The impact of drought on wheat leaf cuticle properties

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1 Bi et l. BMC Plnt Biology (217) 17:85 DOI /s RESEARCH ARTICLE The impt of drought on whet lef utile properties Open Aess Huihui Bi 1,2, Ntliy Kovlhuk 1,2, Peter Lngridge 2, Penny J. Triker 1,2*, Sergiy Lopto 1 nd Nikoli Borisjuk 1,3 Astrt Bkground: The plnt utile is the outermost lyer overing eril tissues nd is omposed of utin nd wxes. The utile plys n importnt role in protetion from environmentl stresses nd gluousness, the luish-white olourtion of plnt surfes ssoited with utiulr wxes, hs een suggested s ontriuting ftor in rop drought tolerne. However, the utile struture nd omposition is omplex nd it is not ler whih spets re importnt in determining role in drought tolerne. Therefore, we nlysed residul trnspirtion rtes, utile struture nd epiutiulr wx omposition under well-wtered onditions nd drought in five Austrlin red whet genotypes, Kukri, Exliur, Drysdle, RAC875 nd Gldius, with ontrsting gluousness nd drought tolerne. Results: Signifint differenes were deteted in residul trnspirtion rtes etween non-gluous nd drought-sensitive Kukri nd four gluous nd drought-tolernt lines. No simple orreltion ws found etween residul trnspirtion rtes nd the level of gluousness mong gluous lines. Modest differenes in the thikness of utile existed etween the exmined genotypes, while drought signifintly inresed thikness in Drysdle nd RAC875. Wx omposition nlyses showed vrious mounts of C31 β-diketone mong genotypes nd inreses in the ontent of lknes under drought in ll exmined whet lines. Conlusions: The results provide new insights into the reltionship etween drought stress nd the properties nd struture of the whet lef utile. In prtiulr, the dt highlight the importne of the utile s iohemil mkeup, rther thn simple orreltion with gluousness or stomtl density, for wter loss under limited wter onditions. Keywords: Cutiulr wx, β-diketone, Gluousness, Residul trnspirtion rte, Stomtl density, Tritium estivum Bkground Bred whet, Tritium estivum, is the world s most widely grown rop, representing out 3% of the erel ultivtion re nd providing 2% of the lories for the humn popultion. Drought signifintly limits rop prodution nd the durtion of drought periods is inresing due to limte hnge, delining ground nd surfe wter resoures, nd wrming of ir temperture in most of the erel ropping regions round the world [1]. An understnding of the physiologil, iohemil, nd geneti mehnisms llowing plnts to ope with environmentl hllenges is of vitl importne for * Correspondene: penny.triker@delide.edu.u 1 Austrlin Centre for Plnt Funtionl Genomis, PMB1 Glen Osmond, Adelide, South Austrli 564, Austrli 2 Shool of Agriulture, Food nd Wine, University of Adelide, PMB1 Glen Osmond, Adelide, South Austrli 564, Austrli Full list of uthor informtion is ville t the end of the rtile reeding rops with improved stress tolerne nd performne under stress [2]. The plnt utile evolved s n exterior extension of epiderml ell wlls, is ontinuous hydrophoi sheet tht overs the eril surfes of ll plnt orgns nd ts s n interfe in plnts intertions with vrious ioti nd ioti ftors. The utile is ruil rrier tht, in onert with stomt, ontrols plnt wter sttus nd helps plnts survive under drought nd high UV rdition [3]. Struturlly, the whet utile is.1 1 μm thik memrne omposed priniplly of polyester mtrix intertwined with rnge of long hin hydrorons. Bsed on soluility in orgni solvents, the utile omponents re divided into insolule utin nd solule utiulr wxes. Intrutiulr wx is emedded in the underlying utin polyester frmework nd epiutiulr wx is overlid on the utin mtrix nd intrutiulr wx. The wxes re typilly omplex mixture of derivtives of very-long-hin sturted The Author(s). 217 Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. The Cretive Commons Puli Domin Dedition wiver ( pplies to the dt mde ville in this rtile, unless otherwise stted.

2 Bi et l. BMC Plnt Biology (217) 17:85 Pge 2 of 13 liphtis, whih inlude ftty ids, lknes, ldehydes, ketones, nd primry nd seondry lohols [4, 5]. Mny elements of the utile iosynthesis pthwys, inluding utile-relted genes enoding key enzymes nd regultory trnsription ftors, were unovered primrily y hrterising utile mutnts in Aridopsis, tomto, rie, mize, nd rley [6 8]. The iosynthesis of mjor utile onstituents egins with de novo synthesis of C16-C18 ftty ids in the plstids of epiderml ells. The ftty ids re onverted to yl-coas prior to their export to the endoplsmi retiulum, where they re trnsformed into very long hin ftty yl-coas (VLCFAs) y the ftty id elongtion omplex (FAE). These sturted VLCFAs ontining rons n e relesed from the FAE s free ftty ids or serve s preursors for (i) the lohol-forming pthwy for onversion into even-numered primry lohols nd lkyl esters, for (ii) the lkne-forming pthwy whih yields ldehydes, odd-numered lknes, seondry lohols nd ketones, nd for (iii) the β-diketone pthwy whih is less prevlent thn the other two pthwys ut represents n importnt pthwy in some plnts like whet nd rley [9, 1]. It hs een proposed tht the min physiologil role of the utile is the redution of wter loss. The utile delys the onset of ellulr dehydrtion stress under drought nd is therefore onsidered n importnt omponent of protetion from drought [5, 11, 12]. During wter defiit, stomt lose nd nnosle diffusion pthwys rossing the utile eome the primry pth of plnt wter loss. Non-stomtl wter loss through the lef epidermis my ount for up to 5% of totl loss in drought-stressed whet plnts during the dy nd 6% during the night [13]. Aumultion of epiutiulr wxes on plnt surfes often results in luish-white olourtion termed gluousness, whih is visile form of densely distriuted epiutiulr wx rystlloids. Gluousness is formed in whet due to the presene of C31 β-diketones on the surfes of eril orgns [14 17]. Reently, the genes enoding three key enzymes involved in the synthesis of β-diketones hve een reported in whet nd rley [1, 18]. Gluousness inreses rdition refletne nd redues lef tempertures nd trnspirtion, therey enhning lef survivl under wter stress [19, 2]. As lssil geneti mrker nd gronomi trit, gluousness hs een intensively studied in ssoition with drought/het tolerne nd yield in different whet vrieties [21 24]. However, the preise vlue of this trit in reltion to mintenne of plnt iomss nd grin yield under stress remins unertin euse of the omplex iohemil nd geneti nture of this esily visile trit. It hs een ssumed tht the mount nd speifi iohemil mkeup of the wxes to lrge extent define protetive funtions of the utile [25, 26]. Inresed mounts of utiulr wxes in trnsgeni plnts hve een ssoited with improved drought tolerne in plnts suh s Aridopsis [27], lflf [28] nd Cmelin [29]. Breeding rley for higher tolerne nd yield under drought led to inresed mounts of utiulr wxes, further onfirming onnetion etween drought tolerne nd the utile [3]. Another exmple is the glossy 1 2 (gl1 2) mutnt in rie with redued umultion of lknes, ldehydes nd ftty ids. The gl1 2 mutnt plnts exhiited inresed drought sensitivity nd utiulr permeility t the reprodutive stge [31]. The geneti, physiologil nd metoli determinnts influening rop yield nd drought tolerne hve een studied in five Austrlin whet lines, Kukri, Exliur, Drysdle, RAC875 nd Gldius [22, 32 36]. Kukri hs lower level of drought tolerne thn the other four lines whih represent importnt soures of drought tolerne in the southern Austrlin environment. Kukri ws used s the drought-sensitive prent in severl mpping popultions for drought nd yield relted studies [33]. Most reently, two of these lines, Kukri nd RAC875, hve een investigted for utile omposition nd its regultion y drought-responsive trnsription ftors of the MYB fmily [16]. In this work, the five whet lines were used to: (i) exmine lef utile properties s wter loss rrier for whet plnts grown under two different wtering regimes, (ii) unover the stomtl nd utile-relted hrteristis responsile for differenes in wter loss nd the responses of these trits to drought, nd (iii) estlish possile links etween utile omposition, permeility nd drought tolerne of these whet lines. Methods Plnt, growth nd smpling The five genotypes used in this study were ll red for the southern Austrlin environment where yields re generlly in the rnge of 1 to 4 t/h. Under fvourle onditions, where verge yields re over 2 t/h, ll show similr yield ut in more severe environments, Kukri is the poorest performer [33, 37]. There re lso differenes etween genotypes in their generl response to drought stress. Drysdle ws seleted sed on wter use effiieny mesured through ron isotope disrimintion ut responds poorly to the sline nd low nutrient soils in muh of southern Austrli. RAC875 nd Exliur differ in their physiologil response to drought stress with RAC875 showing onstitutive response while Exliur displys n dptive mehnism [22]. Gldius is the most reent of the vrieties, relesed in 27, nd hs oth RAC875 nd Exliur in its prentge ( these

3 Bi et l. BMC Plnt Biology (217) 17:85 Pge 3 of 13 genotypes provide spetrum of lines tht hve een seleted for performne in similr environment ut differ in their stress responses. Drysdle, Exliur, Gldius nd Kukri re relesed vrieties nd RAC875 is reeding line ville, with permission, from the University of Adelide nd Austrlin Grin Tehnologies. Drysdle ws developed y the CSIRO in onjuntion with NSW Agriulture. Exliur nd Kukri were developed y the University of Adelide Roseworthy Cmpus Whet Breeding Tem, nd Gldius ws developed y Austrlin Grin Tehnologies Roseworthy Cmpus Whet Breeding Progrm. All seeds used in these experiments were from our own olletion. Drysdle ws originlly soured for our olletion from AWB seeds Ltd. Sixteen seeds (8 seeds/suplot) of eh of the five seleted genotypes: Kukri, Exliur, Drysdle, RAC875 nd Gldius, were sown in eh of two lrge ontiners ( m) in glsshouse, with 14 h dy/1 h night yle. The two ontiners shred the sme distriution of seeds nd rndomised lok design with 1 suplots distriuted within eh ontiner extly s desried y Amlrj et l. [38]. Eh ontiner ommodted 8 tested plnts flnked y order row (vr. Gldius) on eh short side of the in. One ontiner ws used for eh of the ontrolled well-wtered nd drought onditions with drought imposed s shown in Additionl file 1. To mintin nd monitor wter sttus, ontiners were equipped with n utomti wtering system nd four soil wter tensiometers (gypsum loks) instlled t 1 m nd 3 m soil depths, nd onneted to dt logger for ontinuous monitoring of the soil wter tension. Wtering in the drought in ws terminted 32 dys fter plnting when plnts were t stem elongtion stge, while ontinuous wtering ws pplied to the well-wtered in. The soil wter tension in the drought in grdully nd slowly inresed nd flg leves for ll the experiments onduted in this study were smpled fter the reding of 3 m tensiometer rehed the highest mesurele level of 6 kp in the drought tretment (for detils see Additionl file 1). Aironditioning in the glsshouse ontrolled temperture flututions nd the tempertures, reorded every 15 min, were 19.9 ± 3.3 C (men ± stndrd devition) for the period from plnting to smpling of flg leves for ll the nlyses. Flg leves were seleted for ll the nlyses, s they re the min soure of ssimiltes for grin development. Cutile permeility ssy Flg leves were dethed from plnts grown in the well-wtered ontiner or drought ontiner 2 dys fter nthesis, nd lef weight ws immeditely mesured using n nlytil lne (OHAUS Corportion, Prsippny, USA) efore eing pled into olletion tue. Leves in the opened olletion tues were dehydrted t room temperture (23 C) for 12 h with lef weight mesured every hour. Leves were then dried in 37 C inutor for 72 h fter whih lef weight ws mesured nd tken s the dry weight. Lef surfe imprints The lef surfe imprint method, modified from Sinlir nd Dhingr [39], ws used to estimte stomtl numers per unit re. Flg leves from the min tiller were dethed 2 dys fter nthesis from five plnts of eh ultivr grown under well-wtered onditions or drought, nd the mid-point etween the mjor vein nd the lef mrgin nd hlf-wy long the long xis of the lef were used to mke impressions of the surfes. Cynorylte dhesive (Sup glue; Selleys, Pdstow, NSW, Austrli) ws pplied to the re nd the glued side ws pled ginst mirosope slide. A seond slide ws pled on top of the lef in the sme diretion. The slides were held together with two lrge ulldog lips for 3 minutes, fter whih the two slides were seprted nd the lef ws peeled off. Lef imprints were exmined under the Lei AS LMD mirosope (Lei Mirosystems, Wetzlr, Germny) with differentil interferene ontrst (DIC) t 2 times mgnifition t the Wite Fility of Adelide Mirosopy. Stomtl ounts were mde from eh of three imges of eh of the imprints from five iologil replites. Trnsmission eletron mirosopy (TEM) Flg lef ldes were olleted t dy 24 fter nthesis nd severl segments lose to the mjor vein of pproximtely 5 mm 3 mm in size were ut from the midpoint of lef from eh of three plnts. In fume hood, ut piees were pled into TEM fixtive [4% surose, 1 PBS (phosphte uffered sline), 4% prformldehyde, nd.25% glutrldehyde]. After overnight inution t 4 C, smples were rinsed twie with 1 PBS t 4 C to remove fixtive. Then smples were dehydrted in n ethnol series: 5%, 7%, 9%, 95% nd %, eh hnge fter pproximtely 1 hour, followed y two hnges of % dry ethnol (on moleulr sieve). Smples were stored in % dry ethnol t 4 C until resin infiltrtion. Smples were first infiltrted in 5:5 mix of % ethnol nd LR White Resin (London Resin Co. Ltd., London, UK) for 8 hours, followed y three hnges of % LR White Resin, lso for 8 hours eh. Next, infiltrted speimens were emedded in fresh % LR White Resin in geltin psules. Finlly, emedded smples were polymerised t 6 C for 24 h. Prior to setioning on the ultrmirotome, resin loks were trimmed with rzor lde to mke trpezoid fe. 7 nm thik setions were generted using

4 Bi et l. BMC Plnt Biology (217) 17:85 Pge 4 of 13 dimond knife. Setions were first stined with 4% urnyl ette for 2 min, then with led itrte for 2 min fter three rinses in wter. After nother three rinses in wter nd ir drying, setions were exmined under Philips CM trnsmission eletron mirosope t the Adelide Mirosopy Wite Fility. Smples from one or two plnts of eh genotype were sujeted to TEM. Snning eletron mirosopy (SEM) The epiutiulr wx ws exmined using snning eletron mirosope in the Adelide Mirosopy Unit ( University of Adelide, Austrli). Severl segments of pproximtely 4 mm 3 mm were ut from similr lotion on the sme lef s the TEM smples nd exmined s desried y Bi et l. [16] under Philips XL3 Field Emission Snning Eletron Mirosope, equipped with Gtn CT15 HF Cryo-trnsfer Stge. Composition nlysis of utiulr wxes For the wx omposition nlysis, the 6.5 m long sl portions of the flg lef ldes from eh of three individul plnts were olleted 24 dys fter nthesis. The weight of eh lef setion ws mesured, nd leves were snp frozen in liquid nitrogen nd further stored t 8 C until wx extrtion. Wx extrtion nd GC-MS nlysis were rried out following the sme proedures s desried y Bi et l. [16]. GC-MS nlysis ws onduted in the W.M. Kek Metolomis Reserh Lortory of Iow Stte University (USA) ording to the proedure desried y Ch et l. [4]. Sttistil nlysis of dt Dt on stomtl density nd wx omposition were nlysed using two-wy ANOVA with the Fisher s lest signifint differene post-ho test in GenStt (16th Edition; VSN Interntionl Ltd., Hemel Hempsted, UK). The sttistil signifine of differenes in wter loss rtes ws nlysed using Generl Liner Model repeted mesures ANOVA in IBM SPSS Sttistis v. 24 within nd etween sujets with post ho multiple omprisons for the lest signifint differene etween genotypes t α.5. Cron hin length distriutions of mjor utiulr wxes were nlysed using IBM SPSS Sttistis v. 24 Generl Liner Model multivrite nlyses with tretment nd genotype fixed ftors in the model. Post ho multiple omprisons etween genotypes were performed s efore. Results Exmintion of lef surfe permeility In order to determine the utile s ility to t s rrier ginst wter loss, wter losses were ssessed using dethed flg leves of the five whet lines, whih were grown under well-wtered () onditions or under drought (). As shown in Fig. 1, during the first 2 hours of dehydrtion the Kukri nd Exliur leves lost wter more rpidly thn leves of RAC875, Drysdle nd Gldius. After 2 hours, the wter loss rtes eme stle in ll five lines nd ould e onsidered residul trnspirtion rtes (RTRs), whih re primrily ontriuted y the utile. The RTRs of Kukri plnts were higher thn those of the other four lines (Fig. 1) (P = <.1, Additionl file 2). Wter loss rtes from flg leves dethed from plnts grown under were reltively stle during the entire 12 h-long test (Fig. 1). Similr to results otined for whet plnts, the RTRs of Kukri plnts grown under drought onditions were higher thn those of RAC875, Drysdle or Gldius (Fig. 1), however the differene ws less pronouned ompred to the experiments (Fig. 1). Comprison of RTRs of eh line under ompred with onditions reveled tht RTRs of Kukri, Drysdle, RAC875 nd Gldius were lower (P = <.1, Additionl file 2) under drought wheres the RTR of Exliur ws higher under drought ompred with onditions (P =.1, Additionl file 2) (Fig. 1). Investigtion of stomtl density Stomt re speilized strutures on lef surfes tht ontrol the exhnge of wter nd gses etween plnts nd the environment nd therefore stomtl density my ffet the rte of lef wter loss. More stomt per unit of lef re existed on the dxil sides of flg leves thn on the xil sides, s shown in the representtive non-gluous nd drought sensitive ultivr Kukri (Fig. 2, ) nd the gluous nd drought tolernt Gldius (Fig. 2, d). Kukri hd the lowest numer of stomt per unit of lef surfe re on oth surfes of leves under oth ontrol nd drought tretment (Fig. 2e, f). RAC875 ws the only line in whih no signifint differenes in stomtl density were oserved etween tretments on oth lef sides. Overll, the stomtl density on oth sides of flg leves olleted from plnts grown under well-wtered onditions in the other four lines (Kukri, Exliur, Drysdle nd Gldius) ws % lower thn tht of leves of plnts sujeted to drought. Mirosopi ssessments of lef surfes The thikness of the utile lyer ws exmined on the xil side of flg leves grown under the sme onditions s efore using trnsmission eletron mirosopy. Under onditions, modest differenes were found in the thikness of the utile lyer mongst the five genotypes, wheres differenes etween nd within lines were lrger nd more ovious (Fig. 3). In

5 Bi et l. BMC Plnt Biology (217) 17:85 Pge 5 of 13 Wter loss ( g h 1 g 1 DW) KUK EXC S RAC GL Wter loss (g h 1 g 1 DW) KUK EXC S RAC GL W ter loss (g h 1 g 1 DW) KUK Wter loss (g h 1 g 1 DW) EXC W ter loss (g h 1 g 1 DW) S RAC GL Fig. 1 Wter loss rtes of flg leves dethed from five whet lines grown under well-wtered () onditions nd drought (). Wter loss rtes of flg leves dethed from () nd () plnts over 12 h. Comprison of wter loss rtes of nd plnts. KUK - Kukri, EXC - Exliur, S - Drysdle, RAC - RAC875, GL - Gldius. Wter loss rtes were expressed s weight loss per hour per dry weight (DW) of flg leves. Mens nd stndrd errors (indited y rs) were lulted from four replites prtiulr, the thikness ws gretly inresed y drought on the xil side of Drysdle nd RAC875 leves, slightly inresed in Kukri nd Gldius, nd deresed in Exliur. Aumultion of epiutiulr wxes on lef surfes onfers whitish nd powdery pperne to most modern whet vrieties termed gluousness. Kukri is non-gluous, glossy ultivr, Exliur hs very low level nd Drysdle medium level of gluousness, while RAC875 nd Gldius were the most gluous lines exmined (Fig. 4). Vritions in gluousness were more notiele on the xil sides of leves thn on the dxil sides. When exmined under SEM, it ws found tht wx distriution nd struture were different on the dxil nd xil sides of leves. Compred with vrile wxes on the xil side, the dxil side wxes were more densely nd evenly deposited (Fig. 4). The speifi strutures of wxes were explored under high mgnifition. No differene ws found on the dxil sides of leves: the dense pltelet- shped wx rystlloids were dominnt in ll five lines (Additionl file 3). However, different wx shpes were oserved on the xil side of leves. The plte-shped wx rystlloids previled on the xil side of flg leves of Kukri nd Exliur, mixture of pltes nd tuulr shpes were oserved in Drysdle, while long tuule-like rystlloids were the dominnt wx strutures on the surfes of the RAC875 nd Gldius leves (Fig. 5). Pltelets, supposedly omposed of primry lohols nd

6 Bi et l. BMC Plnt Biology (217) 17:85 Pge 6 of 13 e d e d d d 4 2 d f 12 KUK EXC S RAC GL ef f de de d de 2 KUK EXC S RAC GL Fig. 2 Imges of stomt on the epidermis of flg leves of investigted whet lines. Stomt on the dxil side of flg lef in Kukri. Stomt re indited y rrows. Stomt on the dxil side of flg lef in Gldius. Stomt on the xil side of flg lef in Kukri. d Stomt on the xil side of flg lef in Gldius. e Clulted stomtl density on the dxil side of flg leves. f Clulted stomtl density on the xil side of flg leves. KUK - Kukri, EXC - Exliur, S - Drysdle, RAC - RAC875, GL - Gldius, - well-wtered onditions, - drought. Mens nd stndrd errors were lulted from five plnts. Different letters on top of error rs indite signifint differene t P <.5. Sle rs represent 5 μm lknes, nd tuules, ttriuted to β-diketones [14], re the most frequent types of wx rystlloids found in plnts ording to the lssifition nd terminology proposed y Brthlott et l. [41]. Genotypi vritions in epiutiulr wx omposition nd responses to wter defiit To investigte the ssoition etween wter loss rtes nd epiutiulr wxes, we nlysed the mount nd omposition of epiutiulr wxes on the flg leves of these five whet lines using gs hromtogrphy in omintion with mss spetrometry (GC-MS). We oserved good orreltion in these lines etween the ontent of β-diketones nd the predominnt presene of long tuule-like rystlloids in wx lyers (Fig. 5). The speifi mounts of utiulr wxes of Kukri nd RAC875 were previously presented [16]. Dt shown here for Exliur, Drysdle nd Gldius were generted from whet plnts grown under extly the sme onditions s Kukri nd RAC875, desried y Bi et l. [16]. Under onditions, Gldius hd the highest totl wx lods on flg leves; the totl mounts of wxes deresed in the order of Drysdle nd Exliur (Fig. 6). In terms of wx omposition, primry lohols were the most undnt wx speies in ll three whet lines, omprising from 4. to 51.1% of totl wx lods (Fig. 6). The seond most undnt wx speies were C31 β-diketone in Gldius (28.2%) nd lknes for the other two ultivrs. In Exliur nd Drysdle, the ontents of β- diketones were muh lower, ounting for 1.4% nd 4.6% of totl wx lods, respetively. The ontents of lknes in the totl flg lef wxes of Exliur, Drysdle nd Gldius were 31.1%, 32.1% nd 15.9%, respetively. Among minor omponents of wxes, whih were present on leves of ll exmined lines, were ftty ids ( %), ldehydes ( %) nd resorinols ( %). When ompring totl wx lods of plnts grown under nd onditions, we found tht drought signifintly inresed totl wx lods on flg leves of Drysdle y 48.5% (Fig. 6). The wx lod ws modertely inresed on leves of Exliur (28.%) nd slightly deresed on leves of Gldius ( 6.8%) (Fig. 6). The ontents of lknes were inresed in ll three whet lines under drought while the mounts of β-diketones were inresed in Drysdle ut deresed in Gldius (Fig. 6). The distriution of ron hin lengths of mjor wx speies extrted from flg leves of whet lines were exmined (Fig. 7). The ron hin lengths of primry lohols (Fig. 7) rnge from 2 to 34, the β-diketone (Fig. 7) is 31 ron-long nd lknes (Fig. 7) rnge from 23 to 33. The four most undnt wx omponents on flg leves were C28 primry lohol, C31 β-diketone, nd C29 nd C31 lknes. Sttistil nlyses of utiulr wxes within mjor wx speies n e found in Additionl file 4 nd the ron hin length distriution of three

7 Bi et l. BMC Plnt Biology (217) 17:85 Pge 7 of 13 KUK EXC S RAC GL Fig. 3 Trnsmission eletron mirogrphs of utile lyers on the xil epidermis of flg leves in five whet lines. KUK - Kukri, EXC - Exliur, S - Drysdle, RAC - RAC875, GL - Gldius, - well-wtered onditions, - drought. Cutile lyers re mrked using white lines. Sle rs = nm minor wx speies (ftty ids, ldehydes nd resorinols) n e found in Additionl file 5. Inreses in the mounts of epiutiulr wxes under drought were lso oserved y SEM in the most drought-responsive ultivr, Drysdle (Fig. 8). The SEM imge under high mgnifition shows signifint umultion of the tuule-shped rystlloids under drought, orrelting with the drought-indued inrese of β-diketone in this ultivr s reveled y GC-MS nlysis (Fig. 6). Disussion Gluousness is n esy trit to selet for in reeding progrm sine it n e redily sored visully. Consequently, if this trit were of mjor importne for dpttion to dry environments, we would expet to see lmost exlusive doption of gluous vrieties in these environments. However, this is not the se for whet in drought-prone regions suh s southern Austrli where we see full rnge of gluousness, from low to high, in modern vrieties. On the other hnd, numer of studies hve shown tht utile modifitions ould signifintly inrese plnt drought tolerne. A possile explntion for this pprent ontrdition is tht gluousness is only one spet of utile omposition nd struture nd other properties or hnges in the utile in response to stress my lso e importnt. In this study we sought to tese prt different spets of the lef surfe struture nd utile omposition of five Austrlin whet lines with rnge of lef gluousness nd different utiulr responses under wter-limited onditions. These genotypes were seleted for this study euse they were ll red for similr prodution environment ut differed in their responses to drought nd relted environmentl ftors, inluding het, slt nd nutrient stresses. In previous studies it ws found tht the utile ontriuted more thn 5% of totl trnspirtion when whet plnts were stressed [13], nd tht the residul trnspirtion rte (RTR), defined s wter loss from dethed leves t miniml stomtl perture, plys n importnt role in plnt survivl under severe wter defiit. In our wter loss tests the RTRs of Kukri, the lest drought tolernt nd non-gluous line, were signifintly higher thn those of the other more gluous lines Gldius, RAC875 nd Drysdle. The trend ws oserved irrespetive of whether leves used in the experiment were dethed from plnts ultivted under suffiient or wter limited onditions (Fig. 1, ). Interestingly, the less gluous ultivr Exliur (with intermedite gluousness etween the glossy Kukri nd the other more gluous lines (Figs 5 nd 6)), demonstrted RTRs similr to the gluous RAC875 under wellwtered onditions nd similr to the glossy line Kukri under drought (Fig. 1 nd ). There ws differene etween responses to the tretment within lines for RTR, suh tht the RTR of Exliur leves inresed under drought, while the RTRs of ll other lines deresed with tretment. This fits with the previous oservtions tht Exliur shows n dptive response to drought [22]. The wter loss rte of dethed leves is not n inditor of drought tolerne, defined here s the ility of ultivr to mintin yield under field onditions where wter vilility limits yield to elow 2 t/h. Gluousness, however, hs previously een

8 Bi et l. BMC Plnt Biology (217) 17:85 Pge 8 of 13 Ad A Ad A Kukri Exliur Drysdle RAC875 Gldius Fig. 4 Visulistion of epiutiulr wxes on the flg leves of five whet lines. Wx phenotypes on the dxil (Ad) nd xil (A) sides of flg leves of Kukri, Exliur, Drysdle, RAC875 nd Gldius. Snning eletron mirogrphs of the epiutiulr wxes on the dxil (Ad) nd xil (A) sides of flg leves of Kukri, Exliur, Drysdle, RAC875 nd Gldius. Sle rs = 2 μm ssoited with improved grin yield in oth irrigted red whet [42], nd in rley [19] nd durum whet [24] grown in wter-limited environments. In 16 durum whet genotypes nd rley isolines, gluousness did not signifintly ffet RTRs or utiulr ondutne [19, 24]. It hs een suggested, therefore, tht the ssoition etween gluousness nd yield is n indiret result of improved trnspirtion effiieny under onditions of wter limittion nd/or high evportive demnd. Here we found tht RTRs mong genotypes with rnge of gluousness did differ signifintly with drought tretment, ut tht there ws no strightforwrd orreltion (either positive or negtive) etween visile gluousness nd the response to stress. These results suggest tht ftors ontriuting to the visile gluous phenotype, rther thn gluousness itself, my influene drought response. Over the pst few yers there hve een vrious reports on whet utile nd gluousness relted genes, nd their involvement in protetion from ioti nd ioti stresses [14, 15, 21, 43 47]. The geneti ontrol of whet wxiness is omplex with W1 nd W2 loi (W: wx prodution gene) ontriuting to, nd Iw1 nd Iw2 inhiiting gluousness in ner-isogeni lines. When oth funtionl lleles of W1 nd W2 were present, lines hd signifintly lower rtes of wter loss nd hlorophyll lehing thn the non-gluous nd other gluous NILs (W1w2 nd w1w2) [14]. Agin, this ws not the result of gluousness per se, ut ws ssoited with the prodution nd hydroxyltion of the utile omponent wx β-diketone [14, 15]. Here, we showed orreltion etween the mounts of β-diketones nd long tuule-like wx rystlloids nd thus the level of gluousness in five whet lines (Figs. 4 nd 5). However, the inrese in oth β-diketones nd totl lef wx lods oserved with drought tretment in the ultivr Drysdle ws not found in Exliur or Gldius (Fig. 6). There ws no signifint effet of the tretment on β-diketone quntity ut signifint intertion etween ultivr nd tretment ws oserved (Additionl file 4).

9 Bi et l. BMC Plnt Biology (217) 17:85 Pge 9 of 13 Fig. 5 GC-MS hromtogrms representing iohemil ompositon of extrted utiulr wxes nd SEM imges of wx rystlloids on the xil side of the flg lef in the five whet lines exmined. ALC - primry lohol, ALK lkne, FA - ftty id, IS internl stndrd. Sle rs = 5 μm. Arrows indite C31 β-diketone This indited tht quntittive differenes in this utile wx omponent lone, whilst ontriuting to gluousness, ould not explin different stress responses. In our experiments, the umultion of free ftty ids remined mostly unffeted y drought, while lknes inresed signifintly in ll tested whet lines (Fig. 6 nd [16]). These dt re in greement with results from previous studies. Ftty ids rete reltively poor hydrophoi rriers to wter diffusion through oth nturl nd rtifiil utiulr memrnes. In ontrst, lknes represent n effetive wter permeility rrier [48, 49]. The tivtion y wter stress of iohemil pthwys providing omponents for the iosynthesis of lknes hs een demonstrted for numer of plnt speies [12, 5, 51]. Growing whet plnts under onditions of limited wter did not influene the morphology of wx rystlloids on the surfes of flg leves, lthough n inrese in the density of wxes ws oserved (Fig. 8). However, signifint inrese of utile thikness ws seen for two of the five lines, Drysdle nd RAC875 (Fig. 3). In ddition to the highest drought-indued inreses in utile thikness, these two lines lso demonstrted the most signifint inreses in totl wx lods (48.5% nd 31.5%, respetively) during plnt growth under drought (Fig. 6 nd [16]). In ontrst, we oserved disrepny etween these two prmeters for the utile of flg leves of Exliur (28% inrese in mount of wxes ut derese in utile thikness on the xil side of the lef) nd Gldius (6.8% derese in wx lod versus n inrese in utile thikness on the xil side of the lef). These pprent inonsistenies ould reflet different responses in the xil versus dxil wx deposition, or the lk of regultion of wx deposition in response to environmentl stress. Enhnement of lef utiulr wx prodution nd inreses in utile thikness pper to represent prevlent response to wter defiit ross the terrestril

10 Bi et l. BMC Plnt Biology (217) 17:85 Pge 1 of EXC- EXC- S- S- GL- GL Primry lohols d EXC- EXC- S- S- GL- GL- Alknes β-diketones Ftty ids Aldehydes Resorinols Fig. 6 Wx mounts nd omposition on flg leves of whet lines grown under well-wtered () onditions nd drought (). Totl wx lods. Contents of eh wx speies. EXC - Exliur, S - Drysdle, GL - Gldius. Mens nd stndrd errors (indited y rs) were lulted from three replites. Different smll letters on top of error rs indite signifint differenes t P <.5. Wx lods were lulted per grm of dry lef weight (DW) EXC- EXC- S- S- GL- GL Primry lohol hin length Alkne hin length OH -Diketone hin length Fig. 7 Cron hin length distriution of mjor utiulr wx lsses on flg leves of whet lines grown under well-wtered () onditions nd drought (). Cron hin lengths of primry lohols. Cron hin length of β-diketones. Cron hin lengths of lknes. EXC - Exliur, S - Drysdle, GL - Gldius. Mens nd stndrd errors (indited y rs) were lulted from three replites. Wx lods were lulted per grm of dry lef weight (DW). Tiny mounts of 2-, 22- nd 34-ron primry lohols, nd 23-ron lkne re not shown ut used for lultion of totl wx lods in Fig. 6

11 Bi et l. BMC Plnt Biology (217) 17:85 Pge 11 of 13 Fig. 8 SEM imges of xil flg lef surfes of v. Drysdle grown under well-wtered nd drought onditions t 15X mgnifition (left olumn) nd 8X mgnifition (right olumn) plnt kingdom [12]. For exmple, 75% inrese in totl wx mount per unit of lef re nd 49% inrese in utile thikness were reported in Aridopsis under wter defiit [49], nd, on leves of tree too, drought indued over 15% inrese in totl wx umultion [52]. The hnges in Aridopsis utile were ssoited with deresed utile permeility mesured s redutions in wter loss nd hlorophyll lehing rtes of dethed leves [49]. In ddition, elevted utile memrne thikness in the Aridopsis er9 (eeriferum 9) mutnt orrelted with lower trnspirtion rtes nd improved wter use effiieny [53]. Synthesis of lrger utin frmework ws proposed s mehnism of drought limtistion for Aridopsis [49], wheres the mjor wter permeility rrier of tomto fruit utile ws found to e intrutiulr rther thn epiutiulr wx [26]. Both utile thikness nd stomtl density ppered to e relevnt ontriutors to the drought tolerne of four Europen winter whet ultivrs with ontrsting ehviour under limited wter supply, with the most drought tolernt ultivr demonstrting the lowest stomtl density on oth flg lef surfes [54]. In our work, however, the drought-sensitive ultivr Kukri, whih in lef desition tests hd the highest wter loss rtes ompred to the other four whet lines, hd the lowest stomtl density on oth lef surfes (Figs. 1 nd 2). Our findings were onsistent with those of Shhinni et l. [55] who investigted stomtl trits in the prents nd geneti mpping popultion from ross etween RAC875 nd Kukri. They found tht RAC875 hd higher density of smller stomt thn Kukri, so tht the totl pore surfe per unit lef re did not differ etween these two genotypes. Together with o-loted quntittive trit loi for stomtl trits nd yield in this popultion, their results suggested tht the stomtl density-size reltionship ontriuted to the higher yield under drought of RAC875 ompred with Kukri. Tht is, onsistent wter use due to the higher density ut smller stomtl pore perture might e enefiil in wter-limited environments. In our experiments, ll other lines hd higher stomtl density thn Kukri ut the totl pore re per unit of lef ws not mesured. Nonetheless, drought tretment inresed stomtl density in Kukri, Exliur, Drysdle nd Gldius, ut not in RAC875. Xu nd Zhou [56] found tht moderte wter defiits inresed, while more severe wter defiits redued, stomtl density on leves of perennil grss, Leymus hinensis. Their results lso indited tht n inrese in lef stomtl density ws positively ssoited with lef level wter use effiieny. When ssessed over the entire desition period, inluding oth stomtl (first 2 hours) nd residul trnspirtion (therefter), the differene etween ontrol nd droughttreted leves ws not signifint for the ultivr Exliur. In ontrst, Exliur showed the gretest differene in lef stomtl density with tretment. Although trnspirtion effiieny ws not diretly mesured in our experiments, there ws no indition tht differenes in stomtl density under drought explined differenes in wter loss rtes in these lines. Overll, these results suggested tht gluousness ws defined y β-diketone levels, ut tht its preise vlue for drought tolerne remins unresolved. The smll ut ovious hnges in RTRs of whet lines under drought, espeilly in RTRs of Drysdle nd RAC875, orresponded well with their levels of drought-indued wx umultion nd thikening of utile lyers. However, there ws no similr reltionship found etween stomtl density nd wter loss for the exmined lines, nd no meningful hnges ourred in stomtl numers during plnt growth under drought.

12 Bi et l. BMC Plnt Biology (217) 17:85 Pge 12 of 13 Conlusions The sis for this study ws to develop n understnding of utile struture nd deposition of whet in reltion to drought tolerne sine there is some evidene, nd resonle physiologil expettion, tht the utile will ply n importnt role in regulting wter loss. Although vrying levels of gluousness re found in modern whet vrieties nd gluousness is n esy visul trit to sore, few reeders would regrd it s n importnt seletion trget. Overll, our results show tht onsiderle vrition exists etween genotypes in the omposition nd struture of the utile. The reltionship etween the utile nd drought tolerne is not simple nd gluousness is not unique inditor of tolerne. Rther, speifi wx omposition, in prtiulr the presene of signifint proportions of C31 β-diketone, dptive hnges in omposition nd mount indued y exposure to drought nd wx rystl struture will ll influene lef wter loss under onditions of wter defiit. These fetures of utile omposition nd deposition pper importnt nd should provide useful seletion riteri lthough they re more diffiult to mesure thn gluousness. Additionl files Additionl file 1: Figure S1. Soil wter tension monitored for well-wtered nd drought onditions. Soil wter tension t 1 m nd 3 m depths were shown. (PDF 37 k) Additionl file 2: Tle S1. Results of sttistil nlyses for wter loss rtes test in Fig. 1. (PDF 11 k) Additionl file 3: Figure S2. Snning eletron mirogrphs of epiutiulr wxes on the dxil side of flg leves in five whet lines. (A) Kukri; (B) Exliur; (C) Drysdle; (D) RAC875 nd (E) Gldius. Sle rs represent 5 μm. (PDF 194 k) Additionl file 4: Sttistil nlyses for utiulr wxes within eh of mjor wx speies in Fig. 7. (PDF 68 k) Additionl file 5: Figure S3. Cron hin length distriution of minor utiulr wx speies on flg leves of whet lines grown under wellwtered () ondition nd drought (). (A) Cron hin length of ftty ids. (B) Cron hin length of ldehydes. (C) Cron hin length of resorinols. EXC - Exliur, S - Drysdle, GL - Gldius. Mens nd stndrd errors (indited y r) were lulted from three replites. Wx lods were lulted per grm of dry lef weight (DW). (PDF 96 k) Arevitions ALC: Primry lohol; ALK: Alkne; : Drought; S: Drysdle; EXC: Exliur; FA: Ftty id; GC-MS: Gs hromtogrphy-mss spetrometry; GL: Gldius; IS: Internl stndrd; KUK: Kukri; RAC: RAC875; RTR: Residul trnspirtion rte; SEM: Snning eletron mirosopy; TEM: Trnsmission eletron mirosopy; VLCFA: Very long hin ftty id; : Well-wtered; : drought. Aknowledgements We thnk Gwend Myo for suggestions nd help with eletron mirosopi nlysis, Zhihong Song nd M. Ann Perer (W.M.Kek Metolomis Reserh Lortory, Iow Stte University) for the help with nlysis of wx omposition nd Yuriy Onyskiv for tehnil support in the glsshouse. Funding The Chin Sholrship Counil nd the University of Adelide provided HB with joint postgrdute sholrship. PT s ontriution ws supported y the Austrlin Reserh Counil Industril Trnsforming Reserh Hu for Geneti diversity nd moleulr reeding for whet in hot nd dry limte (projet numer IH13227). Experimentl work ws supported y the Austrlin Centre for Plnt Funtionl Genomis, funded y the Austrlin Reserh Counil, the Grins Reserh & Development Corportion nd the Government of South Austrli. Avilility of dt nd mterils The dtsets supporting the onlusions of this rtile re inluded within the rtile nd its dditionl files, 1, 2, 3, 4 nd 5. Authors ontriutions NB oneived the ide, designed nd supervised the experiments, nlysed nd disussed dt, nd edited the mnusript. HB performed most of the experiments, nlysed dt nd wrote the mnusript with the ontriution of other uthors. NK performed stomtl density nlyses nd reviewed the mnusript. PL, PT nd SL supervised experiments nd nlyses of dt, nd edited the mnusript. All uthors red nd pproved the finl mnusript. Competing interests The uthors delre tht they hve no ompeting interests. Consent for pulition Not pplile. Ethis pprovl nd onsent to prtiipte Not pplile. Pulisher s Note Springer Nture remins neutrl with regrd to jurisditionl lims in pulished mps nd institutionl ffilitions. Author detils 1 Austrlin Centre for Plnt Funtionl Genomis, PMB1 Glen Osmond, Adelide, South Austrli 564, Austrli. 2 Shool of Agriulture, Food nd Wine, University of Adelide, PMB1 Glen Osmond, Adelide, South Austrli 564, Austrli. 3 Present ddress: Shool of Life Sienes, Huiyin Norml University, Huin 2233, Chin. Reeived: 17 Novemer 216 Aepted: 2 April 217 Referenes 1. Loell DB, Gourdji SM. The influene of limte hnge on glol rop produtivity. Plnt Physiol. 212;16: Lngridge P, Reynolds MP. Genomi tools to ssist reeding for drought tolerne. Curr Opin Biotehnol. 215;32: Jvelle M, Vernoud V, Rogowsky PM, Ingrm GC. Epidermis: the formtion nd funtions of fundmentl plnt tissue. New Phytol. 211;189: Jetter R, Kunst L, Smuels AL. Composition of plnt utiulr wxes. In: Riederer M, Muller C, editors. Annul Plnt Reviews Volume 23: Biology of the Plnt Cutile. Oxford: Blkwell Pulishing Ltd; 27. p Smuels L, Kunst L, Jetter R. Seling plnt surfes: utiulr wx formtion y epiderml ells. Annu Rev Plnt Biol. 28;59: Borisjuk N, Hrmov M, Lopto S. Trnsriptionl regultion of utile iosynthesis. Biotehnol Adv. 214;32: Lee SB, Suh MC. Advnes in the understnding of utiulr wxes in Aridopsis thlin nd rop speies. Plnt Cell Rep. 215;34: Yets TH, Rose JK. The formtion nd funtion of plnt utiles. Plnt Physiol. 213;163: Delude C, Moussu S, Jouès J, Ingrm G, Domergue F. Plnt surfe lipids nd epidermis development. In: Nkmur Y, Li-Beisson Y, editors. Lipids in plnt nd lge development. Switzerlnd: Springer Interntionl Pulishing; 216. p Hen-Avivi S, Svin O, Rovit R, Lee WS, Admki N, Mlitsky S, Almekis- Siegl E, Levy M, Vutrin S, Berges H, et l. 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