Salt stress aggravates boron toxicity symptoms in banana leaves by impairing guttationp ce_2572...

Salt stress aggravates boron toxicity symptoms in bananaleaves by impairing guttationpce_2572 275..287

OR SHAPIRA1, YAIR ISRAELI3, URI SHANI2 & AMNON SCHWARTZ1

1The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, and 2The Department of Soil and WaterSciences, Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot 76100, and 3JordanValley Banana Research Station, Zemach 15132, Israel

ABSTRACT

Boron (B) is known to accumulate in the leaf margins ofdifferent plant species, arguably a passive consequence ofenhanced transpiration at the ends of the vascular system.However, transpiration rate is not the only factor affectingion distribution. We examine an alternative hypothesis, sug-gesting the participation of the leaf bundle sheath in con-trolling radial water and solute transport from the xylemto the mesophyll in analogy to the root endodermis. Inbanana, excess B that remains confined to the vascularsystem is effectively disposed of via dissolution in the gut-tation fluid; therefore, impairing guttation should aggravateB damage to the leaf margins. Banana plants were sub-jected to increasing B concentrations. Guttation rates weremanipulated by imposing a moderate osmotic stress. Gut-tation fluid was collected and analysed continuously. Thedistribution of ions across the lamina was determined.Impairing guttation indeed led to increased B damage tothe leaf margins. The kinetics of ion concentration in gut-tation samples revealed major differences between ionspecies, corresponding to their distribution in the laminadry matter. We provide evidence that the distributionpattern of B and other ions across banana leaves dependson active filtration of the transpiration stream and onguttation.

Key-words: apoplast; bundle sheath; hydathodes; laminamarginal vein; symplast; xylem.

INTRODUCTION

One striking characteristic of banana (Musa spp.) plantsgrown in the field under optimal conditions (i.e. warmnights, abundant water and intense mineral nutrition) is thehigh rate of guttation from their leaf margins. Guttationdrops appear soon after sunset and continue to exude untilthe evaporative demand exceeds root pressure-driven flow(usually after sunrise). We sampled guttation fluid collectedduring the night from commercially grown banana leaves toanalyse its chemical composition. Surprisingly, it con-tained high concentrations of boron (B), up to 0.37 mm

(unpublished results), even though the irrigation water andsoil solution in the area contained only 0.0046 mm B.Despite the high B concentration, no visual symptoms of Btoxicity were observed on the plants.

Guttation, the exudation of xylem sap from the aerialparts of the shoot, occurs in a wide range of herbaceousspecies, as well as in a number of woody ones (Curtis 1943;Ivanoff 1963). Guttation fluids have been found to containvarious salts (Curtis 1943; Ivanoff 1963; Dieffenbach,Kramer & Luttge 1980; Chen & Chen 2007), sugars (Curtis& Lersten 1974), amino acids (Pilot et al. 2004) and proteins(Komarnytsky et al. 2000). Although some authors havesuggested that salts are removed from the leaf via theirdissolution in the guttation fluid, a role for guttation inmaintaining ion homeostasis in the leaf has not been dem-onstrated experimentally (Ivanoff 1963; Tester & Daven-port 2003). Dissolved B was found at high concentrations inthe guttation fluid of barley seedlings and it was suggestedthat B efflux via guttation might participate in conferring Btolerance (Oertli 1962). This author demonstrated thatgiven the appropriate atmospheric conditions [close to100% relative humidity (RH)], continuous guttation canliterally clean the barley leaves of excess B (Oertli 1962).Sutton et al. (2007) hypothesized that guttation plays a rolein B tolerance of barley cultivars due to the presence of theB efflux protein Bot1 in association with hydathodes at thetip of the blade (Sutton et al. 2007). More recently, however,Reid & Fitzpatrick (2009) found no significant role for Befflux via guttation in conferring B tolerance among barleycultivars. They argued that even though some B is removedfrom the leaf via guttation, most of it is delivered to themain parts of the leaf (photosynthetic mesophyll) in thetranspiration stream (Reid & Fitzpatrick 2009). B is trans-ported, via the transpiration stream in the xylem, to the leafand at super-optimal concentrations, it tends to accumulatein leaf tips and leaf margins, causing margin damage (Oertli1994; Brown & Shelp 1997; Reid et al. 2004). Although spe-cific B-tolerance mechanisms exist at the leaf level (Reid &Fitzpatrick 2009), some B-tolerant genotypes are able tomaintain lower concentrations of B in their leaves (Nable,Bañuelos & Paull 1997; Hayes & Reid 2004). Bot1, a puta-tive B transporter, was found to be responsible for the Btolerance (low tissue content) in wheat and barley via effluxof B back to the soil solution at the root level (Reid 2007).

Correspondence: A. Schwartz. Fax: +972 89489069; e-mail:[email protected]

Plant, Cell and Environment (2013) 36, 275–287 doi: 10.1111/j.1365-3040.2012.02572.x

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© 2012 Blackwell Publishing Ltd 275

Similarly, the relative B tolerance of certain Brassica napusgenotypes is associated with their reduced net uptake of B,possibly as a result of active exclusion of the element, whichreturns to the soil, and decreased B content in the leavesdue to greater retention of B in the taproot and stem (Kauret al. 2006).

Therefore, generally speaking, B tolerance in crop plantsis not based on specific tissue tolerance but on avoidanceand sequestration in sacrificial tissues. Distribution patternsand specific accumulation of solutes such as B in leaf tipsand margins are poorly understood and are often relatedto passive accumulation at the end of the transpirationstream (Kohl & Oertli 1961; Wimmer et al. 2003). Recently,however, evidence of a more mechanistic cause for the phe-nomenon has been reported by our group. We proposed amodel describing the accumulation of Na+ in the laminamargins of banana plants, suggesting a role for the bundlesheath tissue in active filtration of the transpiration stream,protecting the photosynthetic tissue from toxic ions(Shapira et al. 2009). Na+ that reaches the leaf at super-optimal concentrations is effectively confined to the vascu-lar system, only to be disposed of in the leaf margin, asacrificial tissue. In the present study, we incorporate gutta-tion into our suggested model using B, K and Cl to trace thefate of minerals arriving in the leaf at super-optimal con-centrations. The interaction between salt and B stress isexplored at the leaf level. We show that excess B is effi-ciently confined to the vascular system during the day andthat guttation serves as a means for the disposal of excess Bat night.

MATERIALS AND METHODS

Effect of B concentration supplied to plants onB distribution in the leaf (experiment 1)

Plant material and treatmentsTissue culture-propagated banana plantlets (Musacavandishii cv. Grand Nain AAA), 15 cm tall with four fullyunfurled green leaves, were planted on 20 April 2003 in 40 Lcontainers filled with commercial organic potting mixture.The plants were irrigated twice daily with a nutrient solu-tion containing (in mm) 7.5 N, 2.5 K, 2.4 Ca, 1.1 Mg, 2 Na, 1.7Cl, 1.5 P, 0.54 S, 0.014 Zn, 0.034 Mn, 0.18 Fe, 0.002 Cu, 0.001Mo and 0.0046 B. One week after planting, the plants wereseparated into six treatments, three plants per treatment,and boric acid (H3BO3) was added to the irrigation solutionto final concentrations of 0.0046 (control), 0.23, 0.46, 1.1, 1.6and 2.2 mm. The plants were irrigated with the appropriatenutrient solution two to three times a day until solutiondrained freely from the bottom of the containers. Theplants were grown in an environmentally controlledgreenhouse under natural light with maximal photosyn-thetic photon flux density (PPFD) of 1400 mmol m-2 s-1.Temperature reached a maximum of 34 °C during the dayand a minimum 26 °C at night. RH was maintained ataround 60% during the day and close to 92% at night. Theexperiment was terminated on 29 June 2003.

Mineral analysisAt the end of the experiment, the plants were harvested,and the lamina of the four youngest leaves of each plant(average lamina length 101.3 � 8.6 cm and average laminawidth 50.5 � 3.9 cm, n = 36) were divided lengthwise intofour equal tissue strips comprising different regions of thelamina from the margins (1) to the midrib (4). Lamina partswere washed with distilled water to remove any salt pre-cipitates from the margins and oven-dried for 3 d at 70 °C.B concentration in the different tissues was measured byICP (Spectroflame, Model ARCOS; Spectro GmbH, Kleve,Germany).

Experimental design and statistical analysisThe experiment was arranged in a randomized blockdesign. Non-linear regression methods were applied todescribe the response of B content to increasing distancefrom the midrib (Sigmaplot 9; Systat Software Inc., SanJose, CA, USA). The effects of increasing B concentrationand distance from the midrib were assessed by two-wayanalysis of variance (anova) and the means were separatedby Tukey–Kramer HSD test using JMP 5 (SAS InstituteInc., Cary, NC, USA).

Marginal vein anatomyAnatomical cross sections were cut from fresh leaf tissueembedded in 7% (w/v) low-melting agarose (Conda Labo-ratories, Madrid, Spain), after which they were sectioned tobetween 40 and 60 mm using a vibratome (VT 1000S; Leica,Wetzlar, Germany).

Total xylem volume and leaf-specific weightThe bulk volume of the conducting xylem vessels of the leaf(excluding protoxylem and transverse xylem) was esti-mated by counting the number of parallel veins in 1 cm2 leafsections and measuring the average radius of the singlemetaxylem vessel in each vein. Volume was then calculatedon a leaf area basis by multiplying vein number by theaverage volume of a single cylinder-shaped vessel, accord-ing to House & Findlay (1966). Leaf-specific fresh weightwas determined from the same leaf sections.

Mesophyll flooding and guttation photographyGuttating leaves of 6-month-old field-grown banana plants(average height 1.3 m) were photographed 1 h after sunrise.

Interactive effects of rising salt and Bconcentrations on guttation and Bconcentration in the lamina (experiment 2)

Tissue culture-propagated banana plantlets (cv. Grand NainAAA), 15 cm tall with four fully unfurled green leaves,were planted on 25 August 2006 in 25 L pots filled with

276 O. Shapira et al.

© 2012 Blackwell Publishing Ltd, Plant, Cell and Environment, 36, 275–287

commercial organic potting mixture, in an environmentallycontrolled greenhouse as in experiment 1. The plants wereirrigated daily with nutrient solution containing (in mm): 7.5N, 2.5 K, 2.4 Ca, 1.1 Mg, 2 Na, 1.7 Cl, 1.5 P, 0.54 S, 0.014 Zn,0.034 Mn, 0.18 Fe, 0.002 Cu, 0.001 Mo and 0.0046 B. Treat-ments were begun 4 weeks after planting. KCl was added tothe basic irrigation solution to impose a moderate osmoticstress and B (as H3BO3) was added to the basic solutionsaccording to the different treatments.

A full factorial experiment was established with threesalinity levels: 2.5 mm KCl (control, S1), 20 mm KCl (S2)and 42 mm KCl (S3) and two levels of B: 0.0046 mm(control, B1) and 1.1 mm (B2). Each treatment consisted offour replicates (plants) set up in randomized blocks. Plantswere watered twice a day until the solution drained freelyfrom the base of the pot. The experiment was terminatedafter 70 d.

Collection of guttation fluid from thewhole plantGuttation fluid was collected by first washing the leafmargins with distilled water to remove any salt precipitatesin the morning, then inserting the entire shoot of intactplants into a plastic bag in the late afternoon and laying theplants horizontally for the entire night. The bag was leftopen so that the atmosphere inside it would remain as closeto ambient as possible. Guttation fluid was collected fromthe bag the next morning, its volume was determined and itsmineral composition was analysed by ICP. All plants weresampled the same day Sampling was repeated four times inthe last 4 weeks of the experiment.

Collection of guttation fluid from a single leafGuttation fluid was collected continuously from the thirdfully unfurled leaf for 5 h. The leaf margin was firstwashed with distilled water in the morning to remove saltprecipitates. Sap collection was begun about 40 min beforedarkness when the first drop of guttation appeared. Eachdrop was collected immediately as it appeared using apipette and transferred to an Eppendorf tube. Collectionproceeded in 0.6 mL aliquots, noting the starting time ofcollection into each tube. For the rest of the night, the leafwas enclosed in a plastic bag that was left partially open toapproximate ambient atmosphere. Guttation liquid wascollected from the bag the next morning and its volumerecorded. All guttation samples were analysed for mineralcontent by ICP. Sap collection and analysis were repeatedthree times during the last 4 weeks of the experiment.

Mineral analysis of plant tissueAt the end of the experiment, the laminas of the fouryoungest leaves of each plant were washed with distilledwater, ensuring that all precipitates from evaporation ofguttation drops were removed. The lamina was furtherdivided into a 4-cm-wide marginal strip and the remaining

central lamina. Lamina parts were digested in nitric acidand analysed by ICP for their mineral content. Chloridewas extracted from the dry plant material using double-distilled water and measured using a chloride-specificelectrode (Chloride Analyzer 926, Corning, Halstead,England).

Experimental design and statistical analysisThe experiments were arranged in a randomized blockdesign. Linear and non-linear regression analyses wereperformed using Sigmaplot 9. The effects of increasingKCl and B concentrations on guttation parameters andmineral analysis were assessed by two-way anova andseparation of means by Tukey–Kramer HSD test usingJMP 5 (SAS Institute Inc.). Student’s t-test was used incomparing the ion content between lamina and marginpairs (JMP 5).

RESULTS

Effects of increasing B concentration in theirrigation solution on B distribution in thelamina (experiment 1)

Boron distribution in the laminaThe distribution of B in the dry matter of different parts ofthe lamina was monitored after separating the laminalengthwise into four equal quarters (strips 1 to 4, Fig. 1)from the midrib to the margin. In all treatments, B contentwas significantly higher in strip 4, containing the laminamargin, than in inner-tissue strips 1, 2 and 3. B content in thedry matter of strip 4 was 130 mmol kg-1 dry weight (DW) inplants irrigated with 2.2 mm B, whereas in control plantsreceiving only 0.0046 mm B, the corresponding tissue con-tained only 13 mmol B kg-1 DW. B content in strip 1, next tothe midrib, was 17 mmol kg-1 DW when the plants received2.2 mm B and 2.5 mmol kg-1 DW in plants of the controltreatment. B content in the margin (strip 4) was 8 mmol, 52mmol and 75 mmol higher in the 0.046 mm, 1.1 mm and2.2 mm treatments, respectively, than that expected if Bcontent had risen linearly in the lamina from the midrib(strip 1) to the margin (strip 4; Fig. 1).

Boron damage symptoms and lamina anatomyVisible symptoms of severe and prolonged stress causedby excess supply of B included typical chlorotic laminamargins and the accumulation of white precipitates on theepidermis at the extreme margins above the hydathodes(Fig. 2a,b). The white precipitate was gently scrubbed offthe leaf, collected, dissolved in 1% nitric acid (some of thepowder did not dissolve) and the solution was analysedfor its mineral content by ICP: 14.3% of the powder waselemental B, with other major components being 0.3% Ca,0.2% Na, 0.17% Mg, 0.15% K and 0.12% Mn.

A schematic representation of the venation in a bananaleaf is shown in Fig. 3a. In monocotyledons, the leaf vascular

Leaf venation and boron secretion by guttation 277


network consists of a hierarchical sequence of vertical vas-cular bundles and numerous transverse veins that intercon-nect adjacent vertical veins. The lamina of banana leafshows typical pinnate venation (Skutch 1927). Parallelveins, about 100 mm apart, branch perpendicular to thelamina midrib and continue across the lamina until most ofthem turn acropetally (towards the tip of the lamina) andmerge into a large marginal vein that encircles the entirelamina about 1 mm from its edge. Small transverse veinsinterconnect adjacent parallel veins.The marginal vein con-tains a number of large xylem vessels that occupy most ofthe lamina cross section (Fig. 3b) but unlike the vasculartissue of the parallel mid-lamina veins, the vascular tissue ofthe marginal vein is not surrounded by a defined layer ofbundle sheath cells (Fig. 3c). Figure 3d shows a cross sectionof the different orders of parallel veins. The conductingtissues are enclosed by one to several layers of tightlyarranged bundle sheath parenchyma cells with bundlesheath extensions to the epidermis on both sides of thelamina (Fig. 3e; Skutch 1927; Shapira et al. 2009). The trans-verse veins are enclosed by a bundle sheath but lack thebundle sheath extensions. The bulk conducting volume ofthe leaf xylem was estimated to be 6.41 � 0.73 mL m-2.Leaf-specific weight was 480 � 53 g m-2.


Whole-plant guttationGuttation sap was collected from intact banana plantsduring the night by enclosing the entire shoot in a plastic bagand laying the potted plant horizontally.The plastic bag wasleft fully open on the pot side and the RH inside the bag wasvery close to ambient.The sap was collected from the bag inthe morning, and its volume and B concentration were mea-sured (Fig. 4). The volume of guttation per leaf area forplants that were irrigated with the basic nutrient solutioncontaining only 2.5 mm KCl (control solution; treatmentS1B1) was 432 mL m-2 per night. Addition of 17.5 mm KCl(treatment S2B1) and 39.5 mm KCl (S3B1) to the irrigationsolution reduced the volume of guttated sap to 42 and 17%,respectively, of the volume guttated from control plants.When B was added to the irrigation solution to a finalconcentration of 1.1 mm (treatment S1B2), the volume ofguttation decreased by about 20%.Similarly,addition of B tothe solution with 20 mm and 42 mm KCl (S2B2 and S3B2,respectively) further reduced the guttated sap volume,albeitto a lesser extent (Fig. 4a). Increasing B concentration in theirrigation solution from 0.0046 mm (S1B1) to 1.1 mm (S1B2)increased B concentration in the guttated sap from 0.05 mmto 1.2 mm, while reducing the volume of guttation per leafarea by 20% (Fig. 4a,b). Sap B concentration was 0.05, 1.2,0.065, 2.13, 0.25 and 4.53 mm for treatments with 2.5 mm KCl(S1B1), 1.1 mm B (S1B2), 20 mm KCl (S2B1), 1.1 mmB + 20 mm KCl (S2B2), 42 mm KCl (S3B1), and 1.1 mmB + 42 mm KCl (S3B2), respectively (Fig. 4b).

The amount of B excreted per leaf area during thenight from a whole plant irrigated with nutrient solution

Figure 1. B content gradient across the lamina of banana plantsirrigated with increasing B concentrations. The lamina wasdivided into four strips of equal width, (see Fig. 3a) starting fromthe midrib (strip 1) and proceeding to the lamina margins (strip4). The irrigation solution contained 0.0046 mm B (filled circles,control treatment), 1.1 mm B (empty circles) and 2.2 mm B (filledtriangles). Factorial analysis of variance was significant (R2 = 0.84,P < 0.001) and B concentration in the irrigation solution, distancefrom the midrib and their interaction were highly significant aswell (P < 0.0001, <0.0001 and <0.0001, respectively). Separation ofmeans within each distance: * means for 1.1 and 2.2 mm B aresignificantly different from 0.0046 mm B and ** all means aresignificantly different from each other. Dotted line representspredicted B concentration in lamina margins (strip 4) based onlinear regression R2 > 0.98) of B concentration of the first threestrips had the B accumulation been linear. Each data pointrepresents the mean � SE of three plants.

Figure 2. (a) Typical damage to the photosynthetic tissue at thelamina margins resulting from B accumulation. (b) Accumulationof white salt crystals on the adaxial epidermis of the laminamargin next to the hydathodes. The plant was irrigated withnutrient solution containing 1.6 mm B. Salt crystal accumulationis a result of water evaporation from the B-enriched guttationdrops.



Figure 3. (a) Schematic drawing of the parallel-veined lamina of banana. First- and second-order parallel veins, alternately arranged,merge in the lamina margins into a large vein that encircles the entire lamina (inset). (b) Cross section of the lamina margin of a maturebanana leaf, perpendicular to the marginal vein and parallel to the parallel lamina veins. (c) The vascular bundle of the marginal veincontains a number of large meta-xylem vessels that occupy about half of the lamina cross section and little or no phloem. No bundlesheath tissue is present. (d) In contrast, the parallel veins that cross the lamina from the midrib to the margins are surrounded bywell-defined bundle sheath tissue shown in cross section. (e) Close-up view of major parallel vein showing bundle sheath (BS) andextensions. c.s, cross section; E, epidermis; MW, marginal wing; MX, metaxylem; Ph, phloem; UM, upper mesophyll; WT, water tissue;XV, xylem vessel.



containing 1.1 mm B was 22 times higher than that from thecontrol plants irrigated with nutrient solution containingonly 0.0046 mm B, that is 0.419 � 0.039 mmol m-2 versus0.019 � 0.00065 mmol m-2 (S1B2 and S1B1 treatments,respectively, Fig. 4c). The moderate osmotic stress inducedby increasing the KCl concentration of the irrigation solu-tion to 20 mm had no significant effect on the amount of Bexcreted by the plants compared to the amount excreted bythe control plants (S2B1 and S1B1, respectively). Additionof 20 mm KCl to the irrigation solution containing 1.1 mm B(S2B2) did not significantly reduce the amount of Bexcreted from the whole plant (through volume concentra-tion interaction), but increasing KCl concentration to42 mm (S3B2) reduced the amount of B excreted throughguttation by 44% compared to the amount excreted in thepresence of 1.1 mm B (S1B2) and 20 mm KCl + 1.1 mm B(S2B2; Fig. 4c).

Single-leaf guttationThe rate of guttation and the kinetics of B level in theguttation sap were monitored by collecting and analysingsap samples during the evening and overnight from thethird fully expanded leaf, which is the first fully mature leaffrom the top of the plant. Collection started from the firstdrops that appeared on the lamina margins in the earlyevening (around 1800 h) and continued until about 2300 h.The guttation sap was pipetted into small vials in 0.6 mLaliquots and the time of filling of each vial was noted. Afterabout 5 h of continuous sap collection, the lamina of thethird leaf was enclosed in a partially open plastic bag andthe sap that exuded during the rest of the night was col-lected from the bags in the morning. The change in sap Bconcentration from one collection night (two additionalexperiments are presented in Supporting InformationFig. S1) is presented in Fig. 5. B concentration in the first0.6 mL aliquot (the first sap exuded) collected in theevening was maximal, at 0.37, 0.74, 18 and 32.4 mm for S1B1,S2B1, S1B2 and S2B2, respectively (Fig. 5a–d, respectively).From then on, B concentration decreased sharply, reaching0.05, 0.30, 5.58 and 12.13 mm, respectively, after about 4.5 h.The volume of sap that accumulated in the plastic bag untilthe morning (on a leaf area basis) was 174.4, 35.1, 104.8 and31.9 mL m-2 and its B concentration was 0.03, 0.28, 5.46 and12.8 mm, for treatments S1B1, S2B1, S1B2 and S2B2,respectively, very similar to B concentrations at the end ofthe continuous pipette-sampling period. B concentrationkinetics could be divided into two phases (dotted lines inFig. 4). The first was a sharp linear decrease in B concentra-tion and the second was a steady-state concentration of Bcorrelated with the B concentration in the samples col-lected in the morning, representing the final and steady-state B concentration of the exudates. S1B1 and S1B2plants showed a relatively long steady-state phase (Fig. 5a,c,respectively) compared to the S2B1 and S2B2 plants(Fig. 5b,d, respectively). Plants of the S3B1 and S3B2 treat-ments, which are not presented graphically, exuded only1.2 mL of xylem sap in 4.5 h (in two 0.6 mL aliquots) and

Figure 4. Whole-plant guttation as affected by 40 d of irrigationwith nutrient solutions containing six combinations of KCl and Bconcentrations: S1B1 (control) = 2.5 mm KCl and 0.0046 mm B;S1B2 = 2.5 mm KCl and 1.1 mm B; S2B1 = 20 mm KCl and0.0046 mm B; S2B2 = 20 mm KCl and 1.1 mm B; S3B1 = 42 mmKCl and 0.0046 mm B; and S3B2 = 42 mm KCl and 1.1 mm B.Average whole-plant leaf area was 0.776 � 0.014 m2 for alltreatments and was not significantly different between treatmentsat this stage of the experiment (P = 0.35). (a) Guttation volumeper leaf area. Factorial analysis of variance was significant(R2 = 0.98, P < 0.001), and concentrations of KCl, B and theirinteraction were highly significant as well (P < 0.001, <0.001 and=0.004, respectively). (b) Concentration of B in whole-plantguttation. Factorial analysis of variance was significant (R2 = 0.97,P < 0.001), and concentrations of KCl, B and their interactionwere highly significant as well (P < 0.001, <0.001 and <0.001,respectively). (c) Amount of B discarded daily from the plant viaguttation per leaf area. Factorial analysis of variance wassignificant (R2 = 0.95, P < 0.001) and concentrations of KCl, B andtheir interaction were highly significant as well (P = 0.002, <0.001and =0.002, respectively). Treatments sharing the same uppercaseletter are not significantly different (Tukey HSD, P < 0.05). Eachdata point represents the mean � SE of four plants.



their B concentrations were 7.6 and 10 mm in the S3B1treatment and 153.6 mm and 98.1 mm B in the S3B2 treat-ment for the respective aliquots.

The rate of sap guttation during the first 4.5 h was mark-edly affected by the treatments (Table 1, slope of linearregression calculated from data in Fig. 5, R2 > 0.98). The

overnight volume of guttation fluid from the third leafdecreased by about 50% (from 269 to 131 mL m-2) whenthe irrigation solution was switched from S1B1 to S1B2.Application of 20 mm KCl (S2B1) decreased guttation byabout 75% compared to the S1B1 treatment (Table 1). Thecombined effect of KCl and B (S2B2) further decreased the

Figure 5. Kinetics of B concentration measured in guttation samples collected continuously for 5 h in 0.6 mL aliquots from the thirdfully unfurled leaf. Each data point represents one aliquot. Nutrient solutions were as described in legend to Fig. 4. (a) S1B1. (b) S2B1.(c) S1B2. (d) S2B2. The experiment was repeated three times with results showing similar trends but the total amount of guttation variedgreatly between collection nights (see Supporting Information Fig. S1).

Table 1. Effects of KCl and B concentrations in the nutrient solution on time of guttation initiation in the evening, rate of guttationduring the continuous sampling period, volume of sap exuded overnight and amount of B discarded from the leaf. Sap was collected fromthe third fully unfurled leaf. Nutrient solution treatments were as follows: S1B1 (control) = 2.5 mm KCl and 0.0046 mm B, S1B2 = 2.5 mmKCl and 1.1 mm B, S2B1 = 20 mm KCl and 0.0046 mm B, S2B2 = 20 mm KCl and 1.1 mm B, S3B1 = 42 mm KCl and 0.0046 mm B andS3B2 = 42 mm KCl and 1.1 mm B. Results were compiled from data presented in Fig. 5, including data for treatments S3B1 and S3B2which are not presented graphically in Fig. 5 (see text)

TreatmentStart of guttation(time of day)

Rate of guttation(mL m-2 h-1)

Guttation volume(mL m-2)

Total B discarded(mmol m-2)

S1B1 (control) 1800 14.01 236 0.018S1B2 1815 6.41 125 0.85S2B1 1830 7.77 64 0.039S2B2 1840 4.55 49 0.75S3B1 2110 < < 1 5 0.022S3B2 2110 < < 1 4 0.58



guttation volume, and KCl at a final concentration of 42 mmwith or without additional B decreased the volume evenmore (5 and 4 mL m-2, respectively). The total amount of Bexcreted from the leaf was affected by the treatments, withleaves of the plants treated with S1B1 and S2B1 excretingmarkedly less B than the S1B2 plant (Table 1). Decreasedguttation of the S2B2 plant reduced the amount of excretedB by 12% compared to S1B2. Increasing the final concen-tration of KCl to 42 mm and B to 1.1 mm (S3B2) furtherdecreased the amount of B excreted from the leaf by 33 and22% relative to the S1B2 and S2B2 plants, respectively(Table 1).

Figure 6 shows the concentration kinetics of several otherkey minerals from the same guttation samples. The concen-tration of Fe (Fig. 6a) in the guttation sap of a control plantwas 6 mm in the first sap aliquot collected in the earlyevening. It decreased rapidly to around 1 mm after 30 min(the third aliquot). For the rest of the evening, Fe concen-tration remained low (except for aliquot 8), between 0 and2 mm. In the presence of 20 mm KCl (which resulted inreduced rate and total volume of sap exuded), Fe concen-tration was very similar to that of the control plant althoughit lagged behind by 30 min. The concentration of Mn(Fig. 6b) in the exuded sap was around 30 mm during the4.5 h of continuous sap collection from the control plants,while in the treatment with 20 mm KCl, Mn concentrationdecreased during the same period from about 100 to 40 mm.The two first aliquots of sap contained K+ at about 1 mm,whereas most of the aliquots collected thereafter, until2300 h, contained K at less than 0.4 mm in the control treat-ment and between 0.2 and 0.6 mm when the irrigationsolution was of higher osmotic potential and higher K con-centration (Fig. 6c).The concentration of Ca (Fig. 6d) in thereleased sap increased from around 0.4 mm in the first fouraliquots to around 0.8 mm Ca in the last four aliquots ofthe control treatment. In the presence of 20 mm KCl, the

concentration of Ca decreased from around 0.6 mm in theearly evening to around 0.4 mm after 5 h.

Mineral analysis of the laminaAt the end of the experiment, leaves from which guttationfluid had been collected during the night were detachedfrom the plants and their lamina divided into its middle partand margins (4 cm wide strips of tissue at the margins) todetermine B content in the tissue. The moderate osmoticstress induced by adding KCl to the irrigation solution(accompanied by decreasing guttation rates, see Fig. 4 andTable 1 for effect on guttation) had only a slight negativeeffect on B content in the lamina: 11.3, 8.8 and 8.6 mmolkg-1 DW in the middle of the lamina in the presence of 2.5,20 and 42 mm KCl (S1B1, S2B1 and S3B1 plants, Fig. 7) inthe irrigation solution, respectively. When 1.1 mm B wasadded to the irrigation solution that contained 2.5, 20 or42 mm KCl (S1B2, S2B2 and S3B2 plants, accordingly), Bcontent in the mid-lamina increased to 19.7, 24.9 and22.5 mmol kg-1 DW, respectively. In comparison to the mid-lamina, the levels of KCl and B in the irrigation solutionhad a significant effect on the amount of B in the laminamargins. Increasing the salt levels in the presence of 1.1 mmB increased B content in the lamina margins from76.4 mmol kg-1 DW at 2.5 mm KCl to 125.4 mmol kg-1 DWat 20 mm KCl, and 158.7 mmol kg-1 DW at 42 mm KCl(S1B2, S2B2 and S3B2 plants, respectively; Fig. 7). Whenthe irrigation solution contained only 0.0046 mm B, the

Figure 6. Concentration kinetics during continuous collectionof additional ions measured in samples S1B1 (control, fullcircles) and S2B1 (20 mm KCl, empty circles) presented in Fig. 5.Each data point represents one 0.6 mL aliquot of guttation fluid.(a) Fe. (b) Mn. (c) K. (d) Ca. Kinetics is presented for twoguttation rates (see Table 1).

Figure 7. B content in the tissue from the middle part of thelamina (lamina) and lamina margins (margins) as affected by70 d irrigation with nutrient solutions containing six differentcombinations of KCl and B concentrations (see legend to Fig. 4).Lamina: factorial analysis of variance was significant (R2 = 0.89,P < 0.001). KCl concentration was not significant (P = 0.52). Bconcentration was significant (P < 0.001). Margins: factorialanalysis of variance was significant (R2 = 0.91, P < 0.001), and KClconcentration, B concentration and their interaction were highlysignificant as well (P = 0.005, <0.001 and =0.003,respectively).Treatments sharing the same uppercase letter arenot significantly different (Tukey HSD, P < 0.05). Presented dataare the average of four plants per treatment � SE.



addition of 20 mm or 42 mm KCl did not significantly affectB content in the lamina margins (compare S1B1, S2B1 andS3B1, Fig. 7). B content was significantly affected only in thelamina margins of the plants irrigated with the high B con-centration (S1B2, S2B2 and S3B2). Surprisingly, the contentof Mn in lamina margins of the control treatment (irrigatedwith a balanced nutrient solution, S1B1, containing0.034 mm Mn) was four times higher in the dry matter of thelamina margins (14.1 mm in strip 4) than in the middle partsof the lamina (Fig. 8a). Similar to the effect of decreasedguttation on B content in the margins, in the presence of42 mm KCl (S3B1, resulting in decreased guttation), Mncontent in the lamina margins increased significantly to18.3 mm compared to its concentration in the margins of thecontrol plants (S1B1, P = 0.0177; Fig. 8b). The content of Fewas not significantly different between the lamina and themargins and Zn was equally distributed between the twoparts of the lamina as well (Fig. 8a,b).

Analysis of the spatial distribution of macronutrients Na,Mg, Ca, P, S, in the lamina (strips 1, 2, 3 versus strip 4; Fig. 1)when supplied in a balanced nutrient solution (controltreatment) revealed that in contrast to B and Mn, none ofthese elements were concentrated at the lamina margin(Supporting Information Fig. S2). When KCl concentrationof the nutrient solution was raised to 20 and 42 mm (S2B1and S3B1), the content of K was increased in the mid-partof the lamina from 408.9 mmol kg-1 to 1194.8 mmol kg-1

and 1245.1 mmol kg-1, respectively, and that of Cl from481.7 mmol kg-1 to 977.4 mmol kg-1 and 991.5 mmol kg-1,respectively. K and Cl contents were somewhat lower in themargins (Fig. 9a,b).

Guttation and mesophyll floodingA typical guttation pattern is presented in Fig. 10a. Gutta-tion drops are shown on the adaxial surface at their point ofemergence, usually above the marginal vein. Once theyexceed a threshold size and weight, the drops slide over thelamina surface. Most guttation drops end up falling to the

ground. Only a small proportion of the total guttationevaporates while still on the leaf margins.Another phenom-enon that often occurs at the same time is ‘flooding’ of theintercellular air spaces of the mesophyll in the laminamargins with xylem sap. The positive hydraulic pressurethat exists in the xylem during the night and early morningis released mainly by guttation. However, in banana, it isalso released by flow of sap from the xylem of the marginalvein, which lacks a complete bundle sheath, backward intothe mesophyll, between the parallel veins of the lamina.This phenomenon, termed mesophyll flooding, occurs moreoften in mature leaves, appearing as incursions of darkercoloured tissue progressing from the margins into thelamina between the parallel veins (Fig. 10b).The flow of sapbackward into the mesophyll between the veins is allowedsince the transverse veins that interconnect adjacent paral-lel veins lack bundle sheath extensions (Skutch 1927). Theflooded area advances from the marginal vein towards themidrib and its primary width decreases in well-defined dec-rements until it ends abruptly (Fig. 10b). When the stomataopen and transpiration starts, the spaces between the meso-phyll cells ‘dry out’ and refill with air.

Figure 8. Tissue content of additional microelements inresponse to decreased guttation rate due to irrigation underincreasing osmotic stress (KCl) and B stress. (a) S1B1(control) = 2.5 mm KCl and 0.0046 mm B. (b) S3B1 = 42 mm KCl.For effect on guttation see Table 1. Each bar representsmean � SE (n = 4). Black bars, lamina; white bars, margins.*Significantly different lamina margin pairs (Student’s t-testP < 0.05).

Figure 9. (a) Response of tissue K and (b) Cl contents underincreasing KCl concentration (resulting in decreased guttationrates as shown in Table 1). Filled circles, lamina; empty circles,lamina margins. Presented data include only KCl treatments with0.0046 mm B [S1B1 (control) = 2.5 mm KCl, S2B1 = 20 mm KCland S3B1 = 42 mm KCl]. Each point represents mean � SE(n = 4). *Significantly different lamina margin pairs (Student’st-test P < 0.05).



DISCUSSION

Studies on B-tolerant barley varieties and certain B. napusgenotypes have emphasized avoidance of B toxicity to theleaf by exclusion at the root and stem level, rather than therole of increased tissue tolerance (Hayes & Reid 2004; Kauret al. 2006; Sutton et al. 2007). In this study, we show that inbanana, excess B arriving to the leaf is effectively confinedin the vascular system of the lamina during the day and isdumped into the lamina margins. A significant amount ofthe B accumulated in the vascular system and leaf marginscan then be removed from the leaf via its dissolution in theguttation fluid later at night.

Effect of B concentration supplied to the plantson B distribution in the leaf (experiment 1)

B is not distributed homogeneously throughout the lamina:a significant proportion of it accumulates in the laminamargins (Fig. 1). In the inner areas of the lamina, B contentincreases linearly in the first three tissue strips from themidrib towards the margins when B concentration in theirrigation is increased, but B content in the fourth strip

(marginal tissue) deviates from this linearity, increasing byabout twofold (Fig. 1 dotted lines). Non-homogeneousaccumulation of B in leaf margins has been observed inother species as well. Kohl & Oertli (1961) reported a non-linear increase in B content towards the leaf tip in Liliumlongiflorum. In oil palm, B content increases linearly fromthe base of the leaflet towards the tip and margin (Rajarat-nam 1972). Wimmer et al. (2003) observed increasing Bcontent and damage in the leaf tips but not in the basal leafparts of wheat under B stress. They also reported aggrava-tion of B damage symptoms in the margins under salineconditions. Ion accumulation in the leaf margins has fre-quently been attributed to passive flow of xylem sap to theends of the transpiration stream at the lamina margins(Kohl & Oertli 1961;Wimmer et al. 2003). But the end of thetranspiration stream is actually around the stomatal cavitiesacross the entire lamina surface.Therefore, accumulation ofsolutes in the lamina margins or at the ends of the vascularbundles cannot be purely a consequence of the transpira-tional flow (Canny 1990; Fricke 2004). Leaf margins areconsidered to enhance transpiration rates due to the nar-rower boundary layer around them. Moreover, a highertranspiration rate means that more solutes are left behindand their concentration increases. However, transpirationrates from a leaf depend strongly on stomatal conductanceand in general, stomata tend to have smaller apertures andclose more readily at the leaf margins than at its centre(Willmer & Fricker 1996; see measurements in Shapiraet al. 2009 and Supporting Information Table S1). Fricke(2004) studied the role of transpiration in solute sortingusing barley leaves, and concluded that transpiration onlyenhances the differences in solute concentration that aredependent on specific cell and tissue properties. Canny(1990) showed that the concentration increase of dye at thetranspiring edge of a filter paper wick (simulating themargins of a transpiring leaf) is dependent on the sweepingof dye to the margins and not only on enhanced evapora-tion.Therefore, only those solutes that are actively confinedto the vascular system will tend to concentrate significantlytowards the end of the vascular system. In banana, theentire vascular system of the leaf converges and pours intothe leaky marginal vein (Skutch 1927; Shapira et al. 2009).The deviation from linearity in B content at the leaf margin(Fig. 1) is a result of B-enriched sap being sucked into themarginal mesophyll from the marginal vein during transpi-ration (Shapira et al. 2009). In contrast, solutes that areactively taken up from the xylem may be found at higherconcentrations in the middle of the leaf (Fig. 9; Kerton et al.2009).


The relationship between the amount of B excreted dailyfrom the leaves via guttation and B content in the leaf tissuewas studied by imposing a moderate osmotic stress on aset of plants, to reduce the rate and volume of the daily

Figure 10. (a) Banana leaf showing guttation drops at the pointof emergence (adaxial side). (b) Banana leaf showing mesophyllflooding at the lamina margins (abaxial side). During the nightand early morning, when xylem pressure becomes positive, veinrupture is prevented. Firstly, xylem sap is released throughhydathodes that are located in the adaxial epidermis above themarginal vein and guttation drops appear (a). Secondly, if therate of root pressure-driven flow exceeds the rate of sap releasethrough the hydathodes, sap leaks through the bundle sheathlessmarginal vascular bundle and progresses backwards into theadjacent mesophyll causing marginal ‘mesophyll flooding’ (b).



guttation. The choice of KCl as the preferred osmoticumstemmed from previous work showing that NaCl-basedosmoticum results in severe Na-specific damage to the leafmargins, interfering with the process of guttation. In con-trast, increasing KCl concentration caused no damage tothe margins (Shapira et al. 2009). Importantly, KCl had apronounced effect on guttation rate and volume (Fig. 4 andTable 1) but only a moderate effect on stomatal conduc-tance and transpiration (Supporting Information Table S1).Reduced guttation rate and volume under increasingosmotic stress (Fig. 4a and Table 1) resulted in increased Bconcentration in the guttated sap (Figs 4b & 5). The com-bined salt and B stress led to less guttation (on a leaf areabasis) and less B being discarded from the plant (Fig. 4c andTable 1), leading to more B accumulation, especially in themargins (Fig. 7). Most of the experimental data in recentliterature on the response of plants to the combined stressof excess salinity and high B concentrations have been sum-marized and analysed by Yermiyahu et al. (2008). Thoseauthors describe two contrasting patterns of response tocombined salt and B stress: in the first, salt stress results inincreased B content in the leaves; in the second, plantsdisplay less severe signs of B toxicity and lower B concen-tration in the leaves when they are exposed to high levels ofsalt and B simultaneously (Yermiyahu et al. 2008 and refer-ences therein). Interestingly, separate studies using thesame species yielded opposite results with respect to Bdamage to the leaves in response to salt stress. All of theplants that were found to respond to salt stress withelevated B content in their leaves were plants that areknown to guttate, given the appropriate conditions. Never-theless, guttation has never been reported as a possiblefactor affecting B distribution and content in response tocombined salt and B stress.As we show in banana, salt stressaggravates B toxicity symptoms in the leaves by impairingguttation and reducing the amount of B discarded from theleaves. We also suggest that even very low, possibly unno-ticeable rates of guttation (e.g. 1.2 mL per night with anaverage 100 mm B for plants irrigated with 42 mm KCl and1.1 mm B, Table 1) could result in the excretion of largeamounts of B on a daily basis and have a cumulative effecton B content in the tissue.

The kinetics of B concentration in guttation samplesdemonstrates how guttation cleans the leaf of excess Baccumulated during the diurnal period of transpiration. Bconcentration is maximal in the first guttation samples of alltreatments, followed by a sharp linear decrease to a steady-state value after a few hours (Fig. 5). The first linear stagecan be attributed to replacement of the total xylem volumeof the leaf by fresh incoming sap. According to our mea-surements, the volume of sap exuded during this stage wasin the same range as the bulk xylem volume determined forbanana leaves (6.41 � 0.68 mL m-2). At the end of the day,xylem sap in the leaf is enriched with B compared with saparriving from the roots due to the slow rate of tissue uptakeand ‘filtration’ conferred by the bundle sheath tissue. ExcessB that enters the bundle sheath cells might be expelled backto the xylem by specific B efflux transporters such as those

found in roots and hydathodes of certain barley cultivars(Reid 2007; Sutton et al. 2007). Due to the mass flowtowards the end of the vascular system at the margins, a Bconcentration gradient builds up inside the vessels, increas-ing from the midrib to the margins. This B concentrationgradient in the xylem is mirrored by a sharp linear decreasein B concentration in the guttation sap (Fig. 5). At the endof the continuous pipette sap collection period, B concen-tration was similar to that of the sap accumulated in theplastic bag during the rest of the night, and therefore weassume that B concentration in guttation sap reached asteady-state phase after about 5 h in all treatments. Oertli(1962) has shown that B remains mobile in the leaf and canbe removed via guttation. Accordingly, B concentration inthe steady-state phase of guttation was highly correlatedwith B content of the leaf margins in treatments S1B1,S1B2, S2B1 and S2B2 with r = 0.962. The slope of the rela-tion was 10.77, in range with the dry matter content ofbanana leaves. In this stage, B that was deposited in themesophyll at the lamina margins during the day comes intocontact with the fresh guttation sap pouring into the leakymarginal vein, and some of it is washed out of the plant at arate that correlates with the volume of guttation at thatstage. It is therefore proposed that the B content in the leafmargins is affected by the amount of sap exuded during thenight in the steady-state phase of guttation, and that theamount of B, which is washed out during this stage, makes amajor contribution to B content in the tissue of the laminamargins.

As B remains mobile in the marginal mesophyll, its dis-tribution is further affected by a phenomenon known as‘mesophyll flooding’ (Feild et al. 2005). Mesophyll floodingin banana results from root pressure-driven sap flow fromthe xylem of the marginal vein into the air spaces of thespongy mesophyll. Mesophyll flooding is common in thelamina margins of field-grown banana plants under normalconditions and usually accompanies intense guttation(Fig. 10a,b). In aging leaves, it may be a consequence ofnon-functional hydathodes due to occlusion by precipitat-ing minerals (Takeda, Wisniewski & Glenn 1991). There-fore, like guttation, flooding of the mesophyll in the laminamargins plays a role in dissipating the hydraulic pressurethat may build up in the xylem as a result of ‘root pressure’(Feild et al. 2005). Mesophyll flooding can contribute to Bcontent in the margins, but we hypothesize that its mainoutcome is redistribution of B already present in themargins. During the morning, as transpiration resumes,water from the flooded air spaces evaporates. The fact thatmesophyll flooding occurs only at the lamina margins nextto the bundle sheathless marginal vein and not uniformlyacross the lamina provides support for the role of thebundle sheath in regulating radial flow of water and solutesout of the xylem. Indeed Nardini et al. (2010) pointed to thebundle sheath of Aesculu shippocastanum L. as the majorresistor of radial flow from the xylem to the mesophyll.In the absence of such control, mesophyll flooding wouldappear throughout the lamina when root pressure isintense. Indeed in some species, for example, tobacco,



potato and bean, mesophyll flooding appears over theentire surface of the leaf (stomatal guttation, Komarnytskyet al. 2000).

CONCLUSIONS

Guttation was first reported in the 17th century but its rolein plant physiology has been somewhat neglected. Manyplant species guttate but usually in very small amounts thatoften go unnoticed. As we have shown, guttation fluid canbe highly concentrated due to effective filtration of thetranspiration stream. Toxic ions can be discarded from theleaves on a daily basis from the end of the vascular system.According to our results, the volume of sap exuded fromunstressed banana plants overnight replaces the total leafxylem sap many times over. Previous studies regarding gut-tation required an artificial environment and were usuallycarried out on seedlings. Bananas guttate freely as part oftheir normal growth cycle under commercial growing con-ditions. Therefore, we can state with confidence that gutta-tion plays an important role in the homeostasis of B in thelamina of banana, and possibly other species as well. Highroot pressure, abundant guttation and its large simpleleaves make the banana an attractive model plant for plantphysiologists studying mineral nutrition and water relationsin plants.

ACKNOWLEDGMENTS

The authors wish to thank the Israeli Banana GrowersAssociation for financial support of this research. We alsothank Mrs. Carmit Lifshitz for her technical assistance andfor organizing the guttation collection.

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Received 28 April 2011; received in revised form 20 June 2012;accepted for publication 24 June 2012

SUPPORTING INFORMATION

Additional Supporting Information may be found in theonline version of this article:

Figure S1. Kinetics of B concentration measured in gutta-tion samples from 2 additional collection days, showinggreat variability for S2B1 and S2B2 treatments (filledcircles, first date and empty circles, second date). Sampleswere collected as described in Materials and Methods. Eachdata point represents one aliquot. (a) S1B1 (2.5 mm KCl

and 0.0046 mm B), (b) S2B1 (20 mm KCl and 0.0046 mm B),(c) S1B2 (2.5 mm KCl and 1.1 mm B) and (d) S2B2 (20 mmKCl and 1.1 mm B).Figure S2. Content of key macronutrients in the leaftissue of control-treated plants (S1B1: 2.5 mm KCl and0.0046 mm B). Each bar represents mean � SE (n = 4).Black bars, lamina; empty bars, margins.Table S1. Stomatal conductance (gs) to water vapor asaffected by long-term irrigation with a combination ofincreasing KCl and B. Measurements were performed at thelamina and margins on the third fully unfurled leaf from thetop of the plant. Values are mean � SE (n = 4). S1B1,2.5 mm KCl and 0.0046 mm B; S1B2, 2.5 mm KCl and 1.1 mmB; S2B1, 20 mm KCl and 0.0046 mm B; S2B2, 20 mm KCl and1.1 mm B; S3B1, 42 mm KCl and 0.0046 mm B; S3B2, 42 mmKCl and 1.1 mm B.



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