Tall fescue endophyte effects on tolerance to water-deficit stress

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1 Ngbhyru et l. BMC Plnt Biology 13, 13:17 RESEARCH ARTICLE Tll fescue endophyte effects on tolernce to wter-deficit stress Open Access Pdmj Ngbhyru 1, Rndy D Dinkins, Constnce L Wood 3, Chrles W Bcon nd Christopher L Schrdl 1* Abstrct Bckground: The endophytic fungus, Neotyphodium coenophilum, cn enhnce drought tolernce of its host grss, tll fescue. To investigte endophyte effects on plnt responses to cute wter deficit stress, we did comprehensive profiling of plnt metbolite levels in both shoot nd root tissues of geneticlly identicl clone pirs of tll fescue with endophyte (E+) nd without endophyte (E-) in response to direct wter deficit stress. The E- clones were generted by treting E+ plnts with fungicide nd selectively propgting single tillers. In time course studies on the E+ nd E- clones, wter ws withheld from to 5 dys, during which levels of free sugrs, sugr lcohols, nd mino cids were determined, s were levels of some mjor fungl metbolites. Results: After 3 dys of withholding wter, survivl nd tillering of re-wtered plnts ws significntly greter for E+ thn E- clones. Within two to three dys of withholding wter, significnt endophyte effects on metbolites mnifested s higher levels of free glucose, fructose, trehlose, sugr lcohols, proline nd glutmic cid in shoots nd roots. The fungl metbolites, mnnitol nd loline lkloids, lso significntly incresed with wter deficit. Conclusions: Our results suggest tht symbiotic N. coenophilum ids in survivl nd recovery of tll fescue plnts from wter deficit, nd cts in prt by inducing rpid ccumultion of these comptible solutes soon fter imposition of stress. Keywords: Fungl endophyte, Tll fescue, Wter deficit stress, Metbolites, Neutrl sugrs, Amino cids nd lolines Bckground Tll fescue (Lolium rundinceum = Schedonorus rundinceus = Festuc rundince) is the most widely plnted forge grss in the United Sttes [1] nd it is often infected with the endophytic fungus, Neotyphodium coenophilum. The reltionship between the endophyte nd plnt is generlly considered mutulistic becuse the endophyte significntly improves host plnt tolernce to drought, insects, diseses, nd nemtodes, long with incresed persistence nd vigor; nd in turn the plnt provides the symbiont with nutrients, protection, nd relible nd efficient dissemintion (reviewed in []). Evidence suggests tht tll fescue plnts with the endophyte (E+) grow nd persist longer under stressful conditions, such s wter deficit, compred to endophyte free plnts (E-), nd re, therefore, likely to hve n dptive nd competitive dvntge [3-9]. Mechnisms for endophyte-enhnced drought voidnce or * Correspondence: Schrdl@uky.edu 1 Deprtment of Plnt Pthology, University of Kentucky, Lexington, KY 56-31, USA Full list of uthor informtion is vilble t the end of the rticle tolernce pper complex, nd might involve direct nd indirect effects of the endophyte on metbolism nd other physiologicl chnges in the host plnt [13]. Processes ffected by the tll fescue endophyte include stomtl closure [1], decresed root dimeter nd incresed root hir length [7,15], incresed turgid weight/ dry weight rtios suggesting reduced dmged to cell wlls [1], nd enhnced production of phenolic root exudtes [15]. Lef rolling under drought stress is reported to be much more common in E+ thn E- plnts [3]. Greter cell wll elsticity [1] nd higher wter use efficiency [16] in E+ tll fescue compred to E- plnts under drought stress hve lso been reported. Previous reserch hs lso shown tht E+ tll fescue plnts of some genotypes exhibit lower stomtl conductnce thn E- plnts with more sensitive inducement of stomtl closure in E+ plnts in response to erly stges of wter deficit [179]. Endophyte infection confers popultion stbility in tll fescue during drought stress through improved tiller nd whole plnt survivl [5]. 13 Ngbhyru et l.; licensee BioMed Centrl Ltd. This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided the originl work is properly cited.

2 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge of 17 A correltion between drought tolernce nd ccumultion of comptible solutes such s crbohydrtes, mino cids, nd minerl ions tht contribute to osmotic djustment hs been documented in grsses [-]. In generl, ccumultion of sugrs, sugr lcohols [3], nd proline [,5] in response to wter deficit in grsses hs been reported. A significnt endophyte effect on ccumultion of simple sugrs in leves of E+ tll fescue, ws observed when plnts were osmoticlly stressed by polyethylene glycol [6]. Under wter deficit, E+ tll fescue plnts re reported to exhibit decresed growth nd incresed root nd lef senescence, s well s greter ccumultion of sugrs within the pseudostem, nd decresed wter potentil compred to E- plnts [7]. Effects of the endophyte on levels of other metbolites, such s proline [8] nd other mino cids hve not been well studied. Here we report wht is, to our knowledge, the first comprehensive profiling of shoot nd root metbolite responses to cute wter deficit stress, ssessing the timing of endophyte effects on sugrs, sugr lcohols nd mino cids reltive to the endophyte effects on subsequent plnt recovery. Methods Experimentl design Tll fescue is n obligtely outcrossing grss, so tht isogenic lines cnnot be generted, nd plnts derived from different seeds re necessrily unique genotypes. Therefore, to control for host genotype effects we developed geneticlly identicl clones with endophyte (E+) nd without endophyte (E-) s follows. Rmets of tll fescue Kentucky 31 plnts nturlly infected with Neotyphodium coenophilum were treted with the fungicide propiconzole or tebuconzole to remove the fungus [9,3]. The stock plnts nd fungicide-treted clones were exmined for the presence or bsence of endophyte by tissue print immunoblot [31], PCR [3], nd microscopy. This resulted in E+/E- clone pirs, two of which were used in this study. Lb identifiction numbers 78 (E+) nd 79 (E-) represented one clone pir, nd 67 (E+) nd 68 (E-) represented the other clone pir. Plnts of ech clone pir were rised side-by-side in the greenhouse for more thn one yer prior to being used in the study. Rmets consisting of three tillers of similr size were plnted into cm squre pots in snd, in the greenhouse. Snd ws chosen s the growth medium becuse it llows even, uniform nd rpid drying, nd lso provides for esy hrvesting of roots. Plnts were wtered twice dily for six weeks before subjecting them to experimentl conditions to llow for regenertion nd ccumultion of sufficient biomss for smpling. After sufficient re-growth hd occurred, wter ws withheld from the test group, while control plnts were wtered twice dily. Pots were rndomized once while setting up the experiment nd gin before subjecting them to tretments, in order to control for effects microenvironmentl vrition. Tretments were endophyte-infected wtered controls (E+D-), endophyte-infected wter-deficit stressed (E+ D+), endophyte-free wtered controls (E-D-), nd endophytefree wter-deficit stressed (E-D+). Entire pots were smpled on ech dy from dy to dy 5 of withholding wter. Beyond dy 5 plnts were fully dried nd mostly ded. Three or four replictes were smpled for ech tretment x dy. For the first experiment, which ws conducted with the 78/79 clone pir, smples were hrvested from Februry 7, 7. For the second experiment with clone pir 78/79, smples were hrvested from June 7, 8. The third experiment ws conduced with clone pir 67/68, smpled from July 1 6, 8. All plnts were grown in the greenhouse under nturl light conditions, with 5-7% reltive humidity rnges, nd tempertures set to 7 C/ C (dy/night). Photoctive rdition (PAR) mesurements were recorded during the three experiments(seeadditionlfile1,pnels,b,c).smples were hrvested between 7:3.m. to 8:3.m. locl time ech dy, immeditely frozen in liquid nitrogen, lyophilized nd subsequently prepred for metbolite nlysis s described below. The smples were divided into shoot (lef long with tiller bse down to 1 cm from crown region) nd root mteril. Tiller recovery experiment Five to six pots subjected to wter-deficit conditions from ech E+/E- clone pir for ech dy of tretment were left unhrvested, nd were plced bck into dily wtering regime in order to determine their bility to recover from the wter-deficit stress. Live tiller numbers were counted fter 6 weeks of recovery. Crbohydrte nlysis by high ph nion exchnge chromtogrphy Sugrs were extrcted in 1 ml of 8% ethnol per 1 mg of ground lyophilized plnt mteril. The smples were incubted t 65 C for 1 hr nd 9 C for 5 min nd the superntnt ws evported in vcuum centrifuge. The residue ws reconstituted in purified wter t C nd filtered through spin-x HPLC. μm nylon filter micro centrifuge (Corning, NY) tubes. Filtered superntnt (1 μl) ws diluted to 1 ml nd used for nlysis on Dionex ICS 3 with either crbopc PA1 column for neutrl sugrs or crbopc MA1 column for polyols. Neutrl sugrs were seprted by n isocrtic progrm with mm NOH, nd sugr lcohols were seprted using 8 mm NOH. The detection ws by pulsed mperometry, using gold working electrode. Pek identity nd sugr quntity

3 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 3 of 17 were determined by comprison with stndrds. The internl stndrd ws -deoxyglucose. Amino cid nlysis by liquid chromtogrphy-mss spectrometry (LC-MS) The yields of free mino cids from plnt smples were compred for different extrction methods using ) 8% ethnol, or b) chloroform: methnol: wter (5:1:3), nd incubting t different tempertures ( C nd 5 C) for 1 hr. However both extrction solvents nd methods resulted in similr extrction efficiency, so the simpler extrction method ws chosen for further nlysis. Finely ground lyophilized plnt shoot nd root mteril (5 mg) ws extrcted with 5 ml of 8% ethnol on ice for 1 hr. The crude extrct ws filtered through. μm centrifuge tubes nd the superntnt ws used for smple clenup nd derivtiztion with EZ fst LCMS kit for free mino cids from Phenomenex, ccording to the kit protocol. Briefly, 1 μl of ech smple ws mixed with 1 μl of internl stndrd contining homorginine, d3- methionine, nd homophenyllnine provided in the kit. Then smple ws loded onto pipet tip pcked with ion exchnge resin on which free mino cids were bound, subsequently wshed nd relesed from resin. The free mino cids were then derivtized by propyl chloroformte nd liquid-liquid extrcted with isooctne. The orgnic phse contining the derivtized mino cids ws removed under strem of high purity nitrogen gs nd the residue ws redissolved in μl :1mobilephseofA:B(A:1mMmmoniumformte in wter nd B: 1 mm mmonium formte in methnol). Anlysis ws performed by liquid chromtogrphy mss spectrometry with dul pump ProStr 1 HPLC with 1 L qudrupole MS-MS (Vrin). Loline lkloid nlysis Loline lkloids were extrcted from smples using chloroform under lkline conditions [33]. Quinoline ws used s n internl stndrd nd the lolines were quntified by gs chromtogrphy (Vrin CP-38) interfced with Vrin Sturn ion trp mss spectrometer. Loline mounts were clculted s the totl of loline, N-methylloline, N-formylloline, N-cetylloline nd N- cetylnorloline. Sttisticl nlysis Fctoril Anlysis of Vrince ( 6) ws run to nlyze tiller recovery nd metbolite levels in PROC GLM, SAS (SAS Institute Inc., Cry, NC, USA.). Following ANOVA, tiller recovery nd metbolite levels of E+ nd E- clones were compred on ech dy using Estimte Sttements. In order to control the overll α-level for multiple tests, the distribution of the mximum of the bsolute vlue of elements of multivrite (six vrite) t-distribution with μ = nd = I [3], i.e. t-mx, ws used to clculte the significnce levels for ech of the six t-tests. Becuse of the extremely conservtive nture of this procedure, α =.1 is used to determine significnce of differences [p. 71 in ref. [35]. The three fctor ANOVAs of ll metbolites in ll three experiments re given in Tbles 1,, nd 3. For the tiller numbers fter recovery, four biologicl replictions were run for ech tretment in the first experiment on clone pir 78/79, nd five biologicl replictions were run for the other two experiments on clone pir 78/79 nd clone pir 67/68. For metbolites, four biologicl replictions were run for ech tretment in the first experiment, nd three biologicl replictions were run for the other two experiments. In the grphs the significnt differences of vrious metbolites between E+ nd E- plnts in the wter deficit stress tretments were represented bsed on t-mx vlues; * denotes p- vlues = >.1 -.1; ** denotes p-vlues >.1 -.1; *** denotes p- vlues <.1. Results Tiller number nd recovery Overll the E+ plnts survived the stress conditions imposedduringtheexperimentbetterthnthee-plnts when number of tillers produced upon recovery ws used s the mesure. In the first experiment with clone pir 78/79, fter dysofwithholdingwter,e+ plnts produced more tillers thn E- plnts during recovery. However, fter 5 dys withholding wter, none of the E+ or E- plnts recovered (Figure 1). In the second experiment with the sme clone pir, strting t 3 dys of withholding wter, tiller recovery ws significntly higher in E+ clones (Figure 1b). With clone pir 67/68 (Figure 1c), fter 3-dys of wter deficit there ws greter tillering of E+ plnts, which ws mrginlly significnt (p=.11 bsed on t mx, p=.19 bsed on t vlues). Neutrl sugrs The levels of glctose, glucose, fructose, sucrose, rffinose, stchyose, nd trehlose were quntified in the tll fescue clone pirs in response to wter deficit stress nd endophyte infection. Of these, glucose, fructose nd sucrose were the mjor free sugrs identified. In Experiment 1 with clone pir 78/79, E+ shoots ccumulted pproximtely -fold more free glucose nd free fructose t dy 1 compred to E- shoots (Figures nd 3). Similrly in roots, free glucose nd free fructose levels in E+ clones t dy 1 fter withholding wter were significntly higher thn in the E- clones (Figures b nd 3b). In contrst, t dy 1, E+ nd E- clones showed no difference in sucrose levels compred to wtered controls. Sucrose levels incresed in shoots nd roots of both E+ nd E- clones strting from dy fter withholding wter (Figure nd b, Tble 1). Compring combined

4 Tble 1 Three-fctor ANOVA [F df (5,7) ] vlues of ll metbolites in Experiment 1 Metbolite Endophyte Dy Stress Dy * Endophyte Stress * Endophyte Stress * Dy Endophyte * Stress * Dy Shoot glucose 8.89*** 5.96 *** 36.91*** 6.5*** *** 39.5*** Shoot fructose 6.77* *** 13.3***.6*** *** 8.75*** Shoot sucrose 13.18*** 13.97*** 113.3*** ***.58** Shoot GFS *** 6.1*** *** 9.85*** Shoot proline. 18.9*** 178.3*** 1.33***.5** 15.7*** 16.86*** Shoot glutmine *** 33.9*** 3.61** *** 3.** Shoot glutmic cid 6.55* 6.8*** 61.*** 3.56** 11.3*** 9.8*** 5.88*** Shoot sprgine.8 8.7*** 5.16*** ***.7* Shoot sprtic cid *** **.1 1.1*** 1.11*** Shoot tryptophn.9***.9*** ***.6*. 1.*** 9.5*** Shoot phenyllnine 9.9** 78.5*** 56.58*** 7.9*** 36.5*** 56.7*** 17.66*** Shoot tyrosine 3.61*** 19.69*** 67.1*** 9.91*** 5.33* 15.1*** 9.73*** Shoot lolines *** 3.11* 17.8*** 3.11* 17.8*** 9.97*** 9.97*** Root glucose *** *** 7.1*** 6.16*** 1.*** Root fructose. 7.7 ***.17*** 5.39***.93* 18.99*** 15.73*** Root sucrose *** 36.3*** ***.75*** Root GFS *** 5.81*** 5.***.1* 5.8*** 6.*** Root proline *** 9.87*** 7.7*** *** 6.19*** Root glutmine 5.51* 9.58*** 7.53*** 18.69*** *** 1.35 Root glutmic cid 7.13** 5.81*** 86.53*** 3.1*** 5.57*** 1.1*** Root sprgine.63*** 5.1*** 3.3*** 3.9*.75.87* 1.7 Root sprtic cid 5.55* 3.5*** 18.15*** 7.71*** 5.69* 1.*** 1.5*** Root tryptophn 6.35*.88*** * Root phenyllnine 18.***.9*** 8.1** 9.37*** 1.6** 7.83*** 3.* Root tyrosine 13.3*** 7.95***.3.7** 8.11**.6*.3 Root lolines 191.8*** 3.6** 35.1*** 3.6** 35.1*** * denotes p-vlues = >.1 -.5; ** denotes p-vlues = >.1 -.1; *** denotes p-vlues = <.1. Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge of 17

5 Tble Three-fctor ANOVA [F df (5,8) ] vlues of ll metbolites in Experiment Metbolite Endophyte Dy Stress Dy * Endophyte Stress * Endophyte Stress * Dy Endophyte * Stress * Dy Shoot glucose.3*** 11.3 *** 15.13*** * 3.8* Shoot fructose 39.*** 1.8 *** 1.3*** ** 3.35** Shoot sucrose 17.6*** 16.55*** **.68 Shoot GFS 37.8*** 15.85*** 8.37** * 1.67 Shoot proline 5.9*** 1.8*** 581.*** 7.6*** 5.5** 16.*** 7.31*** Shoot glutmine 11.3**.89 *** ***.39*.5 8.5*** 1.9 Shoot glutmic cid 7.19*** 3.68*** ***.3 Shoot sprgine 1.5** 1.8*** 79.38*** ***.38 Shoot sprtic cid *** 6.5* ***. Shoot threonine *** 6.7*** ***.95 Shoot tryptophn *** 5.37*** ***.75 Shoot phenyllnine *** 31.11*** ***.31 Shoot tyrosine 17.3*** 18.31***.79*** 3.35* ***.9 Shoot lolines 86.57*** *** *** 3.5* 3.5* Shoot mnnitol 89.37*** 19.68*** 18.1*** 13.5*** 16.7***.** 3.8** Shoot rbitol 15.7*** 6.5*** 6.9*** 3.6** 1.5*** 5.66*** 3.77*** Shoot sorbitol 1.36**.1** Shoot myo-inositol.98* 17.81*** 1.5*** ** 1. Shoot trehlose 13.11*** 1.5*** 5.65* ** 7.35***.8 Root glucose 8.6*** 5.1***.***.61* 5.1* 3.97**.33* Root fructose 37.16*** 3.3 *** 11.3***.** 9.11*** Root sucrose 5.67* 6.5 *** 8.68*** 5.83*** 5.99* 3.86**.36* Root GFS 11.9** 1.***.5 3.9**.57***.3.9* Root proline 6.51*** 8.7 *** 11.16*** 1.55*** 3.9*** 9.8*** 11.7*** Root glutmine *** 38.96*** *** 1.15 Root glutmic cid 19.9*** 9.***.1.3** Root sprgine 17.3*** 1.58*** Root sprtic cid *** 1.7.** *.19 Root threonine 19.91*** 93.19*** 7.36*** 13.8*** 19.69***.6* Root phenyllnine 78.7*** 9.3*** 177.*** 96.78*** 1.5*** 98.98*** 8.9*** Root lolines 9.61*** *** *** 3.1* 3.1* * denotes p-vlues = >.1 -.5; ** denotes p-vlues = >.1 -.1; *** denotes p-vlues = <.1. Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 5 of 17

6 Tble 3 Three-fctor ANOVA [F df (5,8) ] vlues of ll metbolites in Experiment 3 Metbolite Endophyte Dy Stress Dy * Endophyte Stress * Endophyte Stress * Dy Endophyte * Stress * Dy Shoot glucose *** *** ***.67* Shoot fructose 5.1*.51*** 3.35*** *** 1.56 Shoot sucrose.78* 6.9*** 97.38*** * 17.83***.98* Shoot GFS *** 3.3*** ** 35.35***.1 Shoot proline *** 39.*** ***.81 Shoot glutmine *** *** ***.6* Shoot glutmic cid *** ** ***.67* Shoot sprgine.8 8.3*** 3.99*** ** 1.13 Shoot sprtic cid *** *.5 Shoot tryptophn 5.1*.51*** 3.35*** ** 3.71*** 1.56 Shoot phenyl lnine.78* 6.9*** 97.38*** * 17.83***.98* Shoot tyrosine *** 3.3*** ** 35.35***.1 Shoot threonine *** *** ***.67* Shoot trehlose 3.8*** 1.87*** 69.51***.83* 11.6** 16.6*** 5.79*** Shoot lolines 57.88*** *** *** Root glucose 3.1* ** 1.87 Root fructose. 8. *** 13.*** ***.75* Root sucrose.3* 13.*** 3.1*** *** 1.7 Root GFS *** 7.15*** ***.51* Root proline 9.55*** 59.35*** *** 1.67 Root glutmine *.1*** Root glutmic cid *** 6.7* Root sprgine * Root sprtic cid *** 18.15*** **.3 Root tryptophn.5 5.6*** 56.8*** ***.6 Root phenyllnine *** 5.78*** ***.91 Root tyrosine *** **.77 Root threonine.7 7.3*** 8.63*** *** 1.37 Root lolines 55.13*** 9.6*** 19.8*** 9.6*** 19.8*** * denotes p-vlues = >.1 -.5; ** denotes p-vlues = >.1 -.1; *** denotes p-vlues = <.1. Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 6 of 17

7 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 7 of 17 Tiller number Tiller number Tiller number b c E+D+ E D+ E-D Figure 1 Tiller recovery from wter-deficit stressed E+ nd E- plnts fter plcing them bck into norml wtering regime. () First clone pir 78 (E+)/79 (E-), Experiment 1, error brs re SEM (n = ); (b) First clone pir 78 (E+)/79 (E-), Experiment, error brs re SEM (n = 5); (c) Second clone pir 67(E+)/68 (E-), error brs re SEM (n = 5). E+ D+ = endophyte infected nd wter withheld for the time periods indicted; E-D + = endophyte uninfected nd wter withheld; E+ D- = endophyte infected nd unstressed; E-D- = endophyte uninfected nd unstressed. Symbols indicting sttisticl significnce bsed on t-mx re *** p <.1; ** p >.1 -.1; * p >.1-.1; p =.11 bsed on t-mx; p =.19 bsed on t vlues). totls of glucose, fructose nd sucrose t dy 1, E+ clones hd pproximtely fold higher levels in shoots nd roots compred to their wtered controls, wheres the totls in E- clones did not differ significntly from their wtered controls (see Additionl file, pnels, b). Results in Experiment, lso with clone pir 78/79, were very similr except for one-dy dely in effects on tiller survivl nd metbolites, probbly becuse of overcst skies on the first dy (see Additionl file 1, pnel b). At dy of withholding wter, free glucose nd fructose levels in E+ were pproximtely fold higher thn in wtered controls or in E- stressed plnts (Figures c nd 3c). There were no significnt differences in sucrose levels t dy between E+ nd E- plnts (Figure c, Tble ). In roots, sucrose levels were 3 fold higher in E+ compred to E- roots from dy to dy (Figure d), though there were no significnt differences in glucose or fructose (Figures d nd 3d, Tble, nd see Additionl file, pnels c, d). Compring free glucose nd fructose sugrs in clone pir 67/68 during the wter deficit period, there were significnt differences between E+ nd E- in the roots (Figures f nd 3f); but not in shoots except for fructose t dy 5, where E+ shoots ccumulted fructose to higher levels thn E- shoots (Figures e nd 3e). Root glucose nd fructose concentrtions incresed by dy of withholding wter, nd were significntly higher in E+ thn E- plnts. The level of the discchride, trehlose, ws low in the tll fescue clone pirs. However the trehlose levels were higher in wter deficit tissues compred to the wtered control smples (Tbles nd 3), nd fter 3 dys of withholding wter significnt higher levels of trehlose were observed in the E+ clones compred to the E- clones (Figure 5 nd b). Sugr lcohols/polyols Levels of different sugr lcohols, including myo-inositol, mnnitol, sorbitol, rbitol, glctinol, nd chiro-inositol, were quntified in clone pir 78/79. Significnt increses in myo-inositol were observed (dys nd 3) in response to wter deficit, but there ws no significnt effect of endophyte (Figure 6, Tble ). Mnnitol, fungl metbolite, ws undetectble in E- plnts t most time points, but incresed significntly in E+ plnts t dy 3 fter withholding wter, compred to E+ wter controls (Figure 6b, Tble ). Sorbitol ws found in both E+ nd E- plnts, nd wter deficit nd endophyte did not influence these levels significntly (Figure 6c, Tble ). Arbitol ws not found in either E+ or E- wtered controls, but upon wter deficit stress, rbitol ccumulted with mximum t dy 3 in E+ plnts (Figure 6d, Tble ). Chiro-inositol levels were very low, nd glctinol levels were not significntly ffected by the endophyte or wter deficit sttus (dt not shown). Amino cids A totl of 11 free mino cids were mesured in wtered controls nd stressed shoot nd root tissues of both clone pirs. The mino cids, methionine, rginine, ornithine nd homoserine, were very low or undetectble. Levels of the mino cids vline, tryptophn, tyrosine, threonine, nd phenyllnine were higher in stressed

8 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 8 of b E+D E D+ 1 c E-D d e 3 f Figure Comprison of glucose levels in wter-deficit stressed nd unstressed shoots nd roots of tll fescue. ( nd b) Shoots nd roots, respectively, of the 78/79 clone pir, Experiment 1, error brs re SEM (n = ); (c nd d) shoots nd roots, respectively, of the 78/79 clone pir, Experiment, error brs re SEM (n = 3); (e nd f) shoots nd roots, respectively, of the 67/68 clone pir, error brs re SEM (n = 3). E+ D+ = endophyte infected nd wter withheld for the time periods indicted; E-D + = endophyte uninfected nd wter withheld; E+ D- = endophyte infected nd unstressed; E-D- = endophyte uninfected nd unstressed. Sttisticl significnce is indicted s in Figure 1. plnts from dy to dy 5 compred to wtered controls, but no consistent endophyte effects on these mino-cid levels were observed (Tbles 1, nd 3). Serine levels did not chnge due to endophyte or wter deficit stress. In the first experiment with the 78/79 clone pir, proline levels in shoots nd roots of E+ nd E- clones incresed under wter deficit stress, but not in wtered controls (Figure 7 nd b, Tble 1). At dy 1 of withholding wter, levels of proline incresed pproximtely 6-fold in E+ shoots nd roots, wheres comprble increses in E- plnts were not observed till dy. Thus, levels of proline t dy 1 were significntly greter in E+ thn in E- plnts (Figure 7 nd b). On dy of wter deficit, nd therefter, there were no significnt differences in the levels of proline between E+ nd E- plnts until dy. However, levels in the treted clones remined pproximtely 135-fold higher thn in wtered controls. In this experiment, the elevted levels of proline were ccompnied by slight decrese in glutmine levels (dt not shown), but the totl levels of proline, glutmine, nd glutmic cid, which re metboliclly interrelted, were higher in the stressed tissues. There were no significnt differences in sprgine levels between E+ nd E- plnts upon wter deficit (Tble 1). In the second experiment with clone pir 78/79, increses in mino cid levels strted from dy 3 fter withholding wter. At tht time point, proline levels in E+ clones were significntly higher thn in E- clones both in shoots (Figure 7c) nd roots (Figure 7d). In the experiment with clone pir 67/68, proline levels incresed in shoots of both E+ nd E- plnts by dy of withholding wter, but endophyte effect ws not significnt (Figure 7e, Tble 3). However, levels

9 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 9 of b E+D E D+ 1 c E-D 8 d e Figure 3 Fructose levels in wter-deficit stressed nd unstressed shoots (, c, nd e) nd roots (b, d, nd f) of tll fescue. ( nd b) Shoots nd roots, respectively, of 78/79 clone pir, Experiment 1; (c nd d) shoots nd roots, respectively, of 78/79 clone pir, Experiment ; (e nd f) shoots nd roots, respectively, of 67/68 clone pir. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM of biologicl replictes s indicted in Figure. 8 6 f of glutmine nd glutmic cid, which re metboliclly linked to proline, were higher t dy fter wter deficit (Figure 8 nd c); nd glutmic cid ws significntly higher in E+ compred to E- shoots (Figure 8c). Similrly, in stressed roots, proline levels did not significntly differ between E+ nd E- (Figure 7f, Tble 3), but t dy glutmine reched pproximtely 3-fold higher levels in E+ roots compred to E- stressed roots nd to wtered controls (Figure 8b). Asprgine levels incresed in shoots by dy of withholding wter, but were not significntly different between E+ nd E- shoots (Figure 8e, Tble 3). However, in roots, sprgine levels were significntly higher t dy in E+ compred to E- clones (Figure 8f). Overll, in this genotype, the endophyte effects on metbolites were evident especilly in roots within two dys of withholding wter. Loline lkloids Lolines re the most bundnt lkloids produced by N. coenophilum in tll fescue, where the mjor forms re N- formylloline nd N-cetylloline, lthough N-methylloline nd N-cetylnorloline re lso detected. In Experiment 1 with clone pir 78/79, totl loline lkloid levels in E+ shoot smples were higher in stressed clones from dy to dy of withholding wter (Figure 9, Tble 1). Lower mounts of lolines were detected in root smples compred to shoot smples (Figure 9b). As expected, lolines were undetectble in E- root nd shoot smples. In the second experiment with sme clone pir, lolines incresed in stressed shoots nd were significntly different from wtered controls by dy 3 (Figure 9c nd d, Tble ). Loline levels in clone pir 67/68 showed similr trends (Figure 9e nd f, Tble 3).

10 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 1 of b E+D E D c e Figure Sucrose levels in wter-deficit stressed nd unstressed shoots nd roots of tll fescue. ( nd b) Shoots nd roots, respectively, of 78/79 clone pir, Experiment 1; (c nd d) shoots nd roots, respectively, of 78/79 clone pir, Experiment ; (e nd f) shoots nd roots, respectively, of 67/68 clone pir. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM of biologicl replictes s indicted in Figure. E-D d f ç Discussion We ssessed plnt survivl nd differences in metbolite ccumultion in two tll fescue clone pirs with (E+) or without (E-) symbiotic Neotyphodium coenophilum over time course of wter deficit stress, nd observed tht E+ plnts recovered significntly better thn E- plnts fter 3 dys of withholding wter. Simultneously, the E+ plnts consistently ccumulted more free sugrs, sugr lcohols nd mino cids erly during the onset of stress, compred to E- plnts. The fungl-specific metbolites, mnnitol nd loline lkloids, lso incresed in this time period. The higher metbolite levels in E+ compred to E- plnts over the time course of withholding wter consistently occurred within one dy prior to significnt endophyte effect on plnt recovery, strongly suggesting tht free sugrs, polyols, mino cids, nd fungl metbolites ply roles in endophyte-enhnced tolernce to wter deficit. The production or relese of these substnces my led to osmotic djustment [,36], nd help mintin integrity of cellulr enzymes, proteins, nucleic cids nd membrnes [37], or protect ginst rective oxygen species (ROS) [38,39]. The ccumultion of soluble sugrs is strongly correlted with drought tolernce in plnts []. These sugrs ffect osmotic djustment, which is considered n importnt mechnism to llow mintennce of wter uptke nd cell turgor under stress conditions [1]. Furthermore, hydroxyl groups of sugrs nd polyols cn interct with proteins nd membrnes to prevent denturtion nd help void the crystlliztion of cytoplsm under low-wter stress [,3]. In ddition, these sugrs hve been shown to be importnt regultory molecules in different signling pthwys [,], helping to mintin redox blnce, nd cting s rective oxygen scvengers [5,6]. In generl,

11 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 11 of 17 µ µ b E+D+ E D+ E-D Figure 5 Trehlose levels in shoot. () 78/79 clone pir, Experiment ; (b) 67/68 clone pir. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM (n = 3). endophytic fungi re similr to plnt pthogenic fungi in possessing glucn hydrolse-3 (GH3 invertse) enzymes tht convert sucrose into glucose nd fructose for ctbolism [7]. Fungl invertse ctivity nd presence of invertse gene trnscripts hve been reported in some of the grss endophytes [8,9], so under the conditions imposed in our study, fungl enzymes my ply t lest prtil role in the observed increses in these free sugrs. Mnnitol nd rbitol re common polyols in fungi, nd hve been observed to ccumulte in plnts during infection [5]. We found tht both polyols incresed in response to wter deficit in the E+ tll fescue clones. Our results, re in greement with Richrdson et l. [6] who reported mnnitol in E+ tll fescue plnts, lthough they did not see n effect on the mnnitol levels when the plnts were osmoticlly stressed with polyethylene glycol. Arbitol ccumulted essentilly only under stress (Figure 6) conditions [6]. Most plnts do not normlly contin mnnitol, with some slt tolernt species, such s celery, s exceptions [51]. Note tht the very low levels of mnnitol in some E- plnts ws likely due to the presence of commensl fungi on the plnts, since the plnts were not grown xeniclly. Plnts engineered to produce mnnitol hve shown incresed tolernces to drought, slt, nd temperture stresses [5-55], so mnnitol in the E+ plnts my hve contributed to their tolernce of wter deficit stress. 8 8 b E+D+ E D+ c E-D d Figure 6 Sugr lcohols or polyols in wter-deficit stressed nd unstressed shoots of tll fescue. Shown re, () myo-inositol, (b) mnnitol, (c) sorbitol, (d) nd rbitol in the shoots of unstressed nd wter-deficit stressed plnts in Experiment with the 78/79 clone pir. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM (n = 3).

12 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 1 of 17 1 b E+D E D+ 16 c E-D 6 d e f Figure 7 Proline levels in wter-deficit stressed nd unstressed shoots nd roots of tll fescue. ( nd b) Shoots nd roots, respectively, of 78/79 clone pir, Experiment 1; (c nd d) shoots nd roots, respectively, of 78/79 clone pir, Experiment ; (e nd f) shoots nd roots, respectively, of 67/68 clone pir. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM of biologicl replictes s indicted in Figure. The non-reducing discchride, trehlose, is n importnt osmoprotectnt nd storge crbohydrte in mny orgnisms. In plnts, the trehlose pthwy is ubiquitous nd indispensible, but with few exceptions, such s in resurrection plnts, trehlose typiclly does not ccumulte to high levels, possibly due to trehlse-ctlyzed clevge to glucose. Significnt increses in trehlose ccumultion hve been ccomplished thorough trnsgenic pproches, nd shown to protect plnts from drought nd slt stresses [56-59]. However, the overproduction or ccumultion of high levels of trehlose is lso observed to cuse growth berrtions in some of the trnsgenic experiments [6-63]. In our studies, we observed incresed levels of trehlose fter 3 dys of withholding wter, with significntly higher levels in E+ plnts. Although the overll levels of trehlose observed in the E+ nd E- plnts were very low compred to the other soluble sugrs nd polyols, the observed spike in trehlose ccumultion during stress, nd differences between E+ nd E- plnts in trehlose levels suggest possible functionl role. While it is possible tht the low trehlose levels observed in these plnts could function in stress tolernce [6], it seems more likely tht the trehlose ccumultion is ssocited with the signling/regultion role tht hs been documented [65-7]. Wter deficit hs been shown to increse levels of ROS, so n importnt role of ccumulted metbolites ppers to be scvenging or detoxifying ROS [5,71,7]. Production of phenolics, crbohydrtes, mnnitol, nd proline with ntioxidnt cpcity protects plnts from oxidtive stress under wter-deficit conditions. As reviewed by White nd Torres [73], symbiotic plnts re protected from different biotic nd biotic stresses by production of these ntioxidnts.

13 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 13 of 17 3 b E+D E D+ c E-D 8 d e Figure 8 Some of the metbolite levels in 67/68 clone pir shoots of wter-deficit stressed nd unstressed plnts. () glutmine in the shoot (b) glutmine in the root (c) glutmic cid in the shoot (d) glutmic cid in the root (e) sprgine in the shoot (f) sprgine in the root. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM (n = 3). 5 3 f The timing of metbolite chnges ws lso highly suggestive of their roles in endophyte-enhnced stress tolernce. In ll three experiments we observed endophyteenhnced increses in certin sugrs, sugr lcohols nd mino cids one dy before observing the significnt endophyte effect on recovery of the stressed plnts. Interestingly, endophyte effects on levels of most metbolites were brief, since levels of these metbolites in E+ plnts decresed or plteued over the following dys to levels similr to those in E- plnts. In ddition to enhncing osmotic djustment, it is lso possible tht these ccumulted solutes provided energy, crbon nd nitrogen for the survivl of meristemtic regions, nd helped in regrowth of the plnt fter the wter deficit ws llevited. Levels of severl mino cids hve been shown to increse in drought stressed plnts [7]. In our experiments, the levels of proline, threonine, tryptophn, phenyllnine, tyrosine, nd vline incresed upon wter deficit stress. In ddition, proline ws found to be consistently higher in both shoots nd roots of E+ stressed plnts thn in E- stressed plnts. A correltion between free proline ccumultion nd the performnce of crops in the field t low wter vilbility suggests tht its ccumultion is drought stress dptive response tht enhnces survivl [75]. Proline my serve s n osmoregultor [7] nd lso s ROS scvenger [76]. Loline lkloids re protective secondry metbolites produced by the endophyte in tll fescue nd other cool seson grsses [77,78]. We observed incresed loline lkloid levels in response to wter deficit stress in both clone pirs. Lolines re derived from proline nd sprtte [79]. Conceivbly, proline is depleted by loline production [8], but since no differences in proline levels were observed between E+ nd E- plnts in unstressed conditions, proline levels were pprently djusted in response to loline lkloid synthesis. In the first experiment with clone pir 78/ 79 totl proline nd loline levels in E+ plnts were higher even t dy fter withholding wter compred to E- plnts, though levels of proline (nd the metboliclly closely relted mino cids, glutmic cid nd glutmine)

14 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 1 of 17 b 1 3 µ µ E+D+ c µ d µ e µ Figure 9 Loline levels in wter-deficit stressed nd unstressed shoots nd roots of tll fescue. ( nd b) Shoots nd roots, respectively, of 78/79 clone pir, Experiment 1; (c nd d) shoots nd roots, respectively, of 78/79 clone pir, Experiment ; (e nd f) shoots nd roots, respectively, of 67/68 clone pir. Abbrevitions re s in Figure. Sttisticl significnce is indicted s in Figure 1. Error brs re SEM of biologicl replictes s indicted in Figure. f µ were not different in between E+ nd E- plnts t dy. It is possible tht the proline is converted to loline in the E+ plnts, thus mintining n pprent equl proline level s tht of E- plnts. However, in the other two experiments, the levels of proline in stressed tissues were fr higher compred to mounts of lolines tht ccumulted in those tissues. Although wter deficit hs been reported to increse loline lkloid levels in lef tissues of some tll fescue ccessions [81], direct role of loline lkloids on wter stress tolernce hs not yet been demonstrted. Differences in the timing of metbolite ccumultion were observed between two experiments with the sme clone pir (78/79), with metbolite peks t dy 1 in Experiment 1 nd the corresponding peks occurring t dy or 3 in Experiment. This difference my be becuse of wether nd greenhouse conditions tht differed between these experiments. Specificlly, dy 1 of Experiment ws ccompnied with thunderstorms nd hevily overcst skies, resulting in lower photoctive rdition compred to dy 1 of Experiment 1 (see Additionl file 1, pnel b), pprently delying the onset of drought stress s evidenced by the tiller recovery curves (see Figure 1 nd b). Similrly the observed metbolite differences between the experiments with different clone pirs could be due to plnt genotype effects. Nevertheless, it ws cler tht, in our experiments the endophyte in tll fescue sped up plnt responses to wter deficit by erlier nd fster ccumultion of metbolites compred to uninfected tll fescue plnts. Similr results hve been reported in bcteril endophyte-plnt systems. Bcteril endophyte enhnces cold tolernce of grpevine plnts by ltering sugr metbolism nd photosynthesis [8], nd with higher nd fster ccumultion of stress relted gene trnscripts nd metbolites [83].

15 Ngbhyru et l. BMC Plnt Biology 13, 13:17 Pge 15 of 17 Rsmussen et l. [8] hve conducted comprehensive metbolomic studies in the relted grss, Lolium perenne (perennil ryegrss), nd hve shown significnt effects of the endophyte, Neotyphodium lolii, on primry nd secondry metbolism of tht grss. The need for more reserch to identify robust metbolic trits nd pthwys relting to drought tolernce in forge grsses through integrtion of metbolomic nd trnscriptomic dt hve been emphsized in reviews [85]. From our study it ws evident tht endophyte cn ffect tll fescue plnt metbolism, in response to wter deficit stress. Anlyzing these endophyte effects on host plnts t the moleculr genetic level by trnscriptome profiling is nother pproch, tht we will be exploring further to help elucidte the mechnisms of endophyte-enhnced plnt growth nd survivl under wter deficit conditions. Conclusions In conclusion, enbling the plnt cells to sense nd respond quickly to surrounding environmentl signls or stresses is importnt for their metbolic nd developmentl djustments, nd these responses my be enhnced due either to primry or secondry metbolite signls [86,87]. As we observed in the tll fescue clone pirs, symbiotic fungi in the infected plnts my hve induced, or rpidly ctivted, the plnt biochemicl rections to ccumulte the metbolites erly in stress conditions, nd this my be one of the wys tht the presence of the endophyte helps mitigte the effects of, nd enhnce recovery from, wter deficit stress. The results presented here demonstrte tht symbiosis with endophytes cn significntly enhnce recovery of host plnts from wter deficit stress, nd the effect corresponds in timing with ccumultion of orgnic solutes tht my serve s osmolytes nd cellulr protectnts in leves nd roots. Additionl files Additionl file 1: Photoctive rdition (PAR) t the smpling period. () clone pir 78/79, Experiment 1; (b) clone pir 78/79, Experiment ; (c) clone pir 67/68. Additionl file : Totl mounts of glucose, fructose, sucrose (GFS) in wter-deficit stressed nd unstressed plnts of tll fescue clone pirs. ( nd b) Shoots nd roots, respectively, of clone pir 78/79, Experiment 1; (c nd d) shoots nd roots, respectively, of clone pir 78/79, Experiment ; (e nd f) shoots nd roots, respectively, of clone pir 67/68. Competing interests The uthors declre tht they hve no competing interests. Authors contributions PN designed nd performed ll experiments, nd drfted the mnuscript. RDD helped design experiments, generted nd provided clone pir 67/ 68, nd helped drft the mnuscript. CLW performed sttisticl nlyses. CWB generted nd provided clone pir 78/79, nd helped drft the mnuscript. CLS devised nd supervised experiments nd helped drft the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This reserch is funded by USDA-ARS Specific Coopertive Agreement The uthors re grteful for help nd suggestions from Dr. Bruce A. Downie in HPLC for crbohydrtes. The uthors lso thnk J. Dougls Brown nd W. Troy Bss for mintining plnts, nd Dr. Lowell P. Bush nd Dr. Fnniel F. Fnnin for providing the loline lkloid stndrd. The uthors lso cknowledge the ERTL fcility t the University of Kentucky for llowing use of the LCMS nd for technicl ssistnce. This is publiction number 131 of the Kentucky Agriculturl Experiment Sttion, published with pprovl of the director. Author detils 1 Deprtment of Plnt Pthology, University of Kentucky, Lexington, KY 56-31, USA. USDA-ARS, Forge-Animl Production Reserch Unit, Lexington, KY 56-91, USA. 3 Deprtment of Sttistics, University of Kentucky, Lexington, KY 56-7, USA. USDA-ARS, Toxicology nd Mycotoxin Reserch Unit, Athens, GA 365-7, USA. Received: 19 Februry 13 Accepted: 1 August 13 Published: 9 September 13 References 1. Fribourg HA, Hnnwy DB, West CP: Tll fescue for the twenty-first Century. ASA, CSSA, SSSA: Mdison, WI; 1.. Schrdl CL, Leuchtmnn A, Spiering MJ: Symbioses of grsses with seedborne fungl endophytes. Annu Rev Plnt Biol, 55: Archevlet M, Bcon CW, Hovelnd CS, Rdcliffe DE: Effect of the tll fescue endophyte on plnt response to environmentl stress. Agron J 1989, 81: West CP: Physiology nd drought tolernce of endophyte-infected grsses. In Biotechnology of Endophytic Fungi of Grsses. Edited by Bcon CW, White JF. Boc Rton, FL: CRC Press; 199: West CP, Izekor E, Turner KE, Elmi AA: Endophyte effects on growth nd persistence of tll fescue long wter-supply grdient. Agron J 1993, 85: Bcon CW: Abiotic stress tolernces (moisture, nutrients) nd photosynthesis in endophyte-infected tll fescue. Agric Ecosyst Environ 1993, : Mlinowski DP, Belesky DP: Adpttions of endophyte-infected cool-seson grsses to environmentl stresses: Mechnisms of drought nd minerl stress tolernce. 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