an investigation of boron toxicity in barley using - plant physiology

An Investigation of Boron Toxicity in BarleyUsing Metabolomics1[W]

Ute Roessner*, John H. Patterson, Megan G. Forbes, Geoffrey B. Fincher, Peter Langridge,and Anthony Bacic

Australian Centre for Plant Functional Genomics, School of Botany, University of Melbourne,Victoria 3010, Australia (U.R., J.H.P., M.G.F., P.L., A.B.); and School of Agriculture, Food and Wine,University of Adelaide, Glen Osmond, South Australia 5064, Australia

Boron (B) is an essential micronutrient that affects plant growth at either deficient or toxic concentrations in soil. The aim of thiswork was to investigate the adaptation of barley (Hordeum vulgare) plants to toxic B levels and to increase our understanding ofB toxicity tolerance mechanisms. We used a metabolomics approach to compare metabolite profiles in root and leaf tissues ofan intolerant, commercial cultivar (cv Clipper) and a B-tolerant Algerian landrace (cv Sahara). After exposure to elevated B(200 and 1,000 mM), the number and amplitude of metabolite changes in roots was greater in Clipper than in Sahara. In contrast,leaf metabolites of both cultivars only responded following 1,000 mM treatment, at which B toxicity symptoms (necrosis) werevisible. In addition, metabolite levels were dramatically altered in the tips of leaves of the sensitive cultivar Clipper aftergrowth in 1,000 mM B compared to those of Sahara. This correlates with a gradual accumulation of B from leaf base to tip inB-intolerant cultivars. Overall, there were always greater differences between tissue types (roots and leaves) than between thetwo cultivars. This work has provided insights into metabolic differences of two genetically distinct barley cultivars andinformation about how they respond metabolically to increasing B levels.

Boron (B) is an essential micronutrient for vascularplants. However, when B is present at high concentra-tions in the soil or ground water, plant growth andreproduction can be affected by B toxicity. B toxicityhas been recognized as an important problem limitingcrop production in the low rainfall and on highlyalkaline and saline soils in regions of Australia, WestAsia, and North Africa. Because soil amelioration isimpractical, the development of B-tolerant cultivars isa rational solution to the problem.

B freely diffuses into the roots as boric acid [B(OH)3;pKa 5 9.25] and accumulates in the cytoplasm as theborate anion [B(OH)4

2] due to pH-dependent inter-conversion. An inability to exclude B from the rootsresults in high B concentrations in the tissue. B phy-totoxicity manifests itself in a broad range of physio-logical effects, including decreased shoot and rootgrowth, root cell division and RNA content, reducedleaf chlorophyll, lower photosynthetic rates and sto-matal conductance, and reduced levels of lignin and

suberin (for review, see Nable et al., 1997). Leaf symp-toms of toxicity in barley (Hordeum vulgare) are char-acterized by interveinal chlorotic and/or necroticpatches, generally at the margins and tips of olderleaves. This reflects the accumulation of B at the end ofthe transpiration stream (Nable et al., 1997). Followinglong-term exposure to high B concentrations in thesoil, overall vegetative plant growth is retarded andthis leads to either a reduction in or a complete lack ofseed set.

B is also an essential nutrient, although its role inplant growth, development, and metabolism remainsto be clarified. Originally, B was thought to be essen-tially immobile in the plant and fixed in the apoplast,but recent evidence has shown that, in some species, Bis present as soluble bis-diester complexes with sorbitolor mannitol, which are phloem mobile (Hu et al.,1997). Most of the functional roles ascribed to B arerelated to its capacity to form diester bridges betweenadjacent cis-hydroxyl-containing molecules, such assimple mono- and oligosaccharides, complex sugars,diols, and hydroxyacids (Power and Woods, 1997). Forexample, B plays a major role in maintaining cell wallstructure and membrane function, as well as support-ing metabolic activities. Up to 90% of cellular B is pres-ent in the cell wall fraction (Power and Woods, 1997).When complexes of B-rhamnogalacturonan II (RG-II)pectic polysaccharides have been isolated and charac-terized, B has been shown to cross-link the apioseresidues of the side chains of RGII (Darvill et al., 1978;Thomas et al., 1989; Matoh et al., 1996; Ishii et al., 1999;O’Neill et al., 2001, 2004). A suite of other roles for B in

1 This work was supported by the Australian Centre for PlantFunctional Genomics from the Australian Research Council, theGrain Research and Development Council, and State Governments.

* Corresponding author; e-mail [email protected]; fax61–3–9347–1071.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Ute Roessner ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.106.084053

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planta has been proposed and recently reviewed byBolanos et al. (2004), but definitive data on the biolog-ical functions of B are generally lacking.

Investigation of the genetic control of B tolerancemechanisms in several species has so far allowed ef-ficient approaches for breeding of B-tolerant varieties.In particular, certain wild species are a potential sourceof B tolerance genes for breeding into related culti-vated crops. A RFLP linkage map of a doubled haploidpopulation generated from a cross between an intol-erant Australian cultivar (cv Clipper) and the B toxicity-tolerant landrace (cv Sahara 3771) was used to identifychromosomal regions associated with B tolerance inbarley. Interval regression mapping allowed the iden-tification of four regions on chromosomes 2H, 3H, 4H,and 6H containing the B tolerance traits that control Buptake, root-length response, dry-matter production,and leaf symptom expression (Jefferies et al., 1999).Many tolerant varieties are characterized by a lowerlevel of B in their leaf tissues compared to intolerantvarieties (Nable et al., 1990; Jefferies et al., 1999). This isthought to be due to reduced uptake of B into bothroots and shoots. Recently, Hayes and Reid (2004)demonstrated that the B-tolerant landrace cv Saharawas able to maintain much lower B concentrations inroots (50%), leaves (73%), and xylem (64%) comparedto the intolerant cv Schooner, which displays similar Buptake traits and B sensitivity as cv Clipper underanalysis in this study. They concluded that B must beactively effluxed from the cv Sahara roots and that thismay be the basis for B tolerance in barley. Furthermore,the ability of cv Sahara to maintain low root B con-centrations was constitutive and occurred across awide range of B concentrations (1–5 mM; Hayes andReid, 2004). This mechanism contrasts with those usedby plants that are hyperaccumulators of heavy metalsthrough complexation (Callahan et al., 2006) or thosethat both exclude and/or sequester metals as com-plexes by secreting organic acids (e.g. malate or citrate)to grow under adverse environmental conditions. Awell-described example is the tolerance mechanism ofplants able to grow on acid soils in which aluminum(Al) is present as toxic Al31 ions. To prevent toxicityof these ions, the root tips of tolerant species ex-crete malate and, to a lesser extent, citrate, which formcomplexes that result in detoxification (Ryan et al.,2001).

Two models for this mechanism of active efflux ofthe borate anion have been proposed, involving eitheranion exchange or an anion channel (Hayes and Reid,2004). Both are likely to impose significant conse-quences on cellular metabolism, which can now bemonitored globally using a metabolomics approach(for reviews, see Sumner et al., 2003; Bino et al., 2004;Fernie et al., 2004). Considering that the cell walls onlycontain a small amount of B cross-linking RGII pecticpolysaccharides, we assumed that a potentially alteredcell wall structure in the tolerant cv Sahara would notaccount for a cellular tolerance mechanism, and wetherefore conducted a comprehensive metabolite com-

parison of the tolerant and intolerant cv Sahara and cvClipper. To our knowledge, there are no reports defin-ing differences in the metabolomes of plants charac-terized by different tolerance levels to B toxicity andtheir metabolic responses following exposure to high Blevels. To investigate in more detail to what extent Btoxicity affects plant metabolism, we have comparedthe metabolic complement of an intolerant, commer-cial Australian barley cv Clipper to that of a tolerantAlgerian landrace cv Sahara in control and B stressconditions. A recently described metabolomics approachbased on gas chromatography (GC)-mass spectrome-try (MS) was adopted for determination of low-Mrcompounds in different barley tissues (Roessner-Tunaliet al., 2003). Using this approach we were able to de-termine metabolic differences between the two culti-vars and their responses to increased B concentration.

RESULTS

Morphological Differences between cv Clipperand cv Sahara and the Effect of High B Levelson the Phenotype of Both Cultivars

When two barley cultivars, the B-intolerant cv Clip-per and the B-tolerant landrace Sahara, were grown inhydroponic growth solution with normal (5 mM) Blevels, obvious morphological differences were appar-ent (Fig. 1). In general, the leaf blades of Sahara werelonger and wider, resulting in a greater leaf areacompared to Clipper (Fig. 1, A and C). Sahara plantsalso produced more tillers. Comparison of the rootsshowed that those of Sahara were much thicker andshorter than those of Clipper (Fig. 1, B and D). Fol-lowing exposure to 200 and 1,000 mM B, Clipper plantsbegin to develop leaf symptoms (necrotic lesions at theleaf tip; Fig. 1, E and G) in the youngest leaves after avery short time (2–3 d) in a concentration-dependentmanner. After 2 weeks, almost all the leaves of Clipperplants were affected. In addition, overall shoot growthwas retarded, fewer leaves developed, and roots wereless branched and browner compared to those ofuntreated plants (compare Fig. 1, A, C, and E). Incontrast, exposure to 200 mM B in Sahara showed novisible morphological effects, but, with exposure to1,000 mM B, the tips of younger leaves show yellowing,indicating the beginning of necrosis. However, thiswas after longer treatment than Clipper (Fig. 1E). Thegrowth of the shoots was not retarded, as it was forClipper plants, but the roots of Sahara seemed to pro-duce more lateral branching compared to untreatedroots (Fig. 1F).

To quantify the effect of elevated B on both varietiesin more detail, plants were grown in different concen-trations of B, and B levels in the oldest leaf of eachplant were determined using inductively coupledplasma-optical emission spectrometry (ICP-OES). Dur-ing the course of 2 weeks, samples were taken untilsymptoms first became visible (Fig. 2). When Clipper

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and Sahara plants were grown in low B concentrations(50 mM B; control), the B levels in the oldest leaf weresimilar throughout the course of growth with noapparent symptoms of B phytotoxicity. When Clipperplants were grown in 1,000 mM B, symptoms appearedafter 5 d, when leaf B concentrations reached 178 mgkg21 dry weight (Fig. 2). In contrast, leaf symptomsdid not appear in Sahara plants until 10 d afterexposure to 5,000 mM B, when the concentration ofleaf B was 191 mg kg21 dry weight. These resultsindicated that Sahara could tolerate similar levels ofleaf B for a longer period before visible symptomsappeared, which suggested a greater tissue toleranceto B than in Clipper. These data are consistent withprevious studies (Jefferies et al., 1999; Hayes and Reid,2004), and we therefore undertook a metabolomicexamination of these two cultivars.

Comparison of the Metabolic Profiles of Clipperand Sahara Leaves and Roots under Control BConcentration (5 mM)

In an initial experiment, we compared the metaboliccomplements of roots and leaves of Clipper andSahara plants grown for 3 weeks in control (5 mM) Bconcentration. Resulting metabolite profiles showedthat both the roots and leaves of the two cultivars weredifferent (Supplemental Tables S1 and S2). A numberof amino acids, namely, Ala (1.6-fold), Pro (3-fold), Thr(1.4-fold), b-Ala (2.1-fold), Glu (1.9-fold), and tyramine(4.5-fold) were significantly higher in Sahara rootscompared to those of Clipper, as were a small numberof organic acids (glyceric acid, fumaric acid, and malicacid [each 1.4-fold]). The only exception was erythronicacid, which was dramatically decreased (0.1-fold) inSahara roots compared to those of Clipper. The differ-ences in the levels of sugars and sugar acids includedslight increases in glycerol-3-P and Glc (1.7-fold), Fru(1.4-fold), inositol (2.3-fold), and galactinol (1.5-fold).In addition, the levels of Xyl, inositol-1P (0.7-fold), andgulonic acid (0.2-fold) decreased in the roots of Saharacompared to Clipper.

When the metabolic profiles in leaves of Sahara werecompared to those in Clipper, only a small number of

metabolites (Gly and putrescine [2.0-fold], Asp [0.9-fold],5-oxoproline [1.2-fold], Asn and Gln [0.5-fold], Lysand saccharic acid [0.6-fold], maleic acid [1.6-fold],fumaric acid [1.5-fold], erythronic acid [0.3-fold],threonic acid and maltose [0.7-fold], Xyl, gulonic acidand raffinose [0.4-fold], and Rib [1.4-fold]) were sig-nificantly different between the cultivars (Supplemen-tal Table S2). Most notable, however, was a dramaticincrease of 6 kestose of up to 12.8-fold, although thehigh variation resulted in statistical insignificance dueto quantification problems arising from incompletepeak resolution with raffinose.

When the resulting metabolic profiles from eachreplicate were subjected to hierarchical cluster analysis(HCA), the greatest distance was observed betweenthe roots and leaves of each cultivar by formation oftwo major clusters (data not shown). There was noclear separation of subclusters for each of the cultivarswithin the tissue clusters. Using principal componentanalysis (PCA), a similar picture emerged (Fig. 3),showing separation of the different tissues by the first

Figure 2. Accumulation of B in the oldest leaves of Clipper and Saharagrown hydroponically in control (50 mM) and high B concentrations.Levels of B in mg/kg dry weight were determined with ICP-OES in theoldest leaves of Clipper (black line with triangles) and Sahara (greenline with diamonds) grown in control B concentration (50 mM B),1,000 mM B (Clipper red line with boxes), and 5,000 mM B (Sahara blueline with circles). An asterisk indicates the day of growth when first leafsymptoms (necrosis) became visible.

Figure 1. Barley plants (Clipper and Sahara)grown hydroponically in control (5 mM) and1,000 mM B concentrations. Barley plants weregrown in a nutrient solution for 3 weeks. A,Sahara leaves. B, Sahara roots grown in 5 mM B.C, Clipper leaves. D, Clipper roots grown in 5 mM

B. E, Sahara (left) and Clipper (right) leaves. F,Sahara (left) and Clipper (right) roots grown in1,000 mM B. G, Clipper leaves grown in 5 mM B(right) and 1,000 mM B (left), indicating necroticlesions. H, Sahara leaves grown in 5 mM B (right)and 1,000 mM B (left).

Metabolite Profiling of Barley in Response to Toxic Boron

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component, which represented about 81% of the var-iation between the samples. The second componentdifferentiates the roots of the two cultivars with about6% variability, whereas the leaves do not fall intoseparate clusters. A second important feature of PCAis the ability to assess the importance of each metab-olite (eigenvectors or loadings) for cluster formation.The most important metabolites contributing to thevariation were Glu, maltose, erythronic acid, trehalose,allantoin, galactonic acid, Glc, glyceric acid, and Trp(data not shown).

Comparison of the Metabolic Responses of Clipper andSahara Roots to 200 and 1,000 mM B Concentrations

Both the tolerant and intolerant barley cultivarswere characterized at the metabolite level when grownin control B concentration (5 mM). Plants of both thetolerant landrace Sahara and the intolerant cv Clipperwere also grown at two toxic B concentrations: 200 mM,representing a medium level of stress, and 1,000 mM B.Tissue samples of roots and leaves were taken from3-week-old seedlings after 2 weeks of B treatment.Resulting metabolic profiles were analyzed in two

ways, the first being a comparison of metabolic re-sponses due to increasing B within each cultivar andtissue (Supplemental Tables S1 and S2), and the secondbeing a comparison between the two cultivars within asingle treatment type (Supplemental Tables S3 and S4).

After exposure to 200 mM B, the concentrations of alarge number of metabolites decreased in the roots ofthe intolerant cv Clipper compared to the controlcondition (Fig. 4; Supplemental Table S3). After the1,000 mM B treatment, only Glu (0.2-fold), glutaric acid(0.4-fold), and all the phosphorylated sugars de-creased, but coumaric acid (1.7-fold), saccharic acid(1.6-fold), and Suc (1.2-fold) increased (Fig. 4; Supple-mental Table S3). Sahara roots exhibited the oppositepattern, with many metabolite levels having increasedafter treatment with 200 and 1,000 mM B (Fig. 4;Supplemental Table S3). Only glutaric acid decreased(0.6-fold) at both 200 and 1,000 mM B and 6-phospho-gluconic acid decreased (0.6-fold) only in 1,000 mM B.

Another way of interpreting the metabolic profiles isthrough metabolite differences between the two culti-vars for each B treatment (Supplemental Table S1). Thelevels of the metabolites of the intolerant cv Clipperserved as the reference to which the x-fold levels of

Figure 3. PCA of metabolite profiles of both barley cultivars grown in control (5 mM B). PCA of the metabolic profiles of theanalyzed Clipper (blue) and Sahara (red) leaves and Clipper (yellow) and Sahara (green) roots of plants grown in 5 mM B. Thedistances between these populations were calculated as described in ‘‘Materials and Methods’’ using the log-transformed,normalized data of the single measurements from which the means presented in Supplemental Tables S1 and S2 are derived. PCAvectors span a 9-dimensional space to give best sample separation with each point representing a linear combination of all themetabolites from an individual sample. Vectors 1 and 2 were chosen for best visualization of differences between cultivars andinclude 87% of the information derived from metabolic variances.

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metabolites in Sahara are compared. When grown in200 mM B, the levels of almost all amino acids in Sahararoots were significantly higher (between 2- to 8-fold;Supplemental Table S1) when compared to treatedClipper roots. There were only two exceptions thatshowed a decrease, urea (0.8-fold) and Gly (0.6-fold).A similar pattern was observed when the levels of or-ganic acids were compared between the roots of bothcultivars at 200 mM B. Almost all the metabolites weresignificantly increased with the highest increase(5.8-fold) for a-ketoglutaric acid. The only exceptionwas erythronic acid, which was dramatically decreased(0.1-fold). Similarly, the levels of almost all sugarswere significantly increased. The most pronounceddifferences were found to be in phosphorylated com-pounds, such glycerol-3-P (8.5-fold), Fru-6-P (6.5-fold),

Glc-6-P (6.3), 6-phosphogluconic acid (only detectablein Sahara roots), and in the trisaccharides raffinose(4.9-fold) and 6 kestose (6-fold).

After treatment with 1,000 mM B, similar differenceswere observed to those described for the 200 mM Btreatment, but in slightly fewer metabolites, whenSahara roots were compared to Clipper roots (Supple-mental Table S1). Again, the majority of amino acidssignificantly increased with the largest increase in Glu(8-fold). The levels of urea significantly decreased(0.3-fold) and asparargine decreased (0.5-fold) afterincreasing (2.4-fold) following treatment with 200 mM

B. As with the 200 mM B treatment, the differences inthe levels of organic acids in Sahara roots compared tothose of Clipper increased in a comparable way. Fewerand weaker responses in the levels of metabolites

Figure 4. Mapping of metabolite changes on known pathways for both Clipper and Sahara grown in control and elevatedconcentrations of B. Data from roots and leaves of each cultivar are normalized to the mean response calculated for therespective control samples (Supplemental Tables S3 and S4). x-Fold values are presented as the mean 6 % SE of six independentdeterminations. Those that are not significantly different to control are colored in yellow for root samples and green for leafsamples, and those that are significantly different from control are colored in red for root samples and black for leaf samples. Therespective reference (set to 1) is indicated as a line. Italicized metabolite names indicate not determined. The bars are indicatedas follows: 1, Clipper root grown in 200 mM B; 2, Clipper root grown in 1,000 mM B; 3, Sahara root grown in 200 mM B; 4, Sahararoot grown in 1,000 mM B; 5, Clipper leaf grown in 200 mM B; 6, Clipper leaf grown in 1,000 mM B; 7, Sahara leaf grown in 200 mM

B; 8, Sahara leaf grown in 1,000 mM B.



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following treatment with 1,000 mM B were observed.Erythronic acid again decreased (0.1-fold). The sameresponses were seen in the levels of sugars and sugaracids of Sahara roots compared to Clipper roots aftergrowing in 1,000 mM B, but not as dramatically as in200 mM.

Comparison of Leaf Metabolite Profiles of Clipper and

Sahara following Treatment with 200 and 1,000 mM BCompared to Control Plants

A slight, but significant, increase was found in someof the amino acids (Asp [1.2-fold], Gly [1.4-fold],N-acetyl-glutamate [2.7-fold], 5-oxoproline [1.5-fold],putrescine [1.6-fold], and Val [1.4-fold]) and organicacids (erythronic acid [1.2-fold], fumaric [1.6-fold],glyceric acid [1.6-fold], maleic acid [1.4-fold]) in Clip-per leaves following treatment with 200 mM B whencompared to control Clipper leaves (Fig. 4; Supple-mental Table S4). For all analyzed sugars, only Sucincreased, up to 1.2-fold. When treated with 1,000 mM

B, about one-half of the amino acids and organic acidsshowed significant changes in Clipper leaves whencompared to the control leaves (Fig. 4; SupplementalTable S4). The leaves of Clipper plants grown at1,000 mM B had approximately one-half of the sugarmetabolites increased, including Xyl (1.6-fold), Rib (2.0-fold), Fru (3.8-fold), Glc (2.6-fold), GlcUA (1.8-fold),saccharic acid (1.3-fold), maltose (1.4-fold), and raffi-nose (1.5-fold) when compared to those grown in200 mM B.

Sahara leaves responded in a similar way to 1,000 mM

B as Clipper leaves, which is in contrast to the resultseen in roots, where Sahara had the opposite metabolicresponse (Fig. 4; Supplemental Table S4). Only a smallnumber of amino acids and organic acids in Saharaleaves were increased following treatment with 200 mM

B compared to control Sahara leaves (Fig. 4; Supple-mental Table S4). There was a stronger response inlevels of sugars in Sahara leaves after treatment with200 mM B (Xyl [2.0-fold], Fru [2.3-fold], Glc [5.1-fold],GlcUA [1.9-fold], and galactinol [1.5-fold]), when com-pared to 1,000 mM treatment where only Fru (1.9-fold),Glc (3.3-fold), and GlcUA (1.6-fold) increased. Therewas an apparent dramatic decrease (0.2-fold) in thelevel of 6 kestose, but this was not statistically signifi-cant. In contrast, when grown in 1,000 mM B, more aminoacids were significantly affected than when grown in200 mM B (Fig. 4; Supplemental Table S4). The mostdramatic decrease was in Glu, to almost undetectablelevels, a similar trend as observed in 1,000 mM BClipper leaves. Only a small number of organic acidschanged with 1,000 mM B treatment.

When the metabolite levels between the leaves ofboth cultivars following treatment with 200 mM B in thenutrient solution were compared, there were no dif-ferences in the levels of amino acids, only a single or-ganic acid decreased in Sahara leaves (erythronic acid[0.4-fold]) and a number of sugars (Rib [1.7-fold], Fru[2.0-fold], inositol [0.8-fold], Glc [6-fold], and 6 kestose

[12.4-fold]) were different in Sahara leaves (Supple-mental Table S2). In contrast, many metabolites dif-fered between leaves of both varieties followingtreatment with 1,000 mM B. Almost all of the aminoacids were significantly decreased, with the exceptionof Gly, which increased (3.1-fold) in Sahara leavescompared to Clipper leaves. A similar trend was ob-served in the levels of organic acids and sugars. Morethan one-half of these decreased quite dramatically,with the most pronounced decrease (0.2-fold) both inerythronic acid and raffinose. There were only a fewexceptions, maleic acid (2.2-fold), Glc (2.8-fold), andgalactinol (1.5-fold), which were found to be increased.

Cluster Analysis of Metabolic Profiles of Clipper andSahara Leaves Treated with Different B Concentrations

The resulting metabolic profiles were then subjectedto both HCA and PCA for easier comparison ofsimilarities and differences. HCA of the metabolomesof Clipper and Sahara roots and leaves grown undercontrol (5 mM), medium-stress (200 mM), and high-stress (1,000 mM) B concentrations again clearly delin-eated two major clusters; those representing either allroot (root cluster) or all leaf (leaf cluster) samples (Fig.5A). Within the root cluster, two subclusters wereevident; the first consisting of all Sahara samples bothfrom control and treated samples, with an indepen-dent subcluster of the control Clipper roots; the secondrepresenting both Clipper roots treated with 200 or1,000 mM B. The leaf cluster did not show strongseparation between either the two cultivars or threetreatments; the only obvious formations were smallsubclusters of either Sahara or Clipper leaves treatedwith 1,000 mM B. PCA of this dataset revealed a similarpicture (Fig. 5B). Both root and leaf samples wereclearly separated by the first principal component,which represented 70.4% of the variability between thesamples. Sahara and Clipper root metabolic profileswere further separated by the second principal com-ponent (12.8% variability). Within the two cultivars,there is a distance between the control and treatedroots, but the 200 and 1,000 mM B treatments did notresolve into distinct subclusters. The leaf cluster didnot show any further separation for either of the cul-tivars or treatments. The following metabolites hadthe highest impact on cluster formation: maltose,erythronic acid, trehalose, galactonic acid, allantoin,6-phosphogluconic acid, Glu, Trp, glycerol-3-P, glyc-eric acid, gulonic acid, urea, and tyramine.

Comparison of Metabolic Responses of Clipper andSahara Leaves to Different B Concentrations duringSeedling Development

In this experiment, Clipper and Sahara plants weregrown in parallel for 1 week in control B concentra-tions (5 mM) and samples from the youngest fullydeveloped leaf were harvested (T0). One-half of theplants were subsequently grown in 1,000 mM B and the

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Figure 5. HCA and PCA of metabolite profiles of both barley cultivars grown in three different B concentrations. A, Dendogramobtained following HCA of the metabolic profiles of the analyzed leaves and roots of Clipper and Sahara grown in 5, 200, and1,000 mM B. The distances between the samples were calculated as described in ‘‘Materials and Methods’’ using the normalizeddata of the single measurements from which the means presented in Supplemental Tables S3 and S4 are derived. Wherever



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other one-half maintained at 5 mM B. Leaf sampleswere taken after 1 (T1-treated and control) and 2(T2-treated and control) weeks of growth. Resultingmetabolic profiles were compared (1) within eachvariety through development, with T0 as the reference(Supplemental Table S5); and (2) between the twocultivars for each time point with and without treat-ment, where the respective Clipper samples were usedas the reference data (Supplemental Table S6).

For leaves of control Clipper plants, only a smallnumber of metabolites decreased after 2 (T1, five metab-olites changed) and 3 (T2, four metabolites changed)weeks of growth when compared to T0 (SupplementalTable S5). But a clear metabolic response was observedin Clipper leaves treated with 1,000 mM B for both 1 and2 weeks (T1-treated and T2-treated). A range of me-tabolites increased significantly in both time points,including four amino acids, nine organic acids, andfour sugars. Notably, putrescine was only detectable inleaves of Clipper plants after 3 weeks of growth fol-lowing treatment with high B. In comparison, after1 week of growth in control 5 mM B (T1), only fourmetabolites were changed in Sahara leaves when com-pared to T0 (Supplemental Table S5). However, thisresult was amplified at T2, where 12 metabolites of allclasses were significantly different compared to T0.Sahara plants grown in 1,000 mM B had fewer metab-olites responding than Clipper leaves at both T1-treated(eight metabolites) and T2-treated (11 metabolites)compared to T0 (Supplemental Table S5).

Another way of presenting the resulting metabolicprofiles from this experiment is a direct comparison ofmetabolite levels in Sahara leaves of each time point tothe metabolite levels in the respective Clipper leaves(Supplemental Table S6). Surprisingly, when consid-ering the results from the above-described experi-ments in which both cultivars were found to bemetabolically distinct from each other (SupplementalTable S5), there were few significant differences foundbetween Clipper and Sahara leaves grown in control5 mM B for only 1 week (T0). An obvious difference wasthat putrescine was only detected in Sahara leaves. AtT1, at control (5 mM B), both cultivars are distinguish-able as nine of the metabolites differ significantly.Again, putrescine was only detected in Sahara leaves.At T2, the trend was similar to that at T1, but, due torelatively high variation, many differences were notstatistically significant based on Student’s t test. Asbefore, putrescine was only detected in Sahara leaves.In addition, the levels of a-ketoglutaric acid, 6 kestose,

1 kestose, and raffinose were altered. Following treat-ment with 1,000 mM B, metabolic differences betweenClipper and Sahara became more pronounced. A sim-ilar picture was observed as described before; manymetabolites were decreased in Sahara leaves comparedto Clipper leaves after B treatment. After 1 week oftreatment (T1-treated), the levels of nine metaboliteswere altered in Sahara leaves compared to those ofClipper. A similar, but stronger, pattern was observedafter 2 weeks of treatment (T2-treated) with 12 metab-olites being decreased. In addition, galactinol and quinicacid were increased. Putrescine was now detected inClipper leaves. However, at this stage of developmentand after treatment, the levels of putrescine weresimilar in both cultivars. In all of these measurements,a large number of unidentified compounds, mainlysugars, were analyzed (Supplemental Tables S5 andS6), but these results are not discussed here.

HCA of these data showed that there were twomajor clusters formed, one including four subclustersof Clipper leaves harvested at T0, control Clipperleaves at T1, Sahara leaves at T0, and Clipper leavesat T2 treated with 1,000 mM B (Fig. 6A). The secondmajor cluster had three subclusters, one representingthe metabolic profiles of control Sahara leaves at T2,the second treated Sahara leaves at T2, and the largestsubcluster represented the control Clipper leaves at T2,treated Clipper leaves at T1, control Sahara leaves at T1,and treated Sahara leaves at T2. In contrast, when PCAwas applied, the first principal component (34.0%variability) separated Clipper leaves from Saharaleaves regardless of the treatments, with one excep-tion: Sahara leaves harvested at a very early stage ofdevelopment (T0) clustered with Clipper at T0, indi-cating that, at this stage, the metabolic profiles ofleaves from Clipper and Sahara are very similar (Fig.6B). Within the cluster for Sahara leaves from plantsolder than 1 week, individual clusters for each devel-opmental stage with and without treatment could beassigned. The most important metabolites for clusterseparation were putrescine, melibiose, Glc, Fru, 6kestose, Asp, and erythronic acid (data not shown).

Correlation of Metabolic Profiles on Clipper and Sahara

Leaves with the Gradient of B within a Leaf Bladefollowing Treatment with High B Concentration

It is known that following growth in toxic B con-centrations, B accumulates in the leaf blades, coinciding

Figure 5. (Continued.)possible, individual branches are grouped in brackets for ease of reading. B, PCA of the metabolic profiles of the analyzed leavesand roots of Clipper and Sahara grown in 5, 200, and 1,000 mM B. The distances between these populations were calculated asdescribed in ‘‘Materials and Methods’’ using the log-transformed, normalized data of the single measurements from which themeans presented in Supplemental Tables S3 and S4 are derived. PCA vectors span a 9-dimensional space to give the best sampleseparation, with each point representing a linear combination of all the metabolites from an individual sample. Vectors 1 and 2were chosen for best visualization of differences between cultivars and include 83% of the information derived from metabolicvariances.

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Figure 6. HCA and PCA of metabolite profiles of both barley cultivars grown in 5 and 1,000 mM B through development. A,Dendogram obtained following HCA of the metabolic profiles of the analyzed leaves of Clipper and Sahara grown in 5 and1,000 mM B. Samples were taken after 1 week of growth (only in 5 mM B; T0), 2 weeks (T1), and 3 weeks (T2) of growth. Thedistances between the samples were calculated as described in ‘‘Materials and Methods’’ using the normalized data of the singlemeasurements from which the means presented in Supplemental Tables S3 and S4 are derived. Wherever possible, individualbranches are grouped in brackets for ease of reading. B, PCA of the metabolic profiles of the analyzed leaves of Clipper and



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with a tissue age-associated gradient with highestconcentrations in the leaf tip correlating with B toxicitysymptoms (Reid et al., 2004). To ascertain whether itis possible to correlate the B concentration gradientin the leaf with metabolite levels, and to compare thepattern between Clipper and Sahara, we separatedeach harvested leaf blade into three segments: the base(youngest tissue), the middle portion, and the tip(oldest tissue). Both cultivars were grown in control(5 mM) and 1,000 mM B for 3 weeks. We describe first thecomparison of metabolite level from leaf base throughleaf tip for each cultivar with the respective base as thereference (Supplemental Table S7), and second thecomparison of each portion of the leaf from bothtreated cultivars with the respective control leaf por-tion as the reference (Supplemental Table S8).

When metabolite levels were compared along theleaf blade of plants grown in control conditions, threetypical metabolite abundance patterns appear. Thefirst is an increase in metabolite abundance from thebase to the tip of the leaf (pattern 1). The second isuniform abundance along the length of the leaf blade(pattern 2). The third is an increase in abundance in thebase compared to the other parts of the leaf (pattern 3;Supplemental Table S7). In leaves from Clipper, thegeneral trend is an increase in sugar abundance fromthe base to the tip (pattern 1), with the exception ofxylitol, galactinol, and Suc, which displayed pattern 3distribution. In contrast, sugar distribution along theleaves of Sahara was closer to the uniform, pattern2-type distribution, again with xylitol as an obviousexception. Pattern 1 distribution was also observed forsome amino acids in Clipper leaves, including b-Ala,g-aminobutyrate (GABA), Gln, Ile, Leu, and Pro,whereas for Ala, Asn, Gly, and Phe pattern 3 distribu-tion was displayed. Notably, the levels of Phe wereonly strongly decreased in the tip compared to theother parts of the leaf. In Sahara leaves, a similar dis-tribution was observed, with GABA, Gln, and Gly follow-ing pattern 1 distribution and Ala and Asn followingpattern 3 distribution. Most organic acids in Clipperleaf followed pattern 1 and pattern 2 distribution,whereas in Sahara leaves only threonic acid-1,4-lac-tone displayed pattern 1 distribution and all othermeasured organic acids displayed pattern 2 distribu-tion. Following treatment with 1,000 mM B, the majorityof the distributions in Clipper leaves were similar, butwith much greater amplitude. In Clipper leaves, allmeasured amino acids followed pattern 1 distribution,with the exception of 5-oxoproline, which showedpattern 2 distribution. Similarly, most organic acids

displayed pattern 1 distribution, with the exception ofascorbic acid, which displayed pattern 2 distribution,and shikimic acid, which now displayed pattern 3distribution in contrast to untreated leaves. The samepattern emerged for sugar distribution followingpattern 1, with 3-phosphoglyceric acid, digalactosyl-glycerol, and Suc following pattern 2 distribution. InSahara leaves, only small changes compared to thepatterns of untreated leaves were observed. The onlydifferences were Pro, putrescine, 1,6-anhydro-Glc, Fru,Gal, and Glc, which now displayed pattern 1 distributioncompared to pattern 2 distribution in untreated leaves.

Following PCA analysis, two distinct clusters di-vided by the first component (59.1% of the variability)were formed, one representing the metabolite profilesof 1,000 mM B-treated base, middle, and tip portions ofthe leaves of the intolerant cv Clipper (Fig. 7) and theother major cluster representing all other samples(Clipper base, middle, tip control; Sahara base, middlepart, tip treated, and control). The second component(15.8% of the variability) separated the former clusterinto the tip portions of the Clipper leaf in a subclusterand the base and middle portion of the same leavesinto a second subcluster. The latter major cluster wasseparated by the second component into control Clip-per leaf segments, on one side, and treated and controlSahara leaf segments on the other. There was somedegree of overlap of the Sahara cluster with the controlClipper cluster. Metabolites with the highest impact oncluster formation (loadings) were mainly those onlydetected in either one of the cultivars, segments, or treat-ments (Supplemental Table S7). In addition, Pro, Glu,GABA, homoserine, Met, Gly, and shikimic acid playeda major role in cluster formation (data not shown).

When metabolite profiles resulting from each part ofthe B-treated leaf were compared to the respectivesegment of the control leaf for both cultivars, mostobvious and, in some cases, very strong differenceswere always seen for the treated Clipper segments(Supplemental Table S8), whereas in treated leaf seg-ments of Sahara only a small number of metaboliteswere altered compared to the control segment. After Btreatment, 14 of the 20 analyzed amino acids werestrongly decreased down to 0.02-fold for Glu, Met,Phe, and Pro. Only Asn increased to about 3-fold. Inaddition, four organic acids and five sugars increased.The middle portion shows a similar picture, with 11amino acids decreased, again with Glu and Pro to0.02-fold compared to the control middle part. Asnwas again increased, this time to 4.7-fold. Furthermore,eight organic acids changed significantly, as well as

Figure 6. (Continued.)Sahara grown in 5 and 1,000 mM B. Samples were taken after 1 week of growth (only in 5 mM B; T0), 2 weeks (T1), and 3 weeks (T2)of growth. The distances between these populations were calculated as described in ‘‘Materials and Methods’’ using the log-transformed, normalized data of the single measurements from which the means presented in Supplemental Tables S3 and S4 arederived. PCA vectors span a 9-dimensional space to give best sample separation, with each point representing a linearcombination of all the metabolites from an individual sample. Vectors 1 and 2 were chosen for best visualization of differencesbetween cultivars and include 53.7% of the information derived from metabolic variances.

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almost all of the analyzed sugars. Interestingly, after Btreatment, only seven amino acids were changed in theleaf tip of the treated leaves compared to those of thecontrol. The most pronounced changes were onceagain Pro, which decreased 0.02-fold, and Asn, whichincreased 9.5-fold. Again, eight organic acids werealtered and most of the analyzed sugars increaseddramatically. For example, Ara, Fru, galactinol, Glc,melibiose, and Rib increased more than 5-fold. Nota-bly, 3-phosphoglyceric acid, 6 kestose, raffinose, diga-lactosylglycerol, and the phosphorylated sugars wereonly detectable in each of the treated segments of theClipper leaves.

When the metabolite levels in segments of treatedSahara leaves were compared to those of the untreatedleaves, only a small number of metabolites werealtered (Supplemental Table S8). In the base segmentseven and in the middle part only two metaboliteswere significantly changed, whereas metabolite levelsin the tips of treated Sahara leaves were similar tothose of control Sahara leaves.

In these measurements, there were also 16 majorcomponents analyzed that could not be positivelyidentified, but based on their mass spectra were as-signed to either the sugars or sugar acids. The results

for these compounds follow the same trend as foridentified metabolites.

DISCUSSION

The aim of this study was to investigate the mech-anisms of B toxicity tolerance in more detail. Twoproposed models for a constitutive tolerance mecha-nism in barley were the basis for this work: oneassumes the existence of compounds allowing com-plexing of B once it accumulates to toxic concentra-tions within the cell (Reid et al., 2004) and the otherdescribes an active efflux of B by a transporter (Hayesand Reid, 2004). Comparison of the accumulation of Bin the oldest leaves between Clipper and Sahara plantsand the correlation of these B levels with the appear-ance of visible necrotic regions (Fig. 2) has clearlydemonstrated that Sahara is able to actively exclude Bfrom the tissue. This results in lower B levels in theoldest leaf even when grown at much higher B con-centrations than in Clipper. In addition, leaves fromSahara take longer to develop necrotic symptomswhen comparable tissue B concentrations, relative toClipper, are reached. This may indicate that, in addi-tion to an efflux mechanism, Sahara also exhibits a

Figure 7. PCA of metabolite profiles of leaf segments of both barley cultivars grown in 5 and 1,000 mM B. PCA of the metabolicprofiles of three leaf segments (base, middle, and tip portion) of Clipper and Sahara grown in 5 and 1,000 mM B. The distancesbetween these populations were calculated as described in ‘‘Materials and Methods’’ using the log-transformed, normalized dataof the single measurements from which the means presented in Supplemental Tables S3 and S4 are derived. PCA vectors span a9-dimensional space to give the best sample separation, with each point representing a linear combination of all the metabolitesfrom an individual sample. Vectors 1 and 2 were chosen for best visualization of differences between cultivars and include74.9% of the information derived from metabolic variances.



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cellular/tissue-based mechanism allowing the plantsto tolerate higher tissue concentrations of B before cellmetabolism is affected leading to necrosis and celldeath. This could be either mediated by an apoplasticor vacuolar sequestration or complex formation withspecific metabolites. To examine the latter possibility,we decided to use a recently developed GC-MS-basedmetabolite profiling technology (Roessner et al., 2001;Roessner-Tunali et al., 2003) for a comprehensive com-parison of metabolite levels between the B-intolerantcommercial barley cv Clipper and the tolerant Alger-ian landrace Sahara.

Comparison of the metabolite profiles of the twocultivars has demonstrated that there were more met-abolic differences between the roots of both cultivars,but only a few differences between the leaves whenthey were grown in control (5 mM B; Fig. 3). However,in spite of genetic diversity, the metabolite profiles ofthe two different tissue types, roots and leaves, ex-hibited greater differences between each other thanbetween the two cultivars (Fig. 5). These results sug-gest that there may be a metabolic preadaptation in theroots of tolerant cv Sahara contributing to greatertissue tolerance. There were no sugar alcohols orpolyols, such as mannitol, sorbitol, or pinitol detected,which have been shown to play a role in B complexformation (Hu et al., 1997). Although low levels ofmannitol and sorbitol have been reported for cereals,both were below the detection limit of our GC-MS-based profiling technology.

Clipper and Sahara leaf metabolite profiles weresimilar in the early stages of development (up to 2weeks of growth). The only striking difference wasthat in young leaves the polyamine, putrescine, wasonly detected in Sahara in unstressed conditions,whereas it was only detected in Clipper leaves after3 weeks of growth following treatment with 1,000 mM Bfor 2 weeks. At this stage, it was present in similarquantities in both Clipper and Sahara leaves because itdecreased in Sahara leaves following a 1,000 mM Btreatment compared to control leaves (SupplementalTables S5 and S6). This compound was also found inup to 50-fold higher concentrations in Clipper leaf tips,but only 5-fold in Sahara leaf tips treated with 1,000 mM

B compared to the respective treated leaf base (Sup-plemental Table S7) or 6-fold higher concentrationscompared to the control Clipper tip (SupplementalTable S8). Putrescine belongs to the class of aliphaticpolyamines shown to be involved in both abiotic andbiotic stress responses (Walters, 2003; Capell et al.,2004; Legocka and Kluk, 2005). In tobacco (Nicotianatabacum), putrescine levels were elevated following Bdeficiency (Camacho-Cristobal et al., 2005) and theauthors proposed a potential link between B and pu-trescine. This is supported by our data, which show anincrease in putrescine following 1,000 mM B treatmentin leaves of the sensitive cultivar and a decrease inleaves of the tolerant cultivar. A more detailed inves-tigation of the role of putrescine, and perhaps otherpolyamines, such as spermine and spermidine, in B

toxicity and deficiency will provide insight intowhether these metabolites correlate with B levels inplants or whether they are general stress-responsivemetabolites. The latter is supported by the highly ele-vated levels of putrescine found in treated leaf tips inboth varieties where necrotic lesions were visible andcells were obviously highly stressed.

An interesting finding was that the metabolite pro-files of young, untreated Sahara leaves were moresimilar to Clipper leaves throughout development,regardless of treatment (Fig. 6B), whereas older Saharaleaves, both treated and untreated, form an indepen-dent cluster indicating stronger metabolite differences.This result suggests that Clipper and Sahara plantsmay develop at a different rate.

Following treatment with increasing B concentra-tions, the intolerant cv Clipper showed morphologicalstress responses, such as decreases in root length ornecrotic lesions in leaves, whereas Sahara showedvisible responses at much later stages. The leaf symp-toms were explained by dramatic increases in B withinthe leaves of sensitive cultivars in contrast to tolerantcultivars, which are proposed to either reduce their Buptake or actively efflux it from cells (Hayes and Reid,2004). The comparison of root metabolites to increas-ing B in both a sensitive and a tolerant cultivar clearlydemonstrated a greater response, both in number ofmagnitude of metabolites in roots of the sensitivecultivar, compared to those of Sahara, and also to agreater extent (Fig. 4; Supplemental Table S3). InClipper roots, most of the metabolites were decreased,whereas in Sahara roots, most metabolites were in-creased following B treatment. In leaves, few metab-olites were altered after exposure to 200 mM B in bothcultivars and the pattern of response was very similar(Fig. 4; Supplemental Table S4). In contrast, followinggrowth in 1,000 mM B, a large number of metabolitesfrom all classes were altered, often quite dramaticallyand mainly in Clipper leaves. This is probably notsurprising because at this high B concentration bothcultivars show visible necrotic lesions, especially at thetips of the leaves. Therefore, we postulate that, in theseleaves, we are not measuring B-specific stress re-sponses, but rather apoptosis.

In addition, 6 kestose was dramatically increased intreated leaves of Sahara. 6 Kestose [O-b-D-fructosyl-(2–6)-b-D-fructosyl-(2–1)-a-D-Glc] is an intermediate forfructan biosynthesis. Fructans are synthesized fromSuc by repetitive addition of a Fru moiety. Fructans aresugar polymers made of Fru and have been implicatedin stress responses in grasses (Amiard et al., 2003;Wang et al., 2003). A detailed fructan analysis in bothClipper and Sahara leaves may clarify whether thesecompounds have any direct involvement in B tolerance.

Analysis of metabolite levels in different parts of theuntreated leaves has shown that a large number ofmetabolites occur in a gradient, either increasing ordecreasing, from the base to the tip. This shows theimportance of spatially resolved metabolite profilingrather then analyzing a homogenate of whole leaves.

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Surprisingly, most metabolites were increased in Clip-per leaf tips compared to the base, which is thegrowing zone of grass leaves (Esau, 1977) and, there-fore, where the highest metabolic activities would beexpected. Once plants were treated with 1,000 mM B,most metabolites in Clipper leaf tips were dramaticallyincreased compared to the base. This clearly correlatedwith the response of B-sensitive barley cultivars inaccumulating B in their leaf tips following treatmentwith elevated B concentrations (Hayes and Reid, 2004).However, these tips were already highly necrotic andbrownish lesions were visible, and most possible ef-fects of osmotic imbalances were determined due tohigh B accumulations. Reid et al. (2004) have shownthat many cellular processes are disturbed by high Btissue concentrations in susceptible cultivars, includ-ing respiration, photosynthesis, or protein synthesis.In addition, at high tissue concentrations, B binds toRib moieties of NAD1, NADP1, ATP, ADP, RNA, andDNA, which may result in substantial interruptions ofcellular activities at all levels (Reid et al., 2004). Thismay explain the extensive alterations in the metaboliteprofiles of tips of Clipper leaves when grown in1,000 mM B (Supplemental Table S7). The ability ofSahara to exclude B leading to lower B tissue concen-trations is obviously mirrored in the metabolic pro-files, which did not change substantially in treated tipscompared with either the treated base or control tips(Supplemental Tables S7 and S8). In any case, thesedata demonstrated the value of increasing the spatialresolution of metabolite analysis regardless of thetreatment examined because metabolite profiles alteralong a leaf reflecting changes in development.

This work has provided insight into the metabolicresponses of barley plants (tolerant and intolerant) totoxic B levels. Our data suggest that none of theanalyzed metabolites seem sufficient to explain thecellular tolerance mechanism in the Sahara cultivar.Therefore, we conclude that engineering a functionalefflux mechanism (active transporter) in intolerant cul-tivars might be a more appropriate strategy for in-creasing B tolerance. The proposed transporter (Hayesand Reid, 2004) would be distinct from the recentlyidentified BOR1 plasma membrane protein that is anefflux-type B transporter for loading B into the xylemessential for B translocation from the roots to the shootsunder B-limiting conditions (Noguchi et al., 1997;Takano et al., 2002; Nakagawa-Yokoi et al., 2005). Cur-rently, we are conducting a proteomics approach to in-vestigate the differences between the plasma membraneproteins of Clipper and Sahara roots in an attempt toidentify potential transporters in the tolerant cultivarcapable of actively effluxing B from the tissue.

MATERIALS AND METHODS

Plant Growth

Barley (Hordeum vulgare L. cv Sahara 3771 and cv Clipper) seeds were

surface sterilized with 70% ethanol for 5 min, washed with water (three times),

incubated for 10 min in 0.5% hypochlorite solution, and then rinsed (10 times)

with water. The seeds were then imbibed in water under continuous aeration

for 24 h at room temperature. Germinated seeds were placed onto moist filter

paper for 2 to 3 d until seedlings were 2 to 3 cm in height. Individual seedlings

were placed in a 5-mL plastic pipette tip with a cut end to allow root growth

and transferred to plastic growth containers filled with hydroponic solution.

The nutrient solution contained the following basal macronutrients: 2 mM

Ca(NO3)2, 5 M KNO3, 5 mM NH4NO3, 2 mM MgSO4, 0.1 mM KH2PO4, 0.5 mM

Na2SiO3, and 0.05 mM NaFe(III) EDTA, and the following micronutrients: 5 mM

MnCl2, 5 mM ZnSO4, 0.5 mM CuSO4, and 0.1 mM NaMoO3 with 5 mM H3BO3. The

nutrient solution was aerated continuously and replaced every 3 d. Stress

conditions were applied after 1 week of growth in control conditions by

adding 200 or 1,000 mM H3BO3. Plants were grown in a controlled environment

at 18�C day/13�C night, a photoperiod regime of 14-h d/10-h night at

180 mmol m22 s21 photon flux intensity at the plant level. Root and leaf (the

youngest fully developed leaf) tissues were harvested in 5 to 6 h of the light

period after 3 weeks of growth and for the time course experiment after 1, 2,

and 3 weeks of growth. Samples were immediately frozen in liquid nitrogen

and stored at 280�C until extraction.

Chemicals

All chemicals were purchased from Sigma-Aldrich. N-methyl-N-(trime-

thylsilyl)-trifluoroacetamide was purchased from Biolab.

Determination of B in Leaf Tissues Using ICP-OES

Plants were grown as described above, with a B concentration of 50 mM.

After 7 d, plants were transferred to identical solutions supplemented with B

as described (Fig. 2). At indicated time points (Fig. 2), the oldest leaf was

removed and weighed before and after drying at 70�C for 16 h. The B content

of the dried leaf samples was determined using ICP-OES after tissues were

digested with concentrated nitric acid. Measurements were normalized on a

tissue dry weight basis.

Extraction, Derivatization, and Analysis of Barley Leafand Root Metabolites Using GC-MS

Metabolite analysis was carried out by GC-MS using a modified method of

Roessner-Tunali et al. (2003). Frozen tissues of roots and leaves from barley

plants were homogenized using a mortar and pestle in liquid nitrogen. Frozen

tissue powder (approximately 90–110 mg accurately weighted and recorded)

was extracted with 100% methanol (350 mL) and a polar internal standard

(20 mL of 0.2 mg mL21 ribitol in water) was added. The mixture was extracted

for 15 min at 70�C and then mixed vigorously with 1 volume of water. To

separate polar and nonpolar metabolites, chloroform (300 mL) was added to

the mixture (to generate a biphasic system) and centrifuged at 2,200g for

10 min. The upper methanol-water phase was taken and washed with chlo-

roform (300 mL). Aliquots of the leaf (100 and 5 mL) and root (250 and 5 mL)

polar phases were taken for analysis of high and low abundance metabolites.

The nonpolar phase was discarded. All resulting aliquots were dried under

vacuum. The dried polar residue was redissolved and derivatized for 2 h

at 37�C in methoxyamine hydrochloride (40 mL of 30 mg mL21 in pyridine)

followed by trimethylsilylation for 30 min at 37�C with N-methyl-N-

(trimethylsilyl)-trifluoroacetamide (70 mL). A retention time standard mixture

(10 mL of 0.029% [v/v] n-dodecane, n-pentadecane, n-nonadecane, n-docosane,

n-octacosane, n-dotracontane, n-hexatriacontane dissolved in pyridine) was

added prior to trimethylsilylation. Samples (1 mL) were then injected via the

splitless mode onto a GC column using a hot needle technique.

The GC-MS system comprised an AS 3000 autosampler, a trace GC Ultra,

and a DSQ quadrupole mass spectrometer (Thermo Electron Corporation).

The mass spectrometer was tuned according to the manufacturer’s recom-

mendations using tris-(perfluorobutyl)-amine (CF43). GC was performed on a

30-m VF-5MS column (with 10-m Integra guard column, i.d. 0.25 mm, 0.25-nm

film thickness; Varian). The injection temperature was set at 230�C, the MS

transfer line at 280�C, and the ion source at 250�C. Helium was used as carrier

gas at a flow rate of 1 mL min21. The analysis was performed under the

following oven temperature program: injection at 70�C followed by 1�C min21

oven temperature ramp to 76�C, and then by 6�C min21 to 330�C, and finishing

with 10-min isothermal at 330�C. The GC-MS system was then temperature

equilibrated for 1 min at 70�C prior to injection of the next sample. Mass



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spectra were recorded at two scans per second with a mass-to-charge ratio of

70 to 600 atomic mass units scanning range. Both chromatograms and mass

spectra were evaluated using the Xcalibur program (ThermoFinnigan) and the

resulting data are prepared, normalized, and presented as described by

Roessner et al. (2001). Mass spectra of eluting compounds were identified

using an in-house constructed mass spectra library of authentic standards, the

public domain mass spectra library of the Max-Planck-Institute for Plant

Physiology (http://csbdb.mpimp-golm.mpg.de/csbdb/dbma/msri.html;

Schauer et al., 2005), and the commercial mass spectra library of the National

Institute of Standards and Technology (http://chemdata.nist.gov). All match-

ing mass spectra were additionally coverified by determination of the reten-

tion time and mass spectra by analysis of authentic standards.

A recently developed method for metabolic profiling using GC-MS

(Roessner et al., 2000; Roessner-Tunali et al., 2003; Weckwerth et al., 2004;

Broeckling et al., 2005) was optimized to analyze the levels of metabolites from

leaves and roots of barley plants. First, we tested three different metabolite

extraction protocols described for best performance on barley tissues: (1)

using 100% MeOH at 70�C for 15 min (Roessner-Tunali et al., 2003); (2) using a

chilled MeOH:water:chloroform (2.5:1:1 [v/v/v]) mixture at 4�C for 5 min

(Weckwerth et al., 2004); and (3) using a chilled water:chloroform mixture (1:1

[v/v]), treatment of 1 h at 50�C followed by an incubation at 220�C overnight

(Broeckling et al., 2005). The first method allowed the detection and quanti-

fication of the greatest number of metabolites and, simultaneously, the best

recovery of Suc and phosphorylated compounds and was therefore adopted

for all analyses (data not shown).

The derivatization, GC-MS setup, and chromatogram evaluation proce-

dures were previously described by Roessner-Tunali et al. (2003). Because the

extracts contained compounds differing by several orders of magnitude (high

amounts of malate, Fru, Glc, and Suc), it was necessary to analyze two

different amounts of the polar extracts: first, a small aliquot to integrate high-

abundance compounds in a precise manner, and second, a large aliquot to

allow determination of low-abundance metabolites.

Following deconvolution of resulting chromatograms, more than 400

individual compounds were detected in the polar extracts. Of these approx-

imately 130 polar compounds, including amino acids, organic acids, and

sugars were identified. Further automatic quantification was conducted using

the processing setup method built in the Xcalibur software (ThermoFinnigan)

on more than 70 identified and approximately 20 unknown polar metabolites.

For each targeted metabolite, a specific trace was selected and used for

quantification in each chromatogram. The resulting areas were normalized to

the area of a specific trace of the internal standard ribitol, resulting in relative

response ratios, which were further normalized by the fresh weight of each

sample.

Statistical Analysis

Data were prepared as described in Roessner et al. (2001) and presented as

x-fold compared to a reference, which is set to 1. If two observations are

described in the text as significantly different, this means that their difference

was determined to be statistically significant (P , 0.05) by the performance of

t test algorithms incorporated into Microsoft Excel and these values are

marked in bold in the Supplemental Tables. HCA and PCA were carried out

on the response per gram fresh weight raw data for each individual metabolite

and measurement following a transformation by log10 to allow better com-

parison of large and small numbers as described in Roessner et al. (2001). For

HCA, the Euclidean distance was used to calculate the matrix of all samples

and the complete linkage method for assignment of clusters. The results of the

PCA are presented in a two-dimensional graphical display of the data in which

a single sample is represented by a point in three-dimensional space. Both types

of statistical analyses were carried using Pirouette 3.11 software (Infometrix).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Table S1. Metabolite levels in roots of barley (Clipper and

Sahara) plants grown in 5, 200, and 1,000 mM B.

Supplemental Table S2. Metabolite levels in leaves of barley (Clipper and


Supplemental Table S3. Metabolite levels in roots of barley (Clipper and


Supplemental Table S4. Metabolite levels in leaves of barley (Clipper and


Supplemental Table S5. Metabolite levels in youngest fully developed

leaves of barley (Clipper and Sahara) plants grown in 5 and 1,000 mM B.

Supplemental Table S6. Metabolite levels in youngest fully developed

leaves of barley (Clipper and Sahara) plants grown in 5 and 1,000 mM B.

Supplemental Table S7. Metabolite levels in segments of the youngest

fully developed leaves of barley (Clipper and Sahara) plants grown in

5 and 1,000 mM B.

Supplemental Table S8. Metabolite levels in segments of the youngest

fully developed leaves of barley (Clipper and Sahara) plants grown in

5 and 1,000 mM B.

ACKNOWLEDGMENTS

We would like thank Dr. Tim Sutton, Australian Centre for Plant Func-

tional Genomics, University of Adelaide, Australia, for providing us with

barley cv Clipper and cv Sahara seeds. We are also grateful to Prof. Mark

Tester, Australian Centre for Plant Functional Genomics, University of

Adelaide, Australia, for discussing hydroponic growth of barley plants.

Special thanks to Dr. Ellen Zuther, Max-Planck-Institute for Plant Molecular

Physiology, Golm, Germany, for providing us with purified 6 kestose (Zuther

et al., 2004) for peak identification. U.R. thanks Suganthi Suren for help in

producing Figure 4.

Received May 23, 2006; accepted September 13, 2006; published September 22,

2006.

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an investigation of boron toxicity in barley using - plant physiology

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