Elevated CO2 affects plant responses to variation in boron availability

Transcription

1 University of Nersk - Linoln DigitlCommons@University of Nersk - Linoln Pulitions from USDA-ARS / UNL Fulty U.S. Deprtment of Agriulture: Agriulturl Reserh Servie, Linoln, Nersk 212 Elevted CO2 ffets plnt responses to vrition in oron vilility Ssmit Mishr University of Toledo, ssmit.mishr@utoledo.edu Sott A. Hekthorn University of Toledo, sott.hekthorn@utoledo.edu Jonthn M. Frntz University of Toledo Follow this nd dditionl works t: Mishr, Ssmit; Hekthorn, Sott A.; nd Frntz, Jonthn M., "Elevted CO2 ffets plnt responses to vrition in oron vilility" (212). Pulitions from USDA-ARS / UNL Fulty This Artile is rought to you for free nd open ess y the U.S. Deprtment of Agriulture: Agriulturl Reserh Servie, Linoln, Nersk t DigitlCommons@University of Nersk - Linoln. It hs een epted for inlusion in Pulitions from USDA-ARS / UNL Fulty y n uthorized dministrtor of DigitlCommons@University of Nersk - Linoln.

2 Plnt Soil (212) 35: DOI 1.17/s REGULAR ARTICLE Elevted CO 2 ffets plnt responses to vrition in oron vilility Ssmit Mishr & Sott A. Hekthorn & Jonthn M. Frntz Reeived: 24 My 211 /Aepted: 24 June 211 /Pulished online: 12 July 211 # Springer Siene+Business Medi B.V. 211 Astrt Aim Effets of elevted CO 2 on N reltions re well studied, ut effets on other nutrients, espeilly mironutrients, re not. We investigted effets of elevted CO 2 on response to vrition in oron (B) vilility in three unrelted speies: seed gernium (Pelrgonium x hortorum), rley (Hordeum vulgre), nd wter fern (Azoll rolinin). Methods Plnts were grown t two levels of CO 2 (37, 7 ppm) nd low, medium, nd high B. Tretment effets were mesured on iomss, net photosynthesis (P n ) nd relted vriles, tissue nutrient onentrtions, nd B trnsporter protein BOR1. Results In gernium, there were intertive effets (P<.5) of B nd CO 2 on lef, stem, nd totl plnt mss, root:shoot rtio, lef [B], B uptke rte, root [Zn], nd P n. Elevted CO 2 stimulted growth t 45 μm B, ut deresed it t 45 μm B nd did not ffet it t 4.5 μm B.P n ws stimulted y elevted CO 2 only t 45 μm B nd hlorophyll ws enhned only t 45 μm B. Solule sugrs inresed with high CO 2 Responsile Editor: Roert Reid. S. Mishr (*) : S. A. Hekthorn Deprtment of Environmentl Sienes, University of Toledo, Toledo, OH 4366, USA e-mil: ssmit.mishr@utoledo.edu J. M. Frntz USDA-ARS, University of Toledo, Toledo, OH 4366, USA only t 4.5 nd 45 μm B.HighCO 2 deresed lef [B] nd B uptke rte, espeilly t 45 μm B. Though CO 2 nd B individully ffeted the onentrtion of severl other nutrients, B x CO 2 intertions were evident only for Zn in roots, wherein [Zn] deresed under elevted CO 2. Intertive effets of B nd CO 2 on growth were onfirmed in (1) rley grown t, 3, or 1, μm B, wherein growth t high CO 2 ws stimulted more t 3 μm B, nd (2) Azoll grown t, 1, nd 1, μm B, wherein growth t high CO 2 ws stimulted t nd 1 μm B. Conlusion Thus, low nd high B oth my limit growth stimultion under elevted vs. urrent [CO 2 ], nd B defiieny nd toxiity, lredy ommon, my inrese in the future. Keywords Azoll. Brley. Boron stress. Boron trnsporter protein (BOR1). Gernium. Nutrients. Photosynthesis Introdution The growth of most plnts is enhned t elevted, reltive to urrent, levels of tmospheri CO 2, nd this enhned growth results in greter demnd for minerl nutrients (e.g., Cmpell nd Sge 22; Hgedorn et l. 22). If nutrient vilility or plnt uptke does not inrese to meet this enhned nutrient demnd, then dereses in the onentrtions of nutrients will our in t lest some tissues of plnts grown t elevted CO 2. Consistent with these

3 118 Plnt Soil (212) 35: expettions, severl pst studies hve reported tht high-co 2 -stimultion of plnts under nutrient-defiient onditions is less, or even sent, when ompred to nutrient-suffiient onditions (Cure et l. 1988; Colemn et l. 1993; Zisk23). Beuse elevted CO 2 is expeted to lter plnt tissue nutrient onentrtions, mny studies hve exmined effets of elevted CO 2 on nutrient reltions, ut most previous studies hve foused on mro-nutrients, espeilly N, (e.g., Ehleringer et l. 22; Ellsworth et l. 24; Siher25; Tngetl.26; Tund Wng 28 nd referenes therein), nd only few hve exmined CO 2 effets on miro-nutrient reltions (Nory et l. 1986; O Neill et l. 1987; Mndersheid et l. 1995; Fngmeier et l. 1997; Peñuels et l. 1997, 21; Prior et l. 1998; Blnk nd Derner 24; Pl et l. 24; Luoml et l. 25; Jin et l. 29). In generl, growth of plnts under elevted (vs. urrent) CO 2 typilly dereses the onentrtion of N, espeilly in leves, due lrgely to delines in ruiso (riulose 1,5-isphosphte roxylse/ oxygense) levels in leves, nd high-co 2 dereses re usully greter in C 3 thn C 4 speies (e.g.,. 2 vs. 5% dereses respetively for lef %N; Ehleringer et l. 22). Other mro-nutrients lso often derese in onentrtion with growth in elevted CO 2,though responses n differ mong nutrients within speies (Tle 1, for exmples). Though few studies to dte hve exmined effets of elevted CO 2 on tissue mironutrient onentrtions, the limited results from these studies, summrized in Tle 1, indite (1) tht CO 2 effets will e vrile mong speies, tissues, nd miro-nutrients; (2) tht high CO 2 will often derese miro-nutrient onentrtions; nd (3) dereses in miro- (nd mro-) nutrient onentrtions my e more prevlent in seeds ompred to leves. For exmple, the responses mong relted speies (rley vs. whet), nd mong ultivrs within speies (within rley nd whet seprtely), to mironutrient stress nd elevted CO 2 differed (Mndersheid et l. 1995). There is evidene tht elevted CO 2 hs intertive effets with other spets of nutrition, though this hs not often een exmined. For exmple, in n intertive-effet study in whet, Fngmeier et l. (1997) oserved omplex intertion mong CO 2, nitrogen vilility, nd ozone in spring whet. Similrly, Colemn et l. (1993) oserved CO 2 x N effets in Autilon theophrsti nd Amrnthus retroflexus tht were often medited y effets on development. Hgedorn et l. (22) found tht soil fertility nd CO 2 my hve intertive effets nd these intertions my e speies dependent. Speifilly, on n idi lom soil, CO 2 enrihment suppressed net umultion (totl ontent in iomss) of nine (of 11) investigted minerl nutrients in eeh trees (signifint only for P, S, Zn), ut stimulted it for 1 of 11 nutrients in sprue trees (signifint only for Fe, Zn); in ontrst, on nutrientrih lreous snd, inresed tmospheri CO 2 enhned nutrient umultion in oth speies signifintly (Hgedorn et l. 22). Similrly, Blnk nd Derner (24) oserved intertive effets etween soil fertility (low- nd high-fertility soils) nd CO 2 on vrious spets of plnt nd soil properties in Lepidium ltifolium, inluding effets on plnt nutrient onentrtions tht vried mong the nutrients exmined. To our knowledge, only one previous study hs exmined intertive effets of CO 2 nd mironutrients on plnt growth nd funtion: i.e., Jin et l. (29) exmined intertive effets of CO 2 nd Fe speies (FeEDTA vs. Fe(III) oxide) on tomto, nd found tht the omintion of elevted CO 2 nd low Fe inresed Fe uptke ility, nd tht CO 2 ffeted [Fe] only with Fe oxide. Notly, we n find only six previous studies wherein effets of elevted CO 2 on tissue B onentrtions were investigted (Tle 1; Nory et l. 1986; O Neill et l. 1987; Peñuels et l. 1997, 21; Luoml et l. 25; Liu et l. 27). As hne would hve it, eh of these studies (four reports on trees nd two on shrus) exmined CO 2 -responses in plnts grown in soil hrterized y the uthors s nutrient poor or low in N, nd B soil vilility ws undetermined nd un-mnipulted in these studies. The puity of previous reserh on B x CO 2 effets is striking, sine (1) mong ll essentil plnt nutrients, it is thought tht B hs perhps the nrrowest rnge of tissue onentrtions over whih B levels re dequte nd not stressful (i.e., not limiting or toxi) (e.g., Mrshner 1995), nd (2) B stress is ommon nd eonomilly importnt in griulture world-wide (Shorroks 1997). Though B requirements, s well s thresholds for B defiieny nd toxiity, vry signifintly mong speies nd tegories (e.g., grsses vs. diots; Blevins nd Lukszewski 1998), ville B levels elow. 2 5 μm usully use B defiieny (e.g., El-Shintinwy 1999; Wimmer et l. 25) nd levels ove 1, μm typilly indue toxiity

4 Plnt Soil (212) 35: Tle 1 Summry of pst studies exmining effets of elevted (reltive to urrent) CO 2 on the onentrtion of miro-nutrients Speies Tissue [Nutrient] inrese [Nutrient] derese [Nutrient] no hnge Soure Hereous Hordeum vulgre Leves S N C,Fe,K,Mg,Mn,P,Zn Mndersheid et l Seeds K Fe,N,S,Zn C,Mg,Mn,P Mndersheid et l Tritium estivum Leves P,S K,Mg C,Fe,Mn,N,Zn Mndersheid et l Seeds K C,Fe,Mg,Mn,N,S,Zn P Mndersheid et l Tritium estivum Leves C,K,Mg,Mn,N,P,S,Zn Fe Fngmeier et l Seeds C,Fe,K,Mg,Mn,N,P,S,Zn Fngmeier et l Gossypium hirsutum Leves C,Fe,K,Mg,Mn,N,P,S,Zn Prior et l Seeds Cu,Fe,K,N,Zn C,Mg,Mn,P Prior et l Lepidium ltifolium Shoots Mg C,Fe,K,Mg,Mn,N,P,S Blnk nd Derner 24 Trifolium Leves P N C,Fe Pl et l. 24 lexndrium Lyopersion Shoots Fe (with Fe(III) Fe (with FeEDTA) Jin et l. 29 esulentum oxide) Roots Fe (with Fe(III) Fe (with FeEDTA) oxide) Shrus Citrus urntium Leves B C,N,Mg,Mn Cu,Fe,K,N,P,S,Zn Peñuels et l Eri rore Leves K,S B,B,Sr Al,C,Cd,Co,Cr,Cu,Fe,Mg, Peñuels et l. 21 Mn,Mo,N,N,Ni,P,P,Si, Ti,V,Zn Myrtus ommunis Leves Mg,Mn,S B,B,N,Sr Al,C,Cd,Co,Cr,Cu,Fe,K, Peñuels et l. 21 Mo,N,Ni,P,P,Si,Ti,V,Zn Juniperus ommunis Leves Al,C,Fe,K,Mg,Mn, B,Co B,Cd,Cr,Cu,Mo,N,N, Peñuels et l. 21 S,Ti Ni,P,P,Si,Sr,V,Zn Trees Querus l Wholeseedling Fe B,C,Mg,Mn,N,S,Zn Al,Cu,K,P Nory et l Liriodendron Wholeseedling B,N,S Al,B,C,Cu,Fe,K,Mg, O Neill et l tulipifer Mn,P,Sr,Zn Pinus sylvestris Leves Mn Cu,N,P,S B,C,Fe,K,Mg, Luoml et l. 25 Populus tremul Leves K,P N,B C,S,Mg,Mn,Cu,Fe,Zn Liu et l. 27 Betul ppyrifer Leves K,P N,B C,S,Mg,Mn,Cu,Fe,Zn Liu et l. 27 (though levels s low s 2 μm hve een reported to e stressful in some speies) (Reid et l. 24). To dte, the most-widely-epted role of B in plnts is tht of struturl funtion in plnt ell wlls (Brown et l. 22; Goldh 1997; Koyshi et l. 1996; Mtoh 1997; Power nd Woods 1997). This struturl role of B in ell wlls is due to its pity to form diester ridges etween djent is-hydroxyl ontining moleules, suh s mono-, oligo-, nd polyshrides, nd diols nd hydroxyids (Power nd Woods, 1997). B lso is involved in plnt reprodution, whih my or my not e relted solely to the struturl role of B in ell wlls (Blevins nd Lukszewski, 1998; Mrshner 1995). Other speifi funtions of B hve een postulted s well (Blevins nd Lukszewski 1998; Bolños et l. 24; Dords nd Brown 2), nd oron defiieny n ffet severl metoli proesses; e.g., ell division nd elongtion, metolism of nulei ids, protein synthesis, metolism nd trnsport of rohydrtes,

5 12 Plnt Soil (212) 35: synthesis nd metolism of phenolis, nd photosynthesis (Blevins nd Lukszewski 1998; Goldh 1997; Kouhi 1977; Mishr et l. 29). Reently, the first two B-trnsport memrne proteins hve een identified nd hrterized: one involved in tive trnsport, BOR1, nd one involved in filitted diffusion, the NOD26-like intrinsi protein (NIP), NIP5;1 (Miw et l. 29). BOR1 is B efflux trnsporter expressed in roots nd leves nd is up-regulted under B-defiieny onditions (Tkno et l. 22). The hnnel protein NIP5;1 is ruil for B uptke in plnts under B limittion (Tkno et l. 26). BOR1 inreses B supply to the shoots y loding B from the xylem prenhym into the xylem (Tkno et l. 22). Under toxi onentrtions of oron, BOR1 is degrded vi endoytosis (Tkno et l. 25). Under elevted CO 2, one might expet tht expression levels of BOR1 nd/or NIP5 proteins in roots will hnge, if elevted CO 2 is ltering nutrient demnd. The present study imed to investigte effets of elevted CO 2 on growth, photosynthesis, nd nutrient (espeilly B) reltions in gernium (Pelrgonium hortorum v. Mverik White; diot) plnts grown for 3 dys while supplied with one of three different B onentrtions, rnging from potentilly su-optiml (4.5 μm) to ner-optiml (45 μm) to potentilly suproptiml (45 μm). We tested the priori hypothesis tht elevted CO 2 would (1) exerte B defiieny t low levels of B vilility, nd (2) derese B toxiity t high levels of B; in oth ses, y enhning plnt growth nd thus inresing the dilution of B in tissues. To determine if the CO 2 x B effets oserved in gernium re ommon in other speies, we exmined effets of CO 2 in (1) rley (Hordeum vulgre; monoot) grown t 3 μm B nd trnsferred to, 3 or 1, μm Bnd(2)wterfern(Azoll rolinin) grown t 1 μm B nd trnsferred to, 1, or 1, μm B. For these ltter two speies, we expnded the rnge of B levels to inrese the severity of B defiieny or toxiity. Mterils nd methods Plnt mteril nd B nd CO 2 tretments Seeds of gernium plnts (Pelrgonium x hortorum v. Mverik White) were sown into fom ues (15-mm 15-mm 3-mm eh; LC1-type, Smithers- Osis North Ameri, Kent, Ohio) nd irrigted with omplete fertilizer solution (Hoglnd s). After 4 dys, when seedlings hd 3-to-4 true leves, seedlings were trnsferred to opque 4 L plsti tus filled to volume with omplete Hoglnd s solution, s in Mishr et l. (29). The tus hd opque lids with two evenly-sped 2-m-dimeter holes through whih seedlings were suspended (y wrpping with thin strips of fom round the root-shoot interfe), suh tht the root system ws enlosed in the tus nd shoots were ove the level of the tu lids. Seedlings were then llowed to limte for 1 dys to minimize trnsplnt stress. Plnts were germinted nd grown in greenhouse, under 2 28 C temperture rnge nd with supplementl light to extend the photoperiod to 14 h (6 m 8 pm; minimum PAR=2 μmol m 2 s 1 ). For B nd CO 2 tretments, plnts were trnsferred to (otherwise) omplete nutrient solutions ontining one of three levels of B (4.5, 45, or 45 μm, sed on results in Mishr et l. 29; three replite tus per B level), nd then 9 tus ontining 2 plnts per tu were kept under two different onentrtions of CO 2 (thus 18 tus totl) in ontrolled-environment hmers [one t 37±2 ppm (mient) nd one t 7±2 ppm (elevted) CO 2 ]. Eh tu ontined two plnts, nd these two plnts were verged to generte the vlue for the tu, with men tu vlues eing the experimentl replites. Nutrient solutions, heked regulrly nd mintined t ph 5.6 with ddition of 1 N HCl or KOH, were hnged weekly, whih ws determined in preliminry experiments to e frequent enough to prevent depletion of nutrients. Eh tu ws erted y onstnt uling of nutrient solution to mke it homogeneous. Plnts were grown t 23 C dy/19 C night with unontrolled humidity (typilly>5%), under 16-h photoperiod, nd t light intensity of 3 μmol m 2 s 1 PAR (photosynthetilly tive rdition), whih provided mol m 2 d 1 of PAR. This light level is optiml for this gernium ultivr (Mishr et l. 29). Light levels were monitored twie weekly with line quntum sensor (model LQSV-E, Apogee Instruments, In. Logn, Uth); hmer CO 2 nd temperture levels were monitored severl times dy with lirted nd independent sensors; plnts were rotted within hmers every other dy. Plnts were grown in the ove growth onditions for 3 d, during whih time, plnt iomss inresed in ll tretments.

6 Plnt Soil (212) 35: To onfirm B x CO 2 effets on iomss oserved in the ove experiment, we onduted two dditionl experiments, wherein we grew rley (Hordium vulgre) nd quti fern (Azoll rolinin), respetively. Bsed on results from the gernium experiment, in rley nd A. rolinin, the severity of oth low- nd high-b stress ws inresed y deresing [B] in the low-b tretment nd inresing [B] in the high-b tretment. Brley plnts were grown hydroponilly s ove under three onentrtions of B (, 3 nd 1, μm) nd two levels of CO 2 (37 nd 7 ppm). Seeds were sown into soil, nd fter 15 d, when seedlings hd three to four true leves, they were rinsed to remove soil on roots nd trnsferred to hydroponi tus ontining omplete nutrient solution (inluding 3 μm B) nd llowed to limte for 7 d to minimize trnsplnt stress. At this time, susets of plnts were trnsferred to nutrient solutions ontining or 1, μm B (in otherwise omplete nutrient solution), while ontrol plnts ontinued to reeive 3 μm B. Three replite plnts (in seprte tus) t eh B level were grown under mient (37 ppm) or elevted CO 2 (7 ppm) in growth hmers. Plnts were grown t 25 C dy/ 2 C night, under 16 h photoperiod, nd t light intensity 8 μmol m 2 s 1 PAR. Plnts were kept for 3 dys of tretment prior to hrvest. Nutrient solutions were hnged weekly, nd plnts were rotted within hmers every other dy. For Azoll, plnts were grown for >2 weeks in nutrient solution designed for lge (WC medium: 25 μm CCl 2, 15 μm MgSO 4, 5 μm K 2 HPO 4, 11.7 μm Fe- EDTA,.9 μm MnCl 2,.8 μm ZnSO 4,.5 μm CoCl 2,.4 μm CuSO 4, 1 μm H 3 BO 3, nd.37 μm (NH 4 ) 6 Mo 7 O 24 ). Plnts were trnsferred to plsti-tus (6 ml) with the ove nutrient solution nd one of three B onentrtions (, 1, or 1, μm). Four replites of eh B tretment (=12 tus) were kept under mient CO 2 (37 ppm) nd nother 12 tus under elevted CO 2 (7 ppm) in ontrolled-environment hmers. Prior to hrvest, plnts were grown for 1 dys t 25 C/2 C (dy/ night), under 16 h photoperiod nd 2 μmol m 2 s 1 PAR light intensity. Growth nd nutrient nlysis Entire plnts were hrvested nd then immeditely seprted into roots, stems, nd leves for gernium nd roots nd shoots for rley; intt plnts were nlyzed for Azoll rolinin. Tissues were oven dried t 7 C for 72 h (to onstnt mss) nd then weighed. To determine tissue nutrient ontent, we followed our previously reported method (Mishr et l. 29). Briefly, ll hrvested tissues were rinsed with.1 N HCl, rinsed gin with distilled wter, nd then oven dried in fored-ir oven t 55 C for 72 h. Tissue ws ground y mortr nd pestle into powder nd.15 g ws digested in mirowve digester (MARS Express II, CEM Corp., Mtthews, North Crolin), using modified EPA method (EPA method 351, Nelson 1988; HNO 3 digestion t 2 C with n dditionl peroxide digestion step). Nutrient onentrtion (B, C, Cu, Fe, K, Mg, Mn, P, S, Zn) ws determined with indutively-oupled-plsm optilemission spetrosopy (ICP-OES; Model IRIS Intrepid II, Thermo Corp., Wlthm, MA). Photosynthesis Stedy-stte net photosynthesis (P n ;netco 2 exhnge) of reently fully-expnded intt leves of gernium, whih hd developed fter the exposure to experimentl tretments, ws mesured with portle photosynthesis system with n infrred gs nlyzer (model 64, LiCOR, Linoln, Nersk, USA), equipped with 25-mm 3 lef hmer nd CO 2, light, nd temperture ontrol (s in Mishr et l. 29). Mesurements were mde within one min of insertion of leves in to the uvette, nd fter stiliztion of CO 2 nd H 2 O flux, to ensure tht photosyntheti responses refleted those within the growth hmers. Net photosynthesis of plnts ws mesured t the sme CO 2 levels t whih the plnts were growing (either 37 or 7 ppm CO 2 ) t light level of 3 μmol m 2 s 1 PAR. Chlorophyll nd rohydrte ontent Chlorophyll nd rohydrte ontent ws mesured s in Mishr et l. (29). Briefly, hlorophyll ontent (per fresh mss) in leves ws estimted spetrophotometrilly fter extrtion in dimethyl sulfoxide (DMSO), using the equtions of Brnes et l. (1992). Lef smples were inuted t 65 C for 1 h nd then ooled to room temperture in the drk prior to mesurements. Totl solule rohydrte ontent in root tissue ws estimted y using the phenol-sulfuri

7 122 Plnt Soil (212) 35: id method of Duois et l. (1956), with minor modifition. Fresh tissues (5 mg dry mss) were ground in liquid N 2,ndthenmixedwith2mLof.1M phosphte uffer (ph 7.2) nd re-ground. The homogente ws entrifuged t 21, g, nd then 1 ml of superntnt ws tken nd mixed with 1 ml of 5% queous phenol. Conentrted sulfuri id (5 ml) ws dded, nd sorne t 47 nm ws determined fter 2 min. Gluose ws used for generting stndrd urve. BOR-1 Protein nlysis Totl ell protein ws extrted from frozen root tissues (4 mg fresh weight) y grinding in liquid N 2 in mortr nd pestle, nd then in n extrtion uffer ontining [.5M Tris HCl (ph 8.), 5 mm EDTA,.1 M KCl,.9 M surose nd 2% β merptoethnol]. The homogentes were trnsferred to 15 ml tue nd the sme volume of Tris-uffered phenol (ph 8.) ws dded. After inuting for 1 min on shker t room temperture, smples were entrifuged t 5,5g for 2 min t 4 C to seprte the queous nd orgni phse. The upper phenoli phse ws reovered nd trnsferred to fresh tue. This phenol phse ws wshed with n equl volume of extrtion uffer nd then entrifuged t 5,5g for 2 min t 4 C. The protein-ontining phse ws trnsferred to fresh tue nd preipitted with 5 volumes of.1 M mmonium ette in 1% (v/v) methnol nd inuted overnight t 2 C. The preipitte ws wshed three times with.1 M mmonium ette in 1% methnol followed y three times with 8% etone, nd finl time with 1% etone. The finl protein pellet ws resuspended in smple uffer (Tris HCl ph 6.8, 2% SDS,.5% β-merptoethnol nd glyerol). The totl protein onentrtion of eh smple ws determined in triplite y the Coomssie-dye-inding method of Ghosh et l. (1988), using ovine serum lumin s stndrd. The olorimetri density of protein in smple spots on filter-pper diss ws determined using desktop snner nd densitometry nlysis, using Ntionl-Institutes-of-Helth imging softwre (Sion, Ntionl Institutes of Helth, Bethesd, MD). Proteins were then seprted y 1D SDS-PAGE, trnsferred to nitroellulose y eletro (western) lotting, nd sujeted to immuno-detetion ndquntifitionsinymishretl.(28). BOR1 protein ws deteted using rit polylonl ntiserum generted ginst onserved peptide (GDYPLSATIMSEYANKKTRG) identified from BOR1 mino-id sequenes ville from puli dtses nd the BOR1 sequene identified in gernium (Deng 29). Sttistil nlysis Results were nlyzed sttistilly y two-wy (B x CO 2 ) nlysis-of-vrine (ANOVA), with B nd CO 2 levels s fixed ftors, using JMP softwre (SAS Corp, Cry, NC). Tretment effets were onsidered signifint if P<.5 nd mrginlly signifint if P <.1. Following signifint min-ftor effets y ANOVA, Tukey s test ws used to determine signifint differenes mong tretment levels mong min ftors. Results Though gernium plnts were mesured nd hrvested fter 3 dys of experimentl tretments, tretment effets were visile sooner (not shown). For exmple, fter 2 dys, mild hlorosis ws oserved in leves of plnts grown under low nd high [B] in elevted CO 2 (mostly in the youngest leves t low B, nd in the older leves with high B). However no distint tretment effets were oserved in roots, exept tht they ppered more-rnhed t high B nd high CO 2. There were signifint B x CO 2 intertive effets on dry mss of lef, stem, nd totl mss (similr trend ut non-signifint effets on flower mss) (Fig.1 d; Tle 2). For exmple, pek iomss of leves, stems nd whole-plnts ws oserved t 45 μm t mient CO 2, ut t 45 μm B t elevted CO 2. When ompring effets of elevted vs. mient CO 2 within eh B level, stimultion of growth t high CO 2 ws oserved only t 45 μm B, with no stimultion t 4.5 μm B, nd with deresed iomss with high CO 2 t 45 μm B.NoBx CO 2 effets were oserved for roots, though root mss ws inresed y high CO 2 (Fig. 1e; Tle 2). However, there were intertive effets of B nd CO 2 on root:shoot rtio, nd root:shoot rtio ws inresed y elevted CO 2 (Fig. 1f; Tle 2). As expeted, elevted CO 2 inresed net photosynthesis (P n ), y inresing lef internl CO 2 onentrtion (C i ), ut elevted CO 2 hd no effet on stomtl

8 Plnt Soil (212) 35: Fig. 1 Effet of B (4.5, 45, nd 45 μm) nd CO 2 (mient=37 nd elevted=7 ppm) on iomss of different tissues of gernium. Eh r represents the men (±1SD) of three independent replites. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) Lef dry mss (g) Stem dry mss (g) µm B 45 µm B 45 µm B d e Totl dry mss (g) Root dry mss (g) Flower dry mss (g) Amient Elevted Amient Elevted f.2.1. Root:shoot ondutne (G s ) in gernium (Fig. 2 ; Tle 2). However, when ompring P n in elevted vs. mient CO 2 within eh B level, elevted CO 2 stimulted P n only t 45 μm B.Also,P n ws gretest t 4.5 μm Bt mient CO 2, while B hd no effet on P n t elevted CO 2. Hene, there were intertive effets of B nd CO 2 on P n.nobx CO 2 effets were oserved on totl hlorophyll (Chl tot ), hlorophyll : (Chl :), or solule sugrs (Fig. 2d f; Tle 2). Boron did ffet Chl tot nd Chl :, with mximum Chl tot t 45 μm B, nd with inonsistent effets on Chl :. Solule sugr ontent ws enhned under elevted, ompred to mient, CO 2 oth in lef nd root tissues. As we ntiipted, oth B nd CO 2 hd effets on the onentrtion of B in plnt tissues, nd there ws signifint intertive effet of B nd CO 2 on [B] of lef tissue (Fig. 3,; Tle 2). Lef nd root [B] inresed with inresing B vilility (mrginlly signifint in roots), with lrger inreses in tissue [B] when ompring 45 to 45 μm B thn 45 to 4.5 μm. On verge, ross ll B levels in oth roots nd shoots, elevted CO 2 deresed tissue [B], ut within eh B level individully, this high-co 2 derese ws signifint only in leves t 45 μm B, wherein [B] ws redued y 55% y high CO 2 ; hene, the signifint B x CO 2 intertion in leves. Similr ptterns for [B] were oserved for root-speifi uptke rtes of B (totl g plnt B per g of root); i.e., B uptke rte inresed with B vilility (espeilly t 45 μm B), deresed t high CO 2, nd the high- CO 2 -relted derese ws signifint only t 45 μm B (mient ws 2.5 times tht t elevted CO 2 ), resulting in signifint B x CO 2 effet (Fig. 3; Tle 2). Neither B nor CO 2 hd signifint effets on the reltive ontent of the B trnsporter, BOR1, though elevted CO 2 tended to inrese BOR1 ontent (Fig. 3d; Tle 2). Content of BOR1 remined lmost onstnt in oth CO 2 treted plnts t 45 μm B. Also under elevted CO 2 mong ll the tretments of B, BOR1 delined t 45 μμ. Along with B, the onentrtion of other nutrients (C, Cu, Fe, K, Mg, Mn, P, S, nd Zn) in tissues ws lso mesured in leves nd roots. Beuse effets of elevted CO 2 on nutrient onentrtions hve een shown previously in multiple studies (e.g., Tle 1), we restrit presenttion of results here to nutrients

9 124 Plnt Soil (212) 35: Tle 2 Results from sttistil nlysis (P vlues from ANOVA) of tretment effets of B, CO 2, nd their intertions on vrious response vriles. Gernium plnts were grown t different levels of B (4.5, 45, nd 45 μm) nd CO 2 (37, 7 ppm) Tretment effets Vriles CO 2 B B x CO 2 Biomss: Lef * Stem * Root.11 * Flower Totl * Root:shoot.5 * * P n.1 * * G s C i <.1 * Chlorophyll: Totl.1.7 *.78 Chl /.36 *.16 *.167 Sugr Lef <.1 *.46 *.55 Root.5 * Lef [B].1 * <.1 *.6 * Root [B].82 <.1 *.112 B-uptke rte <.1 *.79 *.43 * BOR * Indites signifint differenes mong tretments t P<.5 tht were ffeted y B or for whih there were signifint B x CO 2 intertions in either leves or roots (Fig. 4 d). However, s in mny pst studies, elevted CO 2 ffeted the onentrtion of nutrients in most instnes here (ll ut P in roots nd shoots, Mg nd S in roots, nd Fe nd Zn in shoots; not shown). B ffeted [P] in leves (deresing [P] t 45 μm B;P=.78), nd oth [Fe] (highest [Fe] t 45 μm B;P=.279)nd[Zn]inroots(highestt 45 μm B in mient CO 2 only; P=.188). Intertive effets of B nd CO 2 were evident only forcuinroots(p=.1); mrginlly-signifint effets were oserved for Zn in roots (P=.655). As with gernium, we oserved B x CO 2 effets on iomss in rley. While elevted CO 2 inresed shoot, root, nd whole-plnt iomss on verge in rley, when ompring elevted vs. mient CO 2 within eh B level, growth ws stimulted signifintly y high CO 2 only t 3 μm B for shoots, roots, nd whole-plnt iomss (Fig. 5 ). In ddition, while shoot, root, nd whole-plnt iomss ws gretest t 3 μm B in elevted CO 2, no deline in iomss ws evident t vs 3 μm B t mient CO 2, nd delines in iomss t 1, vs. 3 μm B were not signifint. A signifint B x CO 2 intertion ws lso oserved for root:shoot iomss in rley, wherein B hd no effet on root:shoot mss t mient CO 2, ut root:shoot mss inresed with inresing B t elevted CO 2 (Fig. 5d). We lso oserved signifint intertive effet of BxCO 2 on dry mss of Azoll rolinin (Fig. 6), nd these effets were similr to those in gernium nd rley. Totl plnt mss in Azoll ws deresed y oth low nd high, reltive to medium, B, nd elevted CO 2 inresed mss. When ompring mss etween elevted nd mient CO 2 within eh B level, iomss ws enhned oth t nd 1 μm B, ut not t 1, μm B. Disussion The present study found tht (1) growth of plnts in elevted, reltive to urrent, tmospheri CO 2 ffeted B reltions, (2) CO 2 nd B hve intertive effets on growth nd funtion, nd (3) elevted CO 2 exerted effets of low B s predited, ut did not minimize effets of high B s expeted; insted, high CO 2 inresed B stress t high levels of B. Regrding effets of elevted CO 2 on B reltions, high CO 2 deresed the onentrtion of B in plnt tissues (espeilly leves), s well s the rte of B uptke y roots, espeilly t high B (45 μm). Though not sttistilly signifint, elevted CO 2 lso tended to inrese the levels of the B trnsport protein, BOR1, t low nd medium B (4.5 nd 45 μm). In gernium, we oserved intertive effets of CO 2 nd B on lef, stem, nd whole-plnt dry mss (flower mss showed similr non-signifint pttern), root:shoot rtio, net photosynthesis, lef [B], B-uptke rte, nd root [Zn]. B x CO 2 effets were lso oserved for shoot, root, nd whole-plnt iomss in rley grown t 3 μm B nd trnsferred to, 3, or 1, μm B, nd for whole-plnt iomss in Azoll grown t 1 μm B nd trnsferred to t, 1 nd 1, μm B. In oth gernium nd rley, sttistillysignifint stimultion of growth y elevted CO 2 ws

10 µ Plnt Soil (212) 35: Fig. 2 Effet of B (4.5, 45, nd 45 μm) nd CO 2 (mient=37 nd elevted=7 ppm) on net photosynthesis (P n ), stomtl ondutne to wter vpor (G s ) internl CO 2 onentrtion (C i ), d totl hlorophyll ontent, e hlorophyll : rtio, nd f solule sugrs of gernium roots. Eh r represents the men (±1SD) of three independent replites. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) G P n ( mol CO 2 m -2 s -1 s (mol H 2 O m -2 s -1 ) µ ) C i ( mol/mol) Amient Elevted 4.5 µm B 45 µm B 45 µm B Amient Elevted d e f Totl hlorophyll ( µ g g -1 ) Chlorophyll / Solule sugrs (mg g -1 ) oserved t medium B levels, ut not t low or high B; in ft, in gernium t high B, elevted CO 2 deresed iomss reltive to mient CO 2 levels. In Azoll, elevted CO 2 stimulted growth nd nd 1 μm B, ut not t 1, μm B. Also, in gernium, pek iomss of leves, stems, nd whole-plnts ws Fig. 3 Effet of B (4.5, 45, nd 45 μm) nd CO 2 (mient=37 nd elevted=7 ppm) in gernium on:, Boron onentrtion in lef nd roots, respetively (dry mss sis), speifi uptke rte of B (totl g plnt B/g dry roots), nd d oron trnsporter protein (Bor1) onentrtion (per unit totl protein, reltive to stndrd). Eh r represents the men (± 1 SD) of three independent replites. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) [B] (mg kg -1 ) [B] (mg kg -1 ) Lef Root Amient Elevted 4.5 µm B 45 µm B 45 µm B Amient Elevted d B-uptke (g B/g root) BOR1 onentrtion

11 126 Plnt Soil (212) 35: Fig. 4 Effet of B (4.5, 45, nd 45 μm) nd CO 2 (mient=37 nd elevted= 7 ppm) on onentrtion (dry mss sis) of seleted nutrients in shoots or roots of gernium. Shown re only those nutrients for whih there were either signifint BorBx CO 2 effets. Eh r represents the men (± 1SD) of three independent replites. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) [ P] mg kg -1 [Zn] mg kg Lef Root Root Root 4.5 µm B 45 µm B 45 µm B [Cu] mg kg -1 [Fe] mg kg -1 Amient Elevted Amient Elevted d 1 oserved t 45 μm tmientco 2, ut t 45 μm B t elevted CO 2. In rley, pek mss ws oserved t nd3μm B in mient CO 2, ut t 3 μm Bin elevted CO 2, while in Azoll, pek mss ws oserved t 1 μm B under oth mient nd elevted CO 2. In gernium, the pttern of B x CO 2 effets on iomss ws not refleted in ny other response vrile mesured, exept for shoot:root iomss (i.e., the inverse pttern for root:shoot results shown), suggesting tht the pttern of B x CO 2 effets on iomss ws likely result of effets on iomss llotion. Notly, the pttern of B x CO 2 effets on iomss ws unrelted to tissue [B]. Hene, though low-b plnts were smller thn medium B plnts t high, ut not low, CO 2, onsistent with our priori predition tht elevted CO 2 would inrese the potentil for B defiieny, this ws not used y simple effets on tissue [B]. In ontrst, we predited priori tht elevted CO 2 would derese the potentil for B toxiity t high B, ut in ft, we oserved the opposite, nd elevted CO 2 used derese in plnt growth t high vs. medium B. Thus, in future high- CO 2 world, B stress my eome more prevlent, t Fig. 5 Effet of B (, 3, nd 1, μm) nd CO 2 (mient=37 nd elevted= 7 ppm) on shoot nd root mss of rley (H. vulgre). Two-week-old seedlings were grown hydroponilly in omplete nutrient solution with 3 μm B,ndthen susets of plnts were trnsferred to, 3, or 1, μm B. Eh r represents the men (± 1 SD) ofthree independent replites. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) Shoot dry mss (g) Root dry mss (g) µm B 3 µm B 1 µm B Amient Elevted Amient Elevted d Totl dry mss (g) Root:shoot

12 Plnt Soil (212) 35: Dry mss (g) µm B 1 µm B 1 µm B Amient Elevted Fig. 6 Effet of B (, 1, nd 1, μm) nd CO 2 (mient= 37 nd elevted=7 ppm) on totl plnt iomss of wter fern (Azoll rolinin). Plnts were grown hydroponilly for mny genertions in omplete nutrient solution with 1 μm B, nd then susets of plnts were trnsferred to, 1, or 1, μm B. Eh r represents the men (± 1 SD) of four independent replites. P vlues from ANOVA for tretments: B=<.1, CO 2 =<.1 nd B x CO 2 =.14. Within eh vrile, different letters ove the rs indite signifint differene mong tretments (P<.5) oth low-b nd high-b stress, though the speifi mehnism for this is not known. The B x CO 2 effets on iomss llotion oserved in this study re similr to those oserved y Siher (25), where the sme trend ws seen in rley roots grown under different P levels nd mient vs. high CO 2. Exposure of plnt nopies to high CO 2 onentrtion often stimultes the growth of oth shoot nd root, ut the question remins whether elevted tmospheri CO 2 onentrtion will ffet roots nd shoots of rop plnts proportionlly. Sine elevted CO 2 n indue hnges in plnt struture nd funtion, there my e differenes in llotion etween root nd shoot, t lest under some onditions (Rogers et l. 1996). It is generlly oserved tht root:shoot rtio responds to defiits in light (Boote 1976), wter (Krmer nd Boyer 1995), nd mjor minerl nutrients (Ckmk et l. 1994; Gutshik 1993), with the root:shoot response to given ftor usully towrds diverting dry weight to the plnt prt tht is the most limiting to growth under previling environmentl onditions (Wilson 1988). However, the effets of elevted tmospheri CO 2 on root-to-shoot re muh less ler (Rogers et l. 1996). The response of root-to-shoot to elevted tmospheri CO 2 is highly vrile mong speies. For exmple, there were signifint inreses in root-to-shoot for soyen (Glyine mx; Rogers et l. 1992) nd in Querus l L. seedlings (Nory et l. 1986) exposed to elevted CO 2, while in otton (Gossypium hirsutum) grown under field onditions, root:shoot mss ppered to e unffeted y CO 2 onentrtion (Prior et l. 1994). Though inreses in photosynthesis nd growth re typil under elevted vs. urrent CO 2 for most C 3 speies, dereses in tissue nutrient onentrtions often our too, nd not just for nitrogen (e.g., Cure nd Aok 1986; Siher nd Bune 1999; Vndermeiren et l. 22; Nory et l. 1986; Roerntz nd Stokfors 1998; Fngmeier et l. 1996; Luomletl.25). The inrese in iomss under elevted CO 2 is lrgely ttriuted to inreses in net photosynthesis nd nutrient limittion hs generlly een found to suppress this response (Conroy 1992; MKee nd Woodwrd, 1994; Lloyd nd Frquhr, 1996; Stitt nd Krpp, 1999). For exmples, when irh (Betul pendul; Pettersson et l. 1993; Silvol nd Ahlholm 1995), lololly pine (Pinus ted; Geuer et l. 1996), rie (Oryz stiv; Zisk et l. 1996), otton (Gossypium hirsutum; Rogers et l. 1993), whet (Tritium estivum; Rogers et l. 1996), nd too (Niotin tum; Geiger et l.1999) were grown t vrious N supplies, elevted CO 2 led to lrge inreses of iomss t the highest N supply, smll inreses t modertely limiting N supply, nd no inrese, or even slight derese, t the lowest N supply. Therefore, nutrient supply nd, onsequently, the nutrient sttus of plnts should e ritil ftor determining growth responses to the elevted CO 2. In this study, growth t elevted CO 2 led to lower tissue B onentrtions in gernium, though this ws sttistilly signifint only in leves t the highest B level, nd to dereses in B uptke rte. Dereses in [B] with growth under elevted CO 2 hve lso een oserved in most (Peñuels et l. 21; Nory et l. 1986; O Neill et l. 1987; Liu et l. 27), ut not ll (Peñuels et l. 1997; Luoml et l. 25), previous studies wherein B ws mesured. Dereses in the uptke rte of B t high CO 2 in this study were unrelted to the presene or sene of high-co 2 - stimultion of growth, nd so re unlikely to e linked to totl plnt demnd for B. Further, B uptke rtes deresed with elevted CO 2 despite tht ft tht B onentrtions in gernium leves deresed under high CO 2 to levels pprohing B defiieny t 4.5 nd 45 μm B (Blevins nd

13 128 Plnt Soil (212) 35: Lukszewski 1998; El-Shintinwy 1999; Wimmer et l. 25). High-CO 2 -relted dereses in B uptke rte were lso unrelted to levels of expression of the B trnsport protein, BOR1, sine BOR1 levels were unffeted y B nd inresed slightly t elevted CO 2 (4.5 nd 45 μm B). In ontrst, Jin et l. (29) reently reported tht severl Fe trnsporter genes were up-regulted more under elevted thn urrent CO 2 levels in tomto plnts grown under iron defiieny onditions. Thus, the reson tht B levels deresed t elevted CO 2 in this study remin unknown. However, we did not exmine effets of B nd CO 2 on levels of the other known mjor B trnsport protein, Nip5;1, nd it is possile tht this protein responds differently thn BOR1. Aknowledgment This reserh ws supported y the U.S. Deprtment of Agriulture, Agriulturl Reserh Servie (SCA to J. Gry nd S.A. Hekthorn). The uthors thnk Dougls Sturtz nd Alyi Pittenger for nutrient nlysis. Referenes Brnes JD, Blguer L, Mnrique E, Elvir S, Dvison AW (1992) A repprisl of the use of DMSO for the extrtion nd determintion of hlorophylls nd in lihens nd higher plnts. Environ Expt Bot 32:85 1 Blnk RR, Derner JD (24) Effets of CO 2 enrihment on plnt-soil reltionships of Lepidium ltifolium. Plnt Soil 262: Blevins DG, Lukszewski KM (1998) Boron in plnt struture nd funtion. Annu Rev Plnt Physiol Plnt Mol Biol 49:481 5 Bolños L, Lukszewski K, Bonill I, Blevins D (24) Why oron? Plnt Physiol Biohem 42: Boote KJ (1976) Root-shoot reltionships. Soil Crop Si So Florid 36:15 23 Brown PH, Bellloui N, Wimmer MA, Bssil ES, Ruiz J, Hu H, Pfeffer H, Dnnel F, Römheld V (22) Boron in plnt iology. Plnt Biol 4: Ckmk I, Hengeler C, Mrshner H (1994) Prtitioning of shoot nd root dry mtter nd rohydrtes in en plnts suffering from phosphorus, potssium nd mgnesium defiieny. J Exp Bot 45: Cmpell CD, Sge RF (22) Intertions etween tmospheri CO 2 onentrtion nd phosphorus nutrition on the formtion of proteoid roots in white lupin. Plnt Cell Environ 25: Colemn JS, MConnughy KDM, Bzzz FA (1993) Elevted CO 2 nd plnt nitrogen-use: is redued tissue nitrogen onentrtion size-dependent? Oeologi 93:195 2 Conroy JP (1992) Influene of elevted tmospheri CO 2 onentrtions on plnt nutrition. Aust J Bot 4: Cure JD, Aok B (1986) Crop responses to ron dioxide douling: literture survey. Agri For Meteorol 38: Cure JD, Rufty TW, Isrel DW (1988) Phosphorus stress effets on growth nd seed yield of nonnodulted soyen exposed to elevted ron dioxide. Agron J 8: Deng Y (29) Biomrkers for the monitoring of oron defiieny in Aridopsis nd Pelrgonium. Thesis, University of Toledo Dords C, Brown PH (2) Permeility of ori id ross lipid ilyers nd ftors ffeting it. J Memr Biol 175:95 15 Duois M, Gilles KA, Hmilton JK, Reers PA, Smith F (1956) Colorimetri method for determintion of sugrs nd relted sustnes. Anl Chem 28: Ehleringer JR, Cerling TE, Dering MD (22) Atmospheri CO 2 s glol hnge driver influening plnt-niml intertions. Integr Comprt Biol 42: Ellsworth D, Reih PB, Numurg ES, Koh GW, Kuiske ME, Smith SD (24) Photosynthesis, roxyltion, nd lef nitrogen responses of 16 speies to elevted CO 2 ross four free-ir CO 2 enrihment experiments in forest, grsslnd nd desert. Glo Chng Biol 1: El-Shintinwy F (1999) Struturl nd funtionl dmge used y oron defiieny in sunflower leves. Photosynth 36: Fngmeier A, Grüters U, Hertstein U, Sndhge-Hofmnn A, Vermehren B, Jäger H-J (1996) Effets of elevted CO 2, nitrogen supply nd tropospheri ozone on spring whet. I Growthnd yield Environ Pollut 91: Fngmeier A, Gruters U, Hogy P, Vermehren B, Jäger H-J (1997) Effets of elevted CO 2, nitrogen supply, nd tropospheri ozone on spring whet-ii. Nutrients (N, P, K, S, C, Mg, Fe, Mn, Zn). Environ Pollut 96:43 59 Geuer RLE, Reynolds JF, Strin BR (1996) Allometri reltions nd growth in Pinus ted: the effet of elevted CO 2 nd hnging N vilility. New Phytol 134:85 93 Geiger M, Hke V, Ludewig F, Sonnewld U, Stitt M (1999) The nitrte nd mmonium nitrte supply hve mjor influene on the response of photosynthesis, ron metolism nd nitrogen metolism nd growth to elevted ron dioxide in too. Plnt Cell Environ 22: Ghosh S, Gepstein S, Heikkil JJ, Dumroff EB (1988) Use of snning densitometer or n ELISA reder for mesurement of nnogrm mount of protein in rude extrts from iologil tissue. Anl Biohem 169: Goldh HE (1997) A ritil review on urrent hypothesis onerning the role of oron in higher plnts: suggestions for further reserh nd methodologil requirements. J Tre Miroproe Teh 15:51 91 Gutshik VP (1993) Nutrients-limited growth rtes: Roles of nutrient-use effiieny nd of dpttion to inrese nutrient uptke. J Exp Bot 44:41 51 Hgedorn F, Lndolt W, Trjn D, Egli P, Buher JB (22) Elevted CO 2 influenes nutrient vilility in young eeh-sprue ommunities on two soil types. Oeologi 132: Jin CW, Du ST, Chen WW, Li GX, Zhng YS, Zheng SJ (29) Elevted ron dioxide improves plnt iron nutrition through enhning the iron-defiieny-indued response

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