 (loadings ≤ -0.5; carbohydrates and CGA) were decreased at high N supply (Fig. 2b).
Most variables (loadings on RF-1 ≥ 0.5) were in fact decreased in the high sugar cultivar AberDove compared to Fennema, but this effect was most prominent on minor AAs and nitrate (Fig. 2d). Interestingly, S was reduced in this cultivar as well, and we note here that the S containing AA L-Met was significantly increased in AberDove (2.4-fold), when that variable is subjected to a standard univariate analysis (Table S1).
Effects of endophyte infection on the magnitude of the standardised univariate responses was strongly strain dependent, with AR1 having the weakest and AR37 the strongest effect on most variables (Fig. 2f). Almost all AAs were reduced in endophyte infected plants, but this effect was most apparent for L-Asn and several minor AAs.
Carbohydrates and CGA were increased in infected plants, but the responses were much weaker for HMW WSCs and CGA. The precursors of aromatic AAs and phenylpropanoids (shikimate and quinate), OMD and ME were strongly increased in Fennema infected with endophyte (especially with AR37; Fig. 3d); this effect was much weaker in AberDove (Fig. 3e). Lipids and malate were increased by endophyte infection in both cultivars to the same degree. The two fatty acids (C17:0 and C18:0), NDF and Mg were reduced in endophyte infected plants, but the magnitude of that effect was dependent on the cultivar. significantly more re-growth, as expected. There was no difference in re-growth between the two EF cultivars, but the endophytic strain AR37 stimulated re-growth more in AberDove compared to AR1 infected plants, and also compared to EF and CS infected Fennema.

Mannitol
Previously (Rasmussen et al., 2007), we reported that a regression of endophyte alkaloid (peramine, lolitrem B, janthitrems) concentrations against fungal concentrations was highly significant. As reported, fungal concentrations were determined by qPCR of two endophyte specific genes, chitinase and a non-ribosomal peptide synthetase, which were highly correlated and condensed to a single principal component PC1. Here, we regressed another fungal metabolite, the sugar alcohol mannitol, to the same PC1 and also found the regression to be highly significant (F 1.114 =190.42, P<0.0001). The untransformed data are shown in Figure 6. Endophytic alkaloid concentrations were affected in the same way and were, in fact, highly correlated with endophyte concentrations. The same plant material used in that previous study was analysed here in detail for metabolic responses to the different treatments (high N supply, high sugar cultivar, and endophyte infection). We discuss possible mechanisms of how these treatments might have caused the described reduction in endophyte concentrations based on the different metabolite profiles. We also discuss the extent to which the observed changes support the notion that endophytes might be a drain (net cost) on plant metabolism or might up-regulate metabolism (cf. sink stimulation). A change in metabolic profiles per se may provide insights into the nature of the grass-endophyte association and may also be critical to understand further multitrophic interactions, e.g. the response of herbivores (insects and grazers) to the ryegrass-endophyte association.

Effects of high nitrogen supply
The effects of high N supply on metabolic profiles in ryegrass blades were most prominent on nitrogenous compounds, as expected. Nitrate levels in blades were approx. 9-fold higher in the high N treatment, indicating that nitrate uptake exceeded the plants' capacity for nitrate assimilation. Although 18 out of 19 analysed amino acids were increased, there was a marked difference in the response of individual amino acids. Major AAs, which represented approx. 85 % of the total free AAs, were much more affected than minor AAs. This is in accordance with findings from a variety of crop plants, where mainly major AAs responded to changes in carbon and nitrogen metabolism, whereas minor AAs were not correlated with these changes and correlated more with each other than with total AA pools (Noctor et al., 2002).
As nitrate assimilation into AAs requires reductants (10 electrons per molecule nitrate), energy (ATP) and carbon skeletons this process is tightly linked with photosynthesis and carbon metabolism (Stitt et al., 2002;Smith and Stitt, 2007). Nitrate supply results in decreased carbohydrate synthesis and accumulation, and a large proportion of carbon is converted via glycolysis and citric acid cycle into organic acids, as was seen in the present study as well -malate, succinate and citrate were all increased at high N supply. These organic acids serve several purposes, the major ones In a discussion of our previous findings that high N supply reduced endophyte concentrations, we hypothesised that this might be due to a 'dilution' effect, i.e. plant growth is increased more than fungal growth under these conditions. This hypothesis is supported by the data set on yield presented here (Fig. 5a) , 2000;Faeth and Sullivan, 2003;Cheplick 2004;Faeth et al., 2004;Hesse et al., 2004;Faeth and Hamilton, 2006;Cheplick 2007). In plants infected with mycorrhizal fungi the increased costs due to carbon flow to the fungus can be off-set by increases in photosynthesis (Wright et al., 1998) and improved plant nutrition (Smith et al., 2001).
Studies of photosynthetic processes in grasses infected with foliar endophytes are not conclusive and rates of net photosynthesis can be increased (Belesky et al., 1987;Amalric et al., 1999), unchanged or decreased (Spiering et al., 2006, depending on the growth phase of the host plants, nutrient status and environmental conditions. Marks and Clay (1996) demonstrated an endophyte by temperature interaction, and Newman et al. (2003) found an endophyte by N interaction on photosynthetic rates. Effects of endophyte infection on growth also strongly depend on host genotype, resource availability and environmental stress (Belesky et al., 1989;Malinowski and Belesky, 2006;Morse et al., 2002;Cheplick & Cho, 2003;Hesse et al., 2003;Zabalgogeazcoa et al., 2006;Cheplick 2007). In our study we saw significant cultivar by endophyte interactions on the re-growth of blades, which was only stimulated in AberDove plants infected with the endophyte strain AR37, clearly demonstrating the importance of specific host plant -endophyte strain interactions and environmental conditions on overall physiological outcomes of the association. The present study is an analysis of combined plant and fungal metabolites and as most metabolites analysed here are likely to be present in both organisms it is impossible to make statements about impacts on plant metabolism only. We can therefore only discuss overall effects of endophyte infection on the symbiotic metabolism as compared to the non-symbiotic endophyte plants.
A major effect of endophyte infection was an approx. 50% reduction in nitrate levels in the blades, which was accompanied by a reduction of several AAs, total N and total protein; such a reduction of nitrogenous compounds has been described earlier for N. coenophialum infected tall fescue (Belesky and Fedders, 1996)  endophytes studied here are absent from the roots, their impacts on plant N uptake and transport are probably more indirect. A study of nitrate transporters, nitrate reductase and root metabolites in endophyte infected plants is needed to understand the mechanisms by which foliar endophytes reduce nitrogenous compounds.
Asparagine was the most reduced amino acid and L-Asn levels are mainly regulated by the C/N status of plants. High levels of organic N and low levels of carbon skeletons result in high levels of L-Asn as this amino acid has a high N to C ratio and acts as an inert and stable N-reserve (Lam et al., 1996). In our study, endophyte infection resulted in an increased C/N ratio -more soluble sugars and less organic N -and this might have negatively affected L-Asn biosynthesis.
As pointed out, sugar levels were increased in endophyte infected plants, it is possible that this increase is caused simply by reduced use of carbon skeletons for AAs and proteins; we also found reduced levels of fibres in endophyte infected plants, which could mean that more of the fixed carbon remains soluble and is not incorporated into cell walls. But higher sugar levels might also, at least partially, be a result of increased 'sink strength' as seen in plants infected with mycorrhizal fungi (Wright et al., 1998;Douds et al., 2000;Graham 2000;Pfeffer et al., 2001). However, as stated above, mycorrhizae have a much higher biomass compared to the foliar endophytes studied here; and furthermore, the tissue we analysed is both, source (photosynthetically active plant tissue) and sink (heterotrophic fungal tissue), at the same time and it is therefore difficult to distinguish specific sink effects.
While the organic acids citrate and succinate were decreased in endophyte infected plants, malate was increased. It has been shown that malate plays a critical role in lipid biosynthesis in filamentous fungi, where it is irreversibly decarboxylated to pyruvate by malic enzyme with the formation of NADPH. Malic enzyme is suggested to be the major NADPH-generating enzyme required for providing reducing power for fatty acid synthase in Aspergillus nidulans and other lipid storing fungi (Wynn and Ratledge, 1997;Wynn et al., 1999;Zhang et al., 2007). Light microscopy studies have shown that N. lolii hyphae accumulate lipid bodies in its hyphae in planta (Christensen et al., 2002), and in the present study lipids were increased in endophyte infected plants. A gene coding for malic enzyme has not been identified in Neotyphodium spp., but it is likely that the identification of this gene and subsequent expression and localisation studies will give further insights into fungal metabolic processes that are linked to the C and N economy of the host plant.