Tall fescue endophyte effects on tolerance to water-deficit stress
© Nagabhyru et al.; licensee BioMed Central Ltd. 2013
Received: 19 February 2013
Accepted: 1 August 2013
Published: 9 September 2013
The endophytic fungus, Neotyphodium coenophialum, can enhance drought tolerance of its host grass, tall fescue. To investigate endophyte effects on plant responses to acute water deficit stress, we did comprehensive profiling of plant metabolite levels in both shoot and root tissues of genetically identical clone pairs of tall fescue with endophyte (E+) and without endophyte (E-) in response to direct water deficit stress. The E- clones were generated by treating E+ plants with fungicide and selectively propagating single tillers. In time course studies on the E+ and E- clones, water was withheld from 0 to 5 days, during which levels of free sugars, sugar alcohols, and amino acids were determined, as were levels of some major fungal metabolites.
After 2–3 days of withholding water, survival and tillering of re-watered plants was significantly greater for E+ than E- clones. Within two to three days of withholding water, significant endophyte effects on metabolites manifested as higher levels of free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots. The fungal metabolites, mannitol and loline alkaloids, also significantly increased with water deficit.
Our results suggest that symbiotic N. coenophialum aids in survival and recovery of tall fescue plants from water deficit, and acts in part by inducing rapid accumulation of these compatible solutes soon after imposition of stress.
Tall fescue (Lolium arundinaceum = Schedonorus arundinaceus = Festuca arundinacea) is the most widely planted forage grass in the United States  and it is often infected with the endophytic fungus, Neotyphodium coenophialum. The relationship between the endophyte and plant is generally considered mutualistic because the endophyte significantly improves host plant tolerance to drought, insects, diseases, and nematodes, along with increased persistence and vigor; and in turn the plant provides the symbiont with nutrients, protection, and reliable and efficient dissemination (reviewed in ). Evidence suggests that tall fescue plants with the endophyte (E+) grow and persist longer under stressful conditions, such as water deficit, compared to endophyte free plants (E-), and are, therefore, likely to have an adaptive and competitive advantage [3–9]. Mechanisms for endophyte-enhanced drought avoidance or tolerance appear complex, and might involve direct and indirect effects of the endophyte on metabolism and other physiological changes in the host plant [10–13].
Processes affected by the tall fescue endophyte include stomatal closure , decreased root diameter and increased root hair length [7, 15], increased turgid weight/dry weight ratios suggesting reduced damaged to cell walls , and enhanced production of phenolic root exudates . Leaf rolling under drought stress is reported to be much more common in E+ than E- plants . Greater cell wall elasticity  and higher water use efficiency  in E+ tall fescue compared to E- plants under drought stress have also been reported. Previous research has also shown that E+ tall fescue plants of some genotypes exhibit lower stomatal conductance than E- plants with more sensitive inducement of stomatal closure in E+ plants in response to early stages of water deficit [17–19]. Endophyte infection confers population stability in tall fescue during drought stress through improved tiller and whole plant survival .
A correlation between drought tolerance and accumulation of compatible solutes such as carbohydrates, amino acids, and mineral ions that contribute to osmotic adjustment has been documented in grasses [20–22]. In general, accumulation of sugars, sugar alcohols , and proline [24, 25] in response to water deficit in grasses has been reported. A significant endophyte effect on accumulation of simple sugars in leaves of E+ tall fescue, was observed when plants were osmotically stressed by polyethylene glycol . Under water deficit, E+ tall fescue plants are reported to exhibit decreased growth and increased root and leaf senescence, as well as greater accumulation of sugars within the pseudostem, and decreased water potential compared to E- plants . Effects of the endophyte on levels of other metabolites, such as proline  and other amino acids have not been well studied. Here we report what is, to our knowledge, the first comprehensive profiling of shoot and root metabolite responses to acute water deficit stress, assessing the timing of endophyte effects on sugars, sugar alcohols and amino acids relative to the endophyte effects on subsequent plant recovery.
Tall fescue is an obligately outcrossing grass, so that isogenic lines cannot be generated, and plants derived from different seeds are necessarily unique genotypes. Therefore, to control for host genotype effects we developed genetically identical clones with endophyte (E+) and without endophyte (E-) as follows. Ramets of tall fescue ‘Kentucky 31’ plants naturally infected with Neotyphodium coenophialum were treated with the fungicide propiconazole or tebuconazole to remove the fungus [29, 30]. The stock plants and fungicide-treated clones were examined for the presence or absence of endophyte by tissue print immunoblot , PCR , and microscopy. This resulted in E+/E- clone pairs, two of which were used in this study. Lab identification numbers 278 (E+) and 279 (E-) represented one clone pair, and 4607 (E+) and 4608 (E-) represented the other clone pair. Plants of each clone pair were raised side-by-side in the greenhouse for more than one year prior to being used in the study.
Ramets consisting of three tillers of similar size were planted into 8.5 × 8.5 cm square pots in sand, in the greenhouse. Sand was chosen as the growth medium because it allows even, uniform and rapid drying, and also provides for easy harvesting of roots. Plants were watered twice daily for six weeks before subjecting them to experimental conditions to allow for regeneration and accumulation of sufficient biomass for sampling. After sufficient re-growth had occurred, water was withheld from the test group, while control plants were watered twice daily. Pots were randomized once while setting up the experiment and again before subjecting them to treatments, in order to control for effects micro-environmental variation.
Treatments were endophyte-infected watered controls (E+D-), endophyte-infected water-deficit stressed (E+D+), endophyte-free watered controls (E-D-), and endophyte-free water-deficit stressed (E-D+). Entire pots were sampled on each day from day 0 to day 5 of withholding water. Beyond day 5 plants were fully dried and mostly dead. Three or four replicates were sampled for each treatment x day. For the first experiment, which was conducted with the 278/279 clone pair, samples were harvested from February 2–7, 2007. For the second experiment with clone pair 278/279, samples were harvested from June 2–7, 2008. The third experiment was conduced with clone pair 4607/4608, sampled from July 21–26, 2008. All plants were grown in the greenhouse under natural light conditions, with 45-70% relative humidity ranges, and temperatures set to 27°C/22°C (day/night). Photoactive radiation (PAR) measurements were recorded during the three experiments (see Additional file 1, panels a, b, c). Samples were harvested between 7:30 a.m. to 8:30 a.m. local time each day, immediately frozen in liquid nitrogen, lyophilized and subsequently prepared for metabolite analysis as described below. The samples were divided into shoot (leaf along with tiller base down to 1 cm from crown region) and root material.
Tiller recovery experiment
Five to six pots subjected to water-deficit conditions from each E+/E- clone pair for each day of treatment were left unharvested, and were placed back into a daily watering regime in order to determine their ability to recover from the water-deficit stress. Live tiller numbers were counted after 6 weeks of recovery.
Carbohydrate analysis by high pH anion exchange chromatography
Sugars were extracted in 1 ml of 80% ethanol per 100 mg of ground lyophilized plant material. The samples were incubated at 65°C for 1 hr and 90°C for 5 min and the supernatant was evaporated in a vacuum centrifuge. The residue was reconstituted in purified water at 4°C and filtered through spin-X HPLC 0.4 μm nylon filter micro centrifuge (Corning, NY) tubes. Filtered supernatant (100 μL) was diluted to 1 ml and used for analysis on a Dionex ICS 3000 with either a carbopac PA1 column for neutral sugars or a carbopac MA1 column for polyols. Neutral sugars were separated by an isocratic program with 24 mM NaOH, and sugar alcohols were separated using 480 mM NaOH. The detection was by pulsed amperometry, using a gold working electrode. Peak identity and sugar quantity were determined by comparison with standards. The internal standard was 2-deoxyglucose.
Amino acid analysis by liquid chromatography-mass spectrometry (LC-MS)
The yields of free amino acids from plant samples were compared for different extraction methods using a) 80% ethanol, or b) chloroform: methanol: water (5:12:3), and incubating at different temperatures (4°C and 45°C) for 1 hr. However both extraction solvents and methods resulted in similar extraction efficiency, so the simpler extraction method was chosen for further analysis. Finely ground lyophilized plant shoot and root material (50 mg) was extracted with 5 ml of 80% ethanol on ice for 1 hr. The crude extract was filtered through 0.4 μm centrifuge tubes and the supernatant was used for sample cleanup and derivatization with EZ faast LCMS kit for free amino acids from Phenomenex, according to the kit protocol. Briefly, 100 μL of each sample was mixed with 100 μL of internal standard containing homoarginine, d3-methionine, and homophenylalanine provided in the kit. Then sample was loaded onto a pipet tip packed with ion exchange resin on which free amino acids were bound, subsequently washed and released from resin. The free amino acids were then derivatized by propyl chloroformate and liquid-liquid extracted with isooctane. The organic phase containing the derivatized amino acids was removed under a stream of high purity nitrogen gas and the residue was redissolved in 200 μL 2:1 mobile phase of A:B (A: 10 mM ammonium formate in water and B: 10 mM ammonium formate in methanol). Analysis was performed by liquid chromatography mass spectrometry with a dual pump ProStar 210 HPLC with 1200 L quadrupole MS-MS (Varian).
Loline alkaloid analysis
Loline alkaloids were extracted from samples using chloroform under alkaline conditions . Quinoline was used as an internal standard and the lolines were quantified by gas chromatography (Varian CP-3800) interfaced with a Varian Saturn 2200 ion trap mass spectrometer. Loline amounts were calculated as the total of loline, N-methylloline, N-formylloline, N-acetylloline and N-acetylnorloline.
Three-factor ANOVA [F df (5,72) ] values of all metabolites in Experiment 1
Day * Endophyte
Stress * Endophyte
Stress * Day
Endophyte * Stress * Day
Shoot glutamic acid
Shoot aspartic acid
Root glutamic acid
Root aspartic acid
Three-factor ANOVA [F df (5,48) ] values of all metabolites in Experiment 2
Day * Endophyte
Stress * Endophyte
Stress * Day
Endophyte * Stress * Day
Shoot glutamic acid
Shoot aspartic acid
Root glutamic acid
Root aspartic acid
Three-factor ANOVA [F df (5,48) ] values of all metabolites in Experiment 3
Day * Endophyte
Stress * Endophyte
Stress * Day
Endophyte * Stress * Day
Shoot glutamic acid
Shoot aspartic acid
Shoot phenyl alanine
Root glutamic acid
Root aspartic acid
Tiller number and recovery
Results in Experiment 2, also with clone pair 278/279, were very similar except for a one-day delay in effects on tiller survival and metabolites, probably because of overcast skies on the first day (see Additional file 1, panel b). At day 2 of withholding water, free glucose and fructose levels in E+ were approximately 2–4 fold higher than in watered controls or in E- stressed plants (Figures 2c and 3c). There were no significant differences in sucrose levels at day 2 between E+ and E- plants (Figure 4c, Table 2). In roots, sucrose levels were 2–3 fold higher in E+ compared to E- roots from day 2 to day 4 (Figure 4d), though there were no significant differences in glucose or fructose (Figures 2d and 3d, Table 2, and see Additional file 2, panels c, d).
Comparing free glucose and fructose sugars in clone pair 4607/4608 during the water deficit period, there were significant differences between E+ and E- in the roots (Figures 2f and 3f); but not in shoots except for fructose at day 5, where E+ shoots accumulated fructose to higher levels than E- shoots (Figures 2e and 3e). Root glucose and fructose concentrations increased by day 2 of withholding water, and were significantly higher in E+ than E- plants.
In the second experiment with clone pair 278/279, increases in amino acid levels started from day 3 after withholding water. At that time point, proline levels in E+ clones were significantly higher than in E- clones both in shoots (Figure 7c) and roots (Figure 7d).
We assessed plant survival and differences in metabolite accumulation in two tall fescue clone pairs with (E+) or without (E-) symbiotic Neotyphodium coenophialum over a time course of water deficit stress, and observed that E+ plants recovered significantly better than E- plants after 2–3 days of withholding water. Simultaneously, the E+ plants consistently accumulated more free sugars, sugar alcohols and amino acids early during the onset of stress, compared to E- plants. The fungal-specific metabolites, mannitol and loline alkaloids, also increased in this time period. The higher metabolite levels in E+ compared to E- plants over the time course of withholding water consistently occurred within one day prior to a significant endophyte effect on plant recovery, strongly suggesting that free sugars, polyols, amino acids, and fungal metabolites play roles in endophyte-enhanced tolerance to water deficit. The production or release of these substances may lead to osmotic adjustment [20, 36], and help maintain integrity of cellular enzymes, proteins, nucleic acids and membranes , or protect against reactive oxygen species (ROS) [38, 39].
The accumulation of soluble sugars is strongly correlated with drought tolerance in plants . These sugars affect osmotic adjustment, which is considered an important mechanism to allow maintenance of water uptake and cell turgor under stress conditions . Furthermore, hydroxyl groups of sugars and polyols can interact with proteins and membranes to prevent denaturation and help avoid the crystallization of cytoplasm under low-water stress [42, 43]. In addition, these sugars have been shown to be important regulatory molecules in different signaling pathways [22, 44], helping to maintain redox balance, and acting as reactive oxygen scavengers [45, 46]. In general, endophytic fungi are similar to plant pathogenic fungi in possessing glucan hydrolase-32 (GH32 invertase) enzymes that convert sucrose into glucose and fructose for catabolism . Fungal invertase activity and presence of invertase gene transcripts have been reported in some of the grass endophytes [48, 49], so under the conditions imposed in our study, fungal enzymes may play at least a partial role in the observed increases in these free sugars.
Mannitol and arabitol are common polyols in fungi, and have been observed to accumulate in plants during infection . We found that both polyols increased in response to water deficit in the E+ tall fescue clones. Our results, are in agreement with Richardson et al.  who reported mannitol in E+ tall fescue plants, although they did not see an effect on the mannitol levels when the plants were osmotically stressed with polyethylene glycol. Arabitol accumulated essentially only under stress (Figure 6) conditions . Most plants do not normally contain mannitol, with some salt tolerant species, such as celery, as exceptions . Note that the very low levels of mannitol in some E- plants was likely due to the presence of commensal fungi on the plants, since the plants were not grown axenically. Plants engineered to produce mannitol have shown increased tolerances to drought, salt, and temperature stresses [52–55], so mannitol in the E+ plants may have contributed to their tolerance of water deficit stress.
The non-reducing disaccharide, trehalose, is an important osmoprotectant and storage carbohydrate in many organisms. In plants, the trehalose pathway is ubiquitous and indispensible, but with a few exceptions, such as in resurrection plants, trehalose typically does not accumulate to high levels, possibly due to trehalase-catalyzed cleavage to glucose. Significant increases in trehalose accumulation have been accomplished thorough transgenic approaches, and shown to protect plants from drought and salt stresses [56–59]. However, the overproduction or accumulation of high levels of trehalose is also observed to cause growth aberrations in some of the transgenic experiments [60–63]. In our studies, we observed increased levels of trehalose after 3 days of withholding water, with significantly higher levels in E+ plants. Although the overall levels of trehalose observed in the E+ and E- plants were very low compared to the other soluble sugars and polyols, the observed spike in trehalose accumulation during stress, and differences between E+ and E- plants in trehalose levels suggest a possible functional role. While it is possible that the low trehalose levels observed in these plants could function in stress tolerance , it seems more likely that the trehalose accumulation is associated with the signaling/regulation role that has been documented [65–70].
Water deficit has been shown to increase levels of ROS, so an important role of accumulated metabolites appears to be scavenging or detoxifying ROS [45, 71, 72]. Production of phenolics, carbohydrates, mannitol, and proline with antioxidant capacity protects plants from oxidative stress under water-deficit conditions. As reviewed by White and Torres , symbiotic plants are protected from different abiotic and biotic stresses by production of these antioxidants.
The timing of metabolite changes was also highly suggestive of their roles in endophyte-enhanced stress tolerance. In all three experiments we observed endophyte-enhanced increases in certain sugars, sugar alcohols and amino acids one day before observing the significant endophyte effect on recovery of the stressed plants. Interestingly, endophyte effects on levels of most metabolites were brief, since levels of these metabolites in E+ plants decreased or plateaued over the following days to levels similar to those in E- plants. In addition to enhancing osmotic adjustment, it is also possible that these accumulated solutes provided energy, carbon and nitrogen for the survival of meristematic regions, and helped in regrowth of the plant after the water deficit was alleviated.
Levels of several amino acids have been shown to increase in drought stressed plants . In our experiments, the levels of proline, threonine, tryptophan, phenylalanine, tyrosine, and valine increased upon water deficit stress. In addition, proline was found to be consistently higher in both shoots and roots of E+ stressed plants than in E- stressed plants. A correlation between free proline accumulation and the performance of crops in the field at low water availability suggests that its accumulation is a drought stress adaptive response that enhances survival . Proline may serve as an osmoregulator  and also as a ROS scavenger .
Loline alkaloids are protective secondary metabolites produced by the endophyte in tall fescue and other cool season grasses [77, 78]. We observed increased loline alkaloid levels in response to water deficit stress in both clone pairs. Lolines are derived from proline and aspartate . Conceivably, proline is depleted by loline production , but since no differences in proline levels were observed between E+ and E- plants in unstressed conditions, proline levels were apparently adjusted in response to loline alkaloid synthesis. In the first experiment with clone pair 278/279 total proline and loline levels in E+ plants were higher even at day 2 after withholding water compared to E- plants, though levels of proline (and the metabolically closely related amino acids, glutamic acid and glutamine) were not different in between E+ and E- plants at day 2. It is possible that the proline is converted to loline in the E+ plants, thus maintaining an apparent equal proline level as that of E- plants. However, in the other two experiments, the levels of proline in stressed tissues were far higher compared to amounts of lolines that accumulated in those tissues. Although water deficit has been reported to increase loline alkaloid levels in leaf tissues of some tall fescue accessions , a direct role of loline alkaloids on water stress tolerance has not yet been demonstrated.
Differences in the timing of metabolite accumulation were observed between two experiments with the same clone pair (278/279), with metabolite peaks at day 1 in Experiment 1 and the corresponding peaks occurring at day 2 or 3 in Experiment 2. This difference may be because of weather and greenhouse conditions that differed between these experiments. Specifically, day 1 of Experiment 2 was accompanied with thunderstorms and heavily overcast skies, resulting in lower photoactive radiation compared to day 1 of Experiment 1 (see Additional file 1, panel b), apparently delaying the onset of drought stress as evidenced by the tiller recovery curves (see Figure 1a and b). Similarly the observed metabolite differences between the experiments with different clone pairs could be due to plant genotype effects. Nevertheless, it was clear that, in our experiments the endophyte in tall fescue sped up plant responses to water deficit by earlier and faster accumulation of metabolites compared to uninfected tall fescue plants. Similar results have been reported in bacterial endophyte-plant systems. Bacterial endophyte enhances cold tolerance of grapevine plants by altering sugar metabolism and photosynthesis , and with higher and faster accumulation of stress related gene transcripts and metabolites .
Rasmussen et al.  have conducted comprehensive metabolomic studies in the related grass, Lolium perenne (perennial ryegrass), and have shown significant effects of the endophyte, Neotyphodium lolii, on primary and secondary metabolism of that grass. The need for more research to identify robust metabolic traits and pathways relating to drought tolerance in forage grasses through integration of metabolomic and transcriptomic data have been emphasized in reviews . From our study it was evident that endophyte can affect tall fescue plant metabolism, in response to water deficit stress. Analyzing these endophyte effects on host plants at the molecular genetic level by transcriptome profiling is another approach, that we will be exploring further to help elucidate the mechanisms of endophyte-enhanced plant growth and survival under water deficit conditions.
In conclusion, enabling the plant cells to sense and respond quickly to surrounding environmental signals or stresses is important for their metabolic and developmental adjustments, and these responses may be enhanced due either to primary or secondary metabolite signals [86, 87]. As we observed in the tall fescue clone pairs, symbiotic fungi in the infected plants may have induced, or rapidly activated, the plant biochemical reactions to accumulate the metabolites early in stress conditions, and this may be one of the ways that the presence of the endophyte helps mitigate the effects of, and enhance recovery from, water deficit stress. The results presented here demonstrate that symbiosis with endophytes can significantly enhance recovery of host plants from water deficit stress, and the effect corresponds in timing with accumulation of organic solutes that may serve as osmolytes and cellular protectants in leaves and roots.
This research is funded by USDA-ARS Specific Cooperative Agreement 200911131030. The authors are grateful for help and suggestions from Dr. Bruce A. Downie in HPLC for carbohydrates. The authors also thank J. Douglas Brown and W. Troy Bass for maintaining plants, and Dr. Lowell P. Bush and Dr. Fanniel F. Fannin for providing the loline alkaloid standard. The authors also acknowledge the ERTL facility at the University of Kentucky for allowing use of the LCMS and for technical assistance. This is publication number 13-12-101 of the Kentucky Agricultural Experiment Station, published with approval of the director.
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