Research Summary• The relationship between cool-season grasses and fungal endophytes is widely regarded as mutualistic, but there is growing uncertainty about whether changes in resource supply and environment benefit both organisms to a similar extent.• Here, we infected two perennial ryegrass ( Lolium perenne ) cultivars (AberDove, Fennema) that differ in carbohydrate content with three strains of Neotyphodium lolii (AR1, AR37, common strain) that differ intrinsically in alkaloid profile. We grew endophyte-free and infected plants under high and low nitrogen (N) supply and used quantitative PCR (qPCR) to estimate endophyte concentrations in harvested leaf tissues.• Endophyte concentration was reduced by 40% under high N supply, and by 50% in the higher sugar cultivar. These two effects were additive (together resulting in 75% reduction). Alkaloid production was also reduced under both increased N supply and high sugar cultivar, and for three of the four alkaloids quantified, concentrations were linearly related to endophyte concentration.• The results stress the need for wider quantification of fungal endophytes in the grassland-foliar endophyte context, and have implications for how introducing new cultivars, novel endophytes or increasing N inputs affect the role of endophytes in grassland ecosystems.

Metabolic channeling has been proposed to occur at the entry point into plant phenylpropanoid biosynthesis. To determine whether isoforms of L-Phe ammonia-lyase (PAL), the first enzyme in the pathway, can associate with the next enzyme, the endomembrane-bound cinnamate 4-hydroxylase (C4H), to facilitate channeling, we generated transgenic tobacco (Nicotiana tabacum) plants independently expressing epitope-tagged versions of two PAL isoforms (PAL1 and PAL2) and C4H. Subcellular fractionation and protein gel blot analysis using epitope-and PAL isoform-specific antibodies indicated both microsomal and cytosolic locations of PAL1 but only cytosolic localization of PAL2. However, both PAL isoforms were microsomally localized in plants overexpressing C4H. These results, which suggest that C4H itself may organize the complex for membrane association of PAL, were confirmed using PAL-green fluorescent protein (GFP) fusions with localization by confocal microscopy. Coexpression of unlabeled PAL1 with PAL2-GFP resulted in a shift of fluorescence localization from endomembranes to cytosol in C4H overexpressing plants, whereas coexpression of unlabeled PAL2 with PAL1-GFP did not affect PAL1-GFP localization, indicating that PAL1 has a higher affinity for its membrane localization site than does PAL2. Dual-labeling immunofluorescence and fluorescence energy resonance transfer (FRET) studies confirmed colocalization of PAL and C4H. However, FRET analysis with acceptor photobleaching suggested that the colocalization was not tight.

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 analyzed 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 analyzed by analysis of variance. At high N supply nitrogenous compounds, organic acids and lipids were increased; WSCs, chlorogenic acid (CGA), and fibers were decreased. The high-sugar cultivar 'AberDove' had reduced levels of nitrate, most minor amino acids, sulfur, and fibers compared to the control cultivar 'Fennema', whereas WSCs, CGA, and methionine were increased. In plants infected with endophytes, nitrate, several amino acids, and, magnesium were decreased; WSCs, lipids, some organic acids, and CGA were increased. Regrowth of blades was stimulated at high N, and there was a significant endophyte 3 cultivar interaction on regrowth. 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 carbon/N ratios and in secondary metabolite production as seen in our study is likely to have impacts on herbivore responses.

Pastoral-based animal production systems are under increasing pressure to provide the high quantity and quality of feed needed for optimal ruminant performance. The capacity of farmers to increase forage yield further, solely by increasing fertilizer inputs or through improved pasture management, is limited. Emerging requirements to balance industry production targets against the need to reduce greenhouse gas emissions and N losses pose further challenges. Plant breeding is being asked to deliver results more urgently than at any time previously, and this review attempts to highlight issues that might limit the prospects for future progress by seeking lessons from four past examples: (i) white clover breeding gains and the need to consider the complexity of the grazed grass-clover mixed sward, with its tendency for cycling in plant species composition; (ii) a systems field trial of new and old grass ⁄ clover cultivars, and how the complexity of growth of perennial forage crops, and the dynamic optimality required for sustainable harvesting might limit our ability to breed for 'yield' per se; (iii) the manipulation of a physiological trait (low 'maintenance' respiration) and the implications of such changes for plant fitness and G · E interactions; and (iv) an hypothesis-driven development of a trait (high-sugar grasses) and the value of 'proof of concept' studies, the requirement of scientific understanding of the mechanisms of trait expression, and how one might in future go about assessing breeding achievements. We discuss the general ecological considerations around shifts in the frequency distribution of traits in new populations, whether altered conventionally or by genetic modification, and how selection for a particular trait might inadvertently reduce both fitness and persistence. A major priority for breeding, we propose, might be to revisit previously abandoned traits that affected the physiological performance of forage species, armed now with a capacity to monitor gene expression at the molecular level, and so unravel ⁄ control the G · E interactions that limited their benefits. We also discuss how a 'loss of yield advantage' of new cultivars, seen when tested several years after sowing, requires urgent investigation and propose this might be associated with fitness costs of perenniality. Finally, we argue for a careful reconsideration of what are realistic expectations for systems field trials and that focus on forage breeding might be shifted more to 'proof of concept' studies, critical experimental design, comparing 'traits' rather than 'cultivars', and the wider ecological assessment of fitness and function of traits in the plant, community and ecosystem.