Q. P. Liu

Grass–endophyte interactions: a note on the role of monosaccharide transport in the Neotyphodium lolii–Lolium perenne symbiosis

Symbiotic mutualistic associations of plants with ectoand endomycorrhizal and endophytic fungi are very common in natural and agricultural ecosystems; these fungi improve nutrient acquisition and ⁄ or tolerance to biotic and abiotic stresses of their hosts (Smith & Gianinazzi-Pearson, 1988; Nehls et al., 2007; Schardl et al., 2007). A major feature of associations between the heterotrophic fungi and autotrophic plants is an exchange of fungus-derived benefits (e.g. phosphorous in ectoand endomycorrhizal associations, or antiherbivorous alkaloids in endophytic Neotyphodium–grass associations) for plant-derived carbohydrates as carbon (C) and energy source for fungal growth and maintenance (Bago et al., 2000; Nehls et al., 2007). Neotyphodium ⁄ Epichloë spp. endophytic fungi occur in 20– 30% of cool-season grass species and are of widespread interest to ecological and agricultural research (Schardl, 2001). These obligate symbiotic fungi are an additional C sink and affect host C metabolism and mobilization (Pan & Clay, 2004; Hunt et al., 2005; Rasmussen et al., 2008, 2009). Under stressful conditions, in particular, such as drought and high temperatures, endophyte symbionts have been shown to enhance host photosynthesis and potentially increase total C reserves (Richardson et al., 1993; Marks & Clay, 1996). It has also been shown that endophyte concentrations are reduced in Lolium perenne cultivars accumulating high amounts of water-soluble carbohydrates compared with control cultivars (Rasmussen et al., 2007; Liu et al., 2011). These studies indicate that endophytic fungi are important regulators of host carbohydrate metabolism; and that carbohydrate supply by hosts and C utilization by endophytes interact with each other. Carbon transfer from host plants to fungal symbionts is catalysed by transporter proteins, and several sugar transporters from mutualistic ectoand endomycorrhizal fungi have been functionally characterized (Schübler et al., 2006; Nehls et al., 2007; Helber et al., 2011). In general, it has been assumed that simple soluble sugars such as glucose and fructose are the major source of C for symbiotic fungi (Bago et al., 2000); however, some of the isolated sugar transporters have been shown to be able to catalyse the uptake of cell wall sugars such as mannose and xylose as well (Schübler et al., 2006; Fajardo López et al., 2008; Helber et al., 2011; Doidy et al., 2012). To provide more information about sugar uptake by endophytic Neotyphodium ⁄ Epichloë fungi from their hosts, we report here the isolation and functional characterization of a monosaccharide transporter from Neotyphodium lolii (MSTN). Initial studies indicated that MSTN preferentially catalyses the uptake of mannose, a monosaccharide mainly found in polymeric cell wall carbohydrates. We therefore hypothesized that N. lolii might be able to hydrolyse cell wall carbohydrates. We tested this hypothesis by quantifying the expression of cell wall carbohydrate hydrolysing fungal mannosidase, cellulase and glucanase, as well as sucrose hydrolysing fungal and plant invertases in cultured N. lolii mycelia and in ryegrass cultivars and tissues differing in their endogenous sugar content.

Functional characterisation and transcript analysis of an alkaline phosphatase from the arbuscular mycorrhizal fungus Funneliformis mosseae.

Alkaline phosphatases (ALP) in arbuscular mycorrhizal (AM) fungi have been suggested to be involved in transfer of phosphate from the mycorrhizal fungus to the host plant, but exact mechanisms are still unknown, partially due to the lack of molecular information. We isolated a full-length cDNA (FmALP) from the AM fungus Funneliformis mosseae (syn. Glomus mosseae) showing similarity with putative ALP genes from Rhizophagus intraradices (syn. Glomus intraradices) and Gigaspora margarita. For functional characterisation FmALP was expressed heterologously in the yeast Pichia pastoris. The recombinant FmALP protein had a pH optimum of 9.5, and catalysed the hydrolysis of glycerolphosphate and, to a lesser extent of glucose-1- and 6-phosphate, confirming it to be an alkaline phosphatase belonging to the family of alkaline phosphomonoesterases (EC 3.1.3.1). FmALP did not catalyse the hydrolysis of ATP or polyP. Relative FmALP transcript levels were analysed in intra- and extraradical hyphae isolated from F. mosseae infected ryegrass (Lolium perenne) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FmALP was highly expressed in intraradical hyphae at low P(i) supply, and its expression was repressed by high P(i) supply. Taken together this study provides evidence for mycorrhizal alkaline phosphatases playing a role in P mobilisation from organic substrates under P starvation conditions.

Transcriptional regulation of phosphate transporters from Lolium perenne and its mycorrhizal symbionts in response to phosphorus supply

Phosphate (P) uptake is critical for plant growth, but to date little is known about P uptake and transport in the pasture grass Lolium perenne L. We have identified a putative P transporter (PT) from L. perenne mycorrhizal roots (LpPT1) and assessed its transcriptional regulation by soil P availability and mycorrhizal colonisation. We also investigated transcript levels of fungal PTs from the two arbuscular mycorrhizal species Rhizophagus intraradices and Funneliformis mosseae. Our analyses indicated that LpPT1 codes for a high affinity PT most likely responsible for direct P uptake from the soil. LpPT1 is highly expressed in roots of plants grown at low P, whereas high P repressed its expression. LpPT1 was not expressed in above-ground plant tissues. Colonisation with R. intraradices did not affect expression of LpPT1 significantly. Transcript levels of the R. intraradices PT were not affected by P availability but the F. mosseae PT was repressed by high P supply, particularly in intraradical hyphae. Our study could assist in deciphering the molecular mechanisms of P uptake in the pasture grass L. perenne.

Does gibberellin biosynthesis play a critical role in the growth of Lolium perenne? Evidence from a transcriptional analysis of gibberellin and carbohydrate metabolic genes after defoliation

Global meat and milk production depends to a large extent on grazed pastures, with Lolium perenne being the major forage grass in temperate regions. Defoliation and subsequent regrowth of leaf blades is a major and essential event with respect to L. perenne growth and productivity. Following defoliation, carbohydrates (mainly fructans and sucrose) have to be mobilized from heterotrophic tissues to provide energy and carbon for regrowth of photosynthetic tissues. This mobilization of reserve carbohydrates requires a substantial change in the expression of genes coding for enzymes involved in carbohydrate metabolism. Here we tested the hypothesis that gibberellins (GA) are at the core of the processes regulating the expression of these genes. Thus, we examined the transcript profiles of genes involved in carbohydrate and GA metabolic pathways across a time course regrowth experiment. Our results show that following defoliation, the immediate reduction of carbohydrate concentrations in growing tissues is associated with a concomitant increase in the expression of genes encoding carbohydrate mobilizing invertases, and was also associated with a strong decrease in the expression of fructan synthesizing fructosyltransferase genes. We also show that the decrease in fructan levels is preceded by increased expression of the GA activating gene GA3-oxidase and decreased expression of the GA inactivating gene GA2-oxidase in sheaths. GA3-oxidase expression was negatively, while GA2-oxidase positively linked to sucrose concentrations. This study provides indicative evidence that gibberellins might play a role in L. perenne regrowth following defoliation and we hypothesize that there is a link between gibberellin regulation and sugar metabolism in L. perenne.