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.
KeywordsFungal endophyte Tall fescue Water deficit stress Metabolites Neutral sugars Amino acids and lolines
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.
- Fribourg HA, Hannaway DB, West CP: Tall fescue for the twenty-first Century. 2010, ASA, CSSA, SSSA: Madison, WIGoogle Scholar
- Schardl CL, Leuchtmann A, Spiering MJ: Symbioses of grasses with seedborne fungal endophytes. Annu Rev Plant Biol. 2004, 55: 315-340. 10.1146/annurev.arplant.55.031903.141735.PubMedView ArticleGoogle Scholar
- Arachevaleta M, Bacon CW, Hoveland CS, Radcliffe DE: Effect of the tall fescue endophyte on plant response to environmental stress. Agron J. 1989, 81: 83-90. 10.2134/agronj1989.00021962008100010015x.View ArticleGoogle Scholar
- West CP: Physiology and drought tolerance of endophyte-infected grasses. Biotechnology of Endophytic Fungi of Grasses. Edited by: Bacon CW, White JF. 1994, Boca Raton, FL: CRC Press, 87-99.Google Scholar
- West CP, Izekor E, Turner KE, Elmi AA: Endophyte effects on growth and persistence of tall fescue along a water-supply gradient. Agron J. 1993, 85: 264-270. 10.2134/agronj1993.00021962008500020019x.View ArticleGoogle Scholar
- Bacon CW: Abiotic stress tolerances (moisture, nutrients) and photosynthesis in endophyte-infected tall fescue. Agric Ecosyst Environ. 1993, 44: 123-141. 10.1016/0167-8809(93)90042-N.View ArticleGoogle Scholar
- Malinowski DP, Belesky DP: Adaptations of endophyte-infected cool-season grasses to environmental stresses: Mechanisms of drought and mineral stress tolerance. Crop Sci. 2000, 40: 923-940. 10.2135/cropsci2000.404923x.View ArticleGoogle Scholar
- Zhang Y, Nan ZB: Growth and anti-oxidative systems changes in Elymus dahuricus is affected by Neotyphodium endophyte under contrasting water availability. J Agron Crop Sci. 2007, 193: 377-386. 10.1111/j.1439-037X.2007.00279.x.View ArticleGoogle Scholar
- Hahn H, McManus MT, Warnstorff K, Monahan BJ, Young CA, Davies E, Tapper BA, Scott B: Neotyphodium fungal endophytes confer physiological protection to perennial ryegrass (Lolium perenne L.) subjected to a water deficit. Environ Exper Bot. 2008, 63: 183-199. 10.1016/j.envexpbot.2007.10.021.View ArticleGoogle Scholar
- White RH, Engelke MC, Morton SJ, Johnsoncicalese JM, Ruemmele BA: Acremonium endophyte effects on tall fescue drought tolerance. Crop Sci. 1992, 32: 1392-1396. 10.2135/cropsci1992.0011183X003200060017x.View ArticleGoogle Scholar
- Carrow RN: Drought avoidance characteristics of diverse tall fescue cultivars. Crop Sci. 1996, 36: 371-377. 10.2135/cropsci1996.0011183X003600020026x.View ArticleGoogle Scholar
- Bayat F, Mirlohi A, Khodambashi M: Effects of endophytic fungi on some drought tolerance mechanisms of tall fescue in a hydroponics culture. Russ J Plant Physiol. 2009, 56: 510-516. 10.1134/S1021443709040104.View ArticleGoogle Scholar
- Hill NS, Pachon JG, Bacon CW: Acremonium coenophialum-mediated short- and long-term drought acclimation in tall fescue. Crop Sci. 1996, 36: 665-672. 10.2135/cropsci1996.0011183X003600030025x.View ArticleGoogle Scholar
- Richardson MD, Hoveland CS, Bacon CW: Photosynthesis and stomatal conductance of symbiotic and nonsymbiotic tall fescue. Crop Sci. 1993, 33: 145-149. 10.2135/cropsci1993.0011183X003300010026x.View ArticleGoogle Scholar
- Malinowski DP, Alloush GA, Belesky DP: Evidence for chemical changes on the root surface of tall fescue in response to infection with the fungal endophyte Neotyphodium coenophialum. Plant Soil. 1998, 205: 1-12. 10.1023/A:1004331932018.View ArticleGoogle Scholar
- Swarthout D, Harper E, Judd S, Gonthier D, Shyne R, Stowe T, Bultman T: Measures of leaf-level water-use efficiency in drought stressed endophyte infected and non-infected tall fescue grasses. Environ Exper Bot. 2009, 66: 88-93. 10.1016/j.envexpbot.2008.12.002.View ArticleGoogle Scholar
- Joost RE, Holder TL: Effect of endophyte infection on ABA content and drought response of tall fescue. Agronomy Abstracts. Madison, WI: American Society of Agronomy: 1994, 140-Google Scholar
- Buck GW, West CP, Elbersen HW: Endophyte effect on drought tolerance in diverse Festuca species. Neotyphodium–Grass Interactions. Edited by: Bacon CW, Hill NS. New York, NY: Plenum Press: 1997,141-143.View ArticleGoogle Scholar
- Elmi AA, West CP: Endophyte Infection effects on stomatal conductance, osmotic adjustment and drought recovery of tall fescue. New Phytol. 1995, 131: 61-67. 10.1111/j.1469-8137.1995.tb03055.x.View ArticleGoogle Scholar
- Chen H, Jiang JG: Osmotic adjustment and plant adaptation to environmental changes related to drought and salinity. Environ Rev. 2010, 18: 309-319. 10.1139/A10-014.View ArticleGoogle Scholar
- Spollen WG, Nelson CJ: Response of fructan to water deficit in growing leaves of tall fescue. Plant Physiol. 1994, 106: 329-336.PubMedPubMed CentralGoogle Scholar
- Hanson J, Smeekens S: Sugar perception and signaling - an update. Curr Opin Plant Biol. 2009, 12: 562-567. 10.1016/j.pbi.2009.07.014.PubMedView ArticleGoogle Scholar
- Loescher WH: Physiology and metabolism of sugar alcohols in higher-plants. Physiol Plant. 1987, 70: 553-557. 10.1111/j.1399-3054.1987.tb02857.x.View ArticleGoogle Scholar
- Abernethy GA, McManus MT: Biochemical responses to an imposed water deficit in mature leaf tissue of Festuca arundinacea. Environ Exper Bot. 1998, 40: 17-28. 10.1016/S0098-8472(98)00017-3.View ArticleGoogle Scholar
- Bandurska H, Jóźwiak W: A comparison of the effects of drought on proline accumulation and peroxidases activity in leaves of Festuca rubra L. and Lolium perenne L. Acta Soc Bot Pol. 2010, 79: 111-116. 10.5586/asbp.2010.015.View ArticleGoogle Scholar
- Richardson MD, Chapman GW, Hoveland CS, Bacon CW: Sugar alcohols in endophyte-infected tall fescue under drought. Crop Sci. 1992, 32: 1060-1061. 10.2135/cropsci1992.0011183X003200040045x.View ArticleGoogle Scholar
- Assuero SG, Tognetti JA, Colabelli MR, Agnusdei MG, Petroni EC, Posse MA: Endophyte infection accelerates morpho-physiological responses to water deficit in tall fescue. N Z J Agri Res. 2006, 49: 359-370. 10.1080/00288233.2006.9513726.View ArticleGoogle Scholar
- Man D, Bao YX, Han LB, Zhang XZ: Drought tolerance associated with proline and hormone metabolism in two tall fescue cultivars. HortScience. 2011, 46: 1027-1032.Google Scholar
- de Battista J, Bouto J, Bacon C, Siegel M: Rhizome and herbage production of endophyte-removed tall fescue clones and populations. Agron J. 1990, 82: 651-654. 10.2134/agronj1990.00021962008200040001x.View ArticleGoogle Scholar
- Bacon C, White J: Stains, media and procedures for analyzing endophytes. Biotechnology of Endophytic Fungi of Grasses. Edited by: Bacon CW, White J. 1994, Boca Raton, FL: CRC Press, 47-56.Google Scholar
- An ZQ, Siegel MR, Hollin W, Tsai HF, Schmidt D, Schardl CL: Relationships among non-Acremonium sp. fungal endophytes in five grass species. Appl Environ Microbiol. 1993, 59: 1540-1548.PubMedPubMed CentralGoogle Scholar
- Takach JE, Mittal S, Swoboda GA, Bright SK, Trammell MA, Hopkins AA, Young CA: Genotypic and chemotypic diversity of Neotyphodium endophytes in tall fescue from Greece. Appl Environ Microbiol. 2012, 78: 5501-5510. 10.1128/AEM.01084-12.PubMedPubMed CentralView ArticleGoogle Scholar
- Yates SG, Petroski RJ, Powell RG: Analysis of loline alkaloids in endophyte-infected tall fescue by capillary gas-chromatography. J Agri Food Chem. 1990, 38: 182-185. 10.1021/jf00091a040.View ArticleGoogle Scholar
- Dutt JE: Computing probability integral of a general multivariate-T. Biometrika. 1975, 62: 201-205.View ArticleGoogle Scholar
- Scheffé H: Analysis of variance. New York: John Wiley & Son, Inc: 1959.Google Scholar
- Burg MB, Ferraris JD: Intracellular organic osmolytes: function and regulation. J Biol Chem. 2008, 283: 7309-7313. 10.1074/jbc.R700042200.PubMedPubMed CentralView ArticleGoogle Scholar
- Bohnert H, Shen B: Transformation and compatible solutes. Sci Hortic (Amsterdam). 1998, 78: 237-240. 10.1016/S0304-4238(98)00195-2.View ArticleGoogle Scholar
- Rodriguez R, Redman R: Balancing the generation and elimination of reactive oxygen species. Proc Natl Acad Sci U S A. 2005, 102: 3175-3176. 10.1073/pnas.0500367102.PubMedPubMed CentralView ArticleGoogle Scholar
- Ahmad P, Sarwat M, Sharma S: Reactive oxygen species, antioxidants and signaling in plants. J Plant Biol. 2008, 51: 167-173. 10.1007/BF03030694.View ArticleGoogle Scholar
- Hoekstra FA, Buitink J: Mechanisms of plant desiccation tolerance. Trends Plant Sci. 2001, 8: 431-438.View ArticleGoogle Scholar
- Morgan J: Osmoregulation and water stress in higher plants. Annu Rev Plant Physiol. 1984, 35: 299-319. 10.1146/annurev.pp.35.060184.001503.View ArticleGoogle Scholar
- Alpert P, Oliver MJ: Drying without dying. Desiccation and Survival in Plants. Edited by: Black M, Prichard HW. Wallingford, UK: CAB International 2002,3-43.View ArticleGoogle Scholar
- Buitink J, Laessens MMAE, Hernmings MA, Hoekstra FA: Influence of water content and temperature on molecular mobility and intracellular glasses in seeds and pollen. Plant Physiol. 1998, 118: 531-541. 10.1104/pp.118.2.531.PubMedPubMed CentralView ArticleGoogle Scholar
- Rolland F, Moore B, Sheen J: Sugar sensing and signaling in plants. Plant Cell. 2002, 14: 185-205.Google Scholar
- Couee I, Sulmon C, Gouesbet G, El Amrani A: Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J Exp Bot. 2006, 57: 449-459. 10.1093/jxb/erj027.PubMedView ArticleGoogle Scholar
- Deryabin A, Sinkevich M, Dubinina I, Burakhanov E, Trunova T: Effect of sugars on the development of oxidative stress induced by hypothermia in potato plants expressing yeast invertase gene. Russ J Plant Physiol. 2007, 54: 32-38. 10.1134/S1021443707010050.View ArticleGoogle Scholar
- Parrent JL, James TY, Vasaitis R, Taylor AF: Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses. BMC Evol Biol. 2009, 9: 148-10.1186/1471-2148-9-148.PubMedPubMed CentralView ArticleGoogle Scholar
- Lam CK, Belanger FC, White JF, Daie J: Mechanism and rate of sugar uptake by Acremonium typhinum, an endophytic fungus infecting Festuca rubra - evidence for presence of a cell wall invertase in endophytic fungi. Mycologia. 1994, 86: 408-415. 10.2307/3760573.View ArticleGoogle Scholar
- Ambrose KV, Belanger FC: SOLiD-SAGE of endophyte-infected red fescue reveals numerous effects on host transcriptome and an abundance of highly expressed fungal secreted proteins. PLoS One. 2012, 7: e53214-10.1371/journal.pone.0053214.PubMedPubMed CentralView ArticleGoogle Scholar
- Solomon PS, Waters OD, Oliver RP: Decoding the mannitol enigma in filamentous fungi. Trends Microbiol. 2007, 15: 257-262. 10.1016/j.tim.2007.04.002.PubMedView ArticleGoogle Scholar
- Keller F, Matile P: Storage of sugars and mannitol in petioles of celery leaves. New Phytol. 1989, 113: 291-299. 10.1111/j.1469-8137.1989.tb02406.x.View ArticleGoogle Scholar
- Tarczynski MC, Jensen RG, Bohnert HJ: Stress protection of transgenic tobacco by production of the osmolyte mannitol. Science. 1993, 259: 508-510. 10.1126/science.259.5094.508.PubMedView ArticleGoogle Scholar
- Hu L, Lu H, Liu QL, Chen XM, Jiang XN: Overexpression of mtlD gene in transgenic Populus tomentosa improves salt tolerance through accumulation of mannitol. Tree Physiol. 2005, 25: 1273-1281. 10.1093/treephys/25.10.1273.PubMedView ArticleGoogle Scholar
- Chan ZL, Grumet R, Loescher W: Global gene expression analysis of transgenic, mannitol-producing, and salt-tolerant Arabidopsis thaliana indicates widespread changes in abiotic and biotic stress-related genes. J Exp Bot. 2011, 62: 4787-4803. 10.1093/jxb/err130.PubMedPubMed CentralView ArticleGoogle Scholar
- Sickler CM, Edwards GE, Kiirats O, Gao ZF, Loescher W: Response of mannitol-producing Arabidopsis thaliana to abiotic stress. Funct Plant Biol. 2007, 34: 382-391. 10.1071/FP06274.View ArticleGoogle Scholar
- Pilon-Smits EAH, Terry N, Sears T, Kim H, Zayed A, Hwang S, van Dun K, Voogd E, Verwoerd TC, Krutwagen RWHH, Goddijn OJM: Trehalose-producing transgenic tobacco plants show improved growth performance under drought stress. J Plant Physiol. 1998, 152: 525-532. 10.1016/S0176-1617(98)80273-3.View ArticleGoogle Scholar
- Karim S, Aronsson H, Ericson H, Pirhonen M, Leyman B, Welin B, Mäntylä E, Palva ET, Dijck P, Holmström K-O: Improved drought tolerance without undesired side effects in transgenic plants producing trehalose. Plant Mol Biol. 2007, 64: 371-386. 10.1007/s11103-007-9159-6.PubMedView ArticleGoogle Scholar
- Garg AK, Kim J-K, Owens TG, Ranwala AP, Choi YD, Kochian LV, Wu RJ: Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses. Proc Natl Acad Sci USA. 2002, 99: 15898-15903. 10.1073/pnas.252637799.PubMedPubMed CentralView ArticleGoogle Scholar
- Redillas MFR, Park S-H, Lee J, Kim Y, Jeong J, Jung H, Bang S, Hahn T-R, Kim J-K: Accumulation of trehalose increases soluble sugar contents in rice plants conferring tolerance to drought and salt stress. Plant Biotechnol Rep. 2012, 6: 89-96. 10.1007/s11816-011-0210-3.View ArticleGoogle Scholar
- Romero C, Belles JM, Vaya JL, Serrano R, CulianezMacia FA: Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco plants: Pleiotropic phenotypes include drought tolerance. Planta. 1997, 201: 293-297. 10.1007/s004250050069.PubMedView ArticleGoogle Scholar
- Yeo ET, Kwon HB, Han SE, Lee JT, Ryu JC, Byun MO: Genetic engineering of drought resistant potato plants by introduction of the trehalose-6-phosphate synthase (TPS1) gene from Saccharomyces cerevisiae. Mol Cells. 2000, 10: 263-268.PubMedGoogle Scholar
- Cortina C, Culianez-Macia FA: Tomato abiotic stress enhanced tolerance by trehalose biosynthesis. Plant Sci. 2005, 169: 75-82. 10.1016/j.plantsci.2005.02.026.View ArticleGoogle Scholar
- Fernandez O, Bethencourt L, Quero A, Sangwan RS, Clement C: Trehalose and plant stress responses: friend or foe?. Trends Plant Sci. 2010, 15: 409-417. 10.1016/j.tplants.2010.04.004.PubMedView ArticleGoogle Scholar
- Goddijn OJM, Verwoerd TC, Voogd E, Krutwagen PWHH, de Graaf PTHM, Poels J, van Dun K, Ponstein AS, Damm B, Pen J: Inhibition of trehalase activity enhances trehalose accumulation in transgenic plants. Plant Physiol. 1997, 113: 181-190. 10.1104/pp.113.1.181.PubMedPubMed CentralView ArticleGoogle Scholar
- Eastmond PJ, van Dijken AJH, Spielman M, Kerr A, Tissier AF, Dickinson HG, Jones JDG, Smeekens SC, Graham IA: Trehalose-6-phosphate synthase 1, which catalyses the first step in trehalose synthesis, is essential for Arabidopsis embryo maturation. Plant J. 2002, 29: 225-235. 10.1046/j.1365-313x.2002.01220.x.PubMedView ArticleGoogle Scholar
- Satoh-Nagasawa N, Nagasawa N, Malcomber S, Sakai H, Jackson D: A trehalose metabolic enzyme controls inflorescence architecture in maize. Nature. 2006, 441: 227-230. 10.1038/nature04725.PubMedView ArticleGoogle Scholar
- Zhang Y, Primavesi LF, Jhurreea D, Andralojc PJ, Mitchell RA, Powers SJ, Schluepmann H, Delatte T, Wingler A, Paul MJ: Inhibition of SNF1-related protein kinase1 activity and regulation of metabolic pathways by trehalose-6-phosphate. Plant Physiol. 2009, 149: 1860-1871. 10.1104/pp.108.133934.PubMedPubMed CentralView ArticleGoogle Scholar
- Jiang Y, Chen XM, Liu YJ, Li YT, Zhang HH, Dyson P, Sheng HM, An LZ: The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures. Arch Microbiol. 2010, 192: 937-943. 10.1007/s00203-010-0625-1.PubMedView ArticleGoogle Scholar
- Paul MJ, Jhurreea D, Zhang Y, Primavesi LF, Delatte T, Schluepmann H, Wingler A: Upregulation of biosynthetic processes associated with growth by trehalose 6-phosphate. Plant Signal Behav. 2010, 5: 386-392. 10.4161/psb.5.4.10792.PubMedPubMed CentralView ArticleGoogle Scholar
- Delatte TL, Sedijani P, Kondou Y, Matsui M, de Jong GJ, Somsen GW, Wiese-Klinkenberg A, Primavesi LF, Paul MJ, Schluepmann H: Growth arrest by trehalose-6-phosphate: An astonishing case of primary metabolite control over growth by way of the SnRK1 signaling pathway. Plant Physiol. 2011, 157: 160-174. 10.1104/pp.111.180422.PubMedPubMed CentralView ArticleGoogle Scholar
- Carvalho M: Drought stress and reactive oxygen species. Plant Signal Behav. 2008, 3: 156-165. 10.4161/psb.3.3.5536.View ArticleGoogle Scholar
- Gill S, Tuteja N: Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem. 2010, 48: 909-930. 10.1016/j.plaphy.2010.08.016.PubMedView ArticleGoogle Scholar
- White JF, Torres MA: Is plant-endophyte defensive mutualism is the result of oxidative stress protection, Review. Physiol Plant. 2010, 138: 440-446. 10.1111/j.1399-3054.2009.01332.x.PubMedView ArticleGoogle Scholar
- Yoshiba Y, Kiyosue T, Nakashima K, YamaguchiShinozaki K, Shinozaki K: Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol. 1997, 38: 1095-1102. 10.1093/oxfordjournals.pcp.a029093.PubMedView ArticleGoogle Scholar
- Verbruggen N, Hermans C: Proline accumulation in plants: a review. Amino Acids. 2008, 35: 753-759. 10.1007/s00726-008-0061-6.PubMedView ArticleGoogle Scholar
- Dickman MB, Chen CB: Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii. Proc Natl Acad Sci U S A. 2005, 102: 3459-3464. 10.1073/pnas.0407960102.PubMedPubMed CentralView ArticleGoogle Scholar
- Blankenship JD, Spiering MJ, Wilkinson HH, Fannin FF, Bush LP, Schardl CL: Production of loline alkaloids by the grass endophyte, Neotyphodium uncinatum, in defined media. Phytochemistry. 2001, 58: 395-401. 10.1016/S0031-9422(01)00272-2.PubMedView ArticleGoogle Scholar
- Schardl CL, Grossman RB, Nagabhyru P, Faulkner JR, Mallik UP: Loline alkaloids: Currencies of mutualism. Phytochemistry. 2007, 68: 980-996. 10.1016/j.phytochem.2007.01.010.PubMedView ArticleGoogle Scholar
- Blankenship JD, Houseknecht JB, Pal S, Bush LP, Grossman RB, Schardl CL: Biosynthetic precursors of fungal pyrrolizidines, the loline alkaloids. Chembiochem. 2005, 6: 1016-1022. 10.1002/cbic.200400327.PubMedView ArticleGoogle Scholar
- Zhang DX, Nagabhyru P, Schardl CL: Regulation of a chemical defense against herbivory produced by symbiotic fungi in grass plants. Plant Physiol. 2009, 150: 1072-1082. 10.1104/pp.109.138222.PubMedPubMed CentralView ArticleGoogle Scholar
- Belesky DP, Stringer WC, Hill NS: Influence of endophyte and water regime upon tall fescue accessions. 1. growth-characteristics. Ann Bot. 1989, 63: 495-503.Google Scholar
- Fernandez O, Theocharis A, Bordiec S, Feil R, Jacquens L, Clement C, Fontaine F, Barka EA: Burkholderia phytofirmans PsJN acclimates grapevine to cold by modulating carbohydrate metabolism. Mol Plant Microbe Interact. 2012, 25: 496-504. 10.1094/MPMI-09-11-0245.PubMedView ArticleGoogle Scholar
- Theocharis A, Bordiec S, Fernandez O, Paquis S, Dhondt-Cordelier S, Baillieul F, Clement C, Barka EA: Burkholderia phytofirmans PsJN primes Vitis vinifera L. and confers a better tolerance to low nonfreezing temperatures. Mol Plant Microbe Interact. 2012, 25: 241-249. 10.1094/MPMI-05-11-0124.PubMedView ArticleGoogle Scholar
- Rasmussen S, Parsons AJ, Fraser K, Xue H, Newman JA: Metabolic profiles of Lolium perenne are differentially affected by nitrogen supply, carbohydrate content, and fungal endophyte infection. Plant Physiol. 2008, 146: 1440-1453. 10.1104/pp.107.111898.PubMedPubMed CentralView ArticleGoogle Scholar
- Rasmussen S, Parsons AJ, Jones CS: Metabolomics of forage plants: a review. Ann Bot. 2012, 110: 1281-1290. 10.1093/aob/mcs023.PubMedPubMed CentralView ArticleGoogle Scholar
- Chaves M, Maroco J, Pereira J: Understanding plant responses to drought – from genes to the whole plant. Funct Plant Biol. 2003, 30: 239-264. 10.1071/FP02076.View ArticleGoogle Scholar
- Chaves MM, Flexas J, Pinheiro C: Photosynthesis under drought and salt stress: regulation mechanisms from whole plant to cell. Ann Bot. 2009, 103: 551-560.PubMedPubMed CentralView ArticleGoogle Scholar
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