Plant Physiol. (1979) 64, 460-4660032-0889/79/64/0460/07/$00.50/0

Amino Acids Translocated from Turgid and Water-stressedBarley LeavesI. PHLOEM EXUDATION STUDIES12

Received for publication February 23, 1979 and in revised form May 25, 1979

RAYMOND E. TULLY3 AND ANDREW D. HANSONMSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

The phloem exudation technique of King and Zeevaart (Plant Physiol1974 53: 96-103) was modified for use with barley plants, to investigate theeffect of water stress upon amino acid translocation at seedling and grain-filled stages.

Seedling leaves and flag leaves from unstressed and moderately water-stressed plants exuded "CO2 assimilates, sugars, and amino acids whentheir sheaths were cut and immersed in a 5 millimolar solution ofNa2EDTA(pH 7.0). By including PEG 6000 (-10 bars) in the Na2EDTA solution,leaves severed from moderately water-stressed plants could be maintainedin a wilted state. Such leaves produced about as much exudate as turgidleaves of unstressed plants.The foUlowing observations suggest a phloem origin for most of the

exudate. Exudation was markedly stimulated by light and by CO2 enrich-ment. The release of N03- declined after cutting, and did not parallelexudation of "CO2 assimilates, sugar, and amino acids. The relativequantities and specific radioactivities of sugars and amino acids in theexudate differed from those of sugars and amino acids extracted fromsheath tissue.

Major amino acids in exudate from unstressed seedling and flag leaveswere glutamine, glutamate, serine, alanine, and aspartate; proline wasvirtually absent. Exudate from water-stressed leaves contained relativelymore serine, and also some proline and y-aminobutyric acid.

While sucrose is well established as the principal sugar trans-ported in the phloem of cereals (e.g. 3), there are few previousreports on the nitrogenous phloem constituents in these crops ( 18).The N-compounds moving in the phloem of cereals may originatefrom either or both of the following sources: (a) recent N assimi-lation (18, 23 and refs. cited therein), and (b) hydrolysis of leafproteins, which occurs during senescence (1) and episodes ofwaterstress (23). Although it is clear that the relative importance ofsources (a) and (b) changes both with developmental stage andwith environmental conditions, little is known about the chemicalforms of N which these sources make available for phloem trans-port. Such descriptive information on the N-compounds in thephloem of cereals is pertinent to understanding the striking ca-pacity of these plants to remobilize N from leaves and stems,sometimes directing over 70%'o of total plant N to the grains; it isalso needed for quantitative treatments of partitioning of assimi-

' Research carried out under United States Department of EnergyContract EY-76-C-02-1338.

2 Michigan Agricultural Experiment Station Journal Article No. 891 1.3Present address: Boyce Thompson Institute, Cornell University, Ithaca,

New York 14853.

lates (e.g. 6), and bears on calculations of the bioenergetic costs ofseed protein reserves (1).

In a recent paper (23) we reported that export of organic Nfrom second leaves of barley proceeded at a similar rate in bothturgid plants and in plants water-stressed for I day. In the turgidplants the source of the exported N was mainly recently assimi-lated N03, while in wilted plants it was net protein breakdown.Our results implicated proline as a minor transport form of N instressed plants, but left open the question of the major transportforms in both stressed and turgid plants. In this and the followingpaper, we report on the amino acids in the phloem of unstressedand stressed barley plants, at both the three- to four-leaf stage andduring grain-filling. Here, we summarize results obtained with aphloem exudation technique based on that of King and Zeevaart(1 1). In the following paper (9), we describe supporting evidencederived from '3N and 'C tracer studies.

MATERIALS AND METHODS

Plant Material and Water Stress Regimes. Young barley plants(Hordeum vulgare L. cv. Proctor, CI 11806) were grown in Perlitein a growth chamber employing conditions given by Tully et al.(23), and were used at 19 days (three-leaf stage), or at 21 to 24days (four-leaf stage). Such plants were water-stressed by floodingthe roots with polyethylene glycol (PEG 6000) solution 1 day priorto use, as described previously (23). Plants for experiments withflag leaves were grown in the greenhouse in a soil mixture (7) (oneor two plants per pot). Flag leaves were taken from tillers fromears which were in the mid- or late grain-filling stage. Water stresstreatments involved withholding irrigation for 2 days. Onset ofwater stress was detectable by a rise in diffusive resistance of theupper surface of the flag leaves, measured with a Li-Cor modelLI-65 Autoporometer (Lambda Instruments Corp., Lincoln,Nebr.). For one experiment, flag leaves were harvested from afield plot of Proctor; this plot had been prepared, fertilized, andsown according to standard Michigan practice and was not water-stressed.

Leaf water potential (Ile'ie4) was measured on 13-mm slices ofleaves taken at the midpoint of the blades, using a Wescor dewpoint microvoltmeter equipped with C-52 sample chambers, anda 2-h equilibration time (16).

Labeling with 14CO2. Plants at the three- or four-leaf stage wereexposed for 30 min to 14CO2 in a sealed 20-liter glass tank placedunder a bank of four fluorescent tubes (Westinghouse Agro-Lite,20 w: photosynthetic irradiance about I mw-cm-2). Batches of 12to 38 intact plants or plants trimmed to second and third leaveswere supplied with doses of either 10 or 20 ,uCi 14CO2, generatedwith 50%o (v/v) lactic acid from Na2 4CO3 (Amersham, 60 ,uCi/

4Abbreviations: MCW: methanol-chloroform-water, I,le:f leaf waterpotential; TLE: thin layer electrophoresis.

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AMINO ACIDS IN BARLEY PHLOEM. I

umol) or 260 to 280 CiL'4CO2 released in the same way fromBa'4CO3 (51 ,uCi/,umol, New England Nuclear). The air in thetank was agitated with a magnetic stirring bar (75 x 12 mm)throughout the 30-min "CO2 exposure; after this, the tank waspurged with air. The plants were held for a further 30 min underthe bank of fluorescent lights before use.

Attached flag leaves were fed '4C02 in inverted 250-ml Erlen-meyer flasks, each equipped with a small side arm closed with aserum stopper. The flasks contained three leaves, the sheaths ofwhich were passed through a notch in the stopper and sealed inplace with Vaseline. The flasks were illuminated under the bankof four fluorescent lamps, and each received 500 .Ci of 14CO2 (51,uCi/,umol) which was liberated from Ba'4CO3 in a 5-ml syringeand injected as a gas through the serum stopper on the side arm.After a 30-min exposure period, the leaves were removed fromthe flasks and illuminated for an additional 30 min in air beforeuse.Phloem Exudation Experiments. Organs for exudation experi-

ments (whole shoots, young leaves, flag leaves) were excised, atpositions specified in the text, under water or a solution of thedesired concentration of Na2EDTA, adjusted to pH 7.0 withNaOH (11). Before use they were recut with a razor 5 to 30 mmabove the first cut. Batches of organs (e.g. four whole shoots, or 12leaves) were placed in small vials with about a 1-cm length of theircut ends immersed in I or 2 ml of exudation medium (see text forexudation media used). The batches of organs were transferred tofresh exudation medium at 2 and 4 h, and experiments wereterminated at 6 h. Incubation during the 6-h exudation period wasin 20-liter glass tanks, at about 22 C, in atmospheres saturatedwith water vapor; in some experiments the tanks were illuminatedunder the bank of four fluorescent tubes described above. Furtherdetails are jiven in the text. Samples of the exudate solution weretaken for ' C assay by liquid scintillation counting and for thequalitative and quantitative chemical analyses described below.For experiments in which comparisons were made between 14C

exudation and 14C translocation in intact plants, or between theamount of 14C exuded and the amount present in the exudingorgans, plant parts were frozen in liquid N2 and dried at about 70C. The 14C content of the dry tissue was determined by liquidscintillation counting following combustion in a Packard Tri-Carbmodel 306 sample oxidizer. In experiments in which the compo-sition of the exudate was compared with the soluble products ofsheath tissue, sections of sheath were extracted with the simplifiedMCW technique described by Hanson et al. (7). The aqueousphases were evaporated to dryness and redissolved in H20 forfractionation and analysis.Chemical Assays. The ninhydrin method of Rosen (19) was

used to determine total a-amino acids, using a glycine standard.This method is well suited for amino acid mixtures because itgives essentially the same color yield (molar basis) for all commona-amino acids. Proline was estimated by the acid ninhydrinmethod as described by Hanson et al. (7), except that the Permutittreatment was omitted. Carbohydrate was assayed with anthrone(22) using a sucrose standard; it was verified that an invert mixturegave the same color as the equivalent amount of sucrose. Nitratewas determined with the soybean bacteroid technique of Loweand Hamilton (13). When using the above assays with phloemexudate, small amounts of EDTA and PEG were sometimespresent; it was found that this did not interfere with any of theassays.

Separation and Identification of Amino Acids and Sugars.Aqueous phases of MCW extracts and exudate samples werebrought to 5 ml with water and separated into cationic, anionic,and neutral fractions using Dowex 50-H+ and Dowex 1-acetateresins (2). Cationic fractions (eluted from Dowex 50 with I NNH40H) were evaporated to dryness and taken up in a smallvolume of water. The amino acids in this fraction were thenseparated by high voltage TLE on glass-backed 0.I-mm cellulose

plates in 70 mm sodium tetraborate buffer (pH 9.3) (8). Radioac-tivity on TLE plates was detected with a Packard radiochroma-togram scanner or by autoradiography, and amino acids werevisualized with ninhrdrin spray reagent. The contribution ofaspartate to the total 4C activity in the aspartate-glutamate peakin radiochromatogram scans was estimated by rescanning theplates after ninhydrin spraying, with and without aluminum foil(Reynolds 624) exactly covering the glutamate zone. Preliminaryidentifications of amino acids were based on coelectrophoresiswith authentic markers in sodium tetraborate buffer. These iden-tifications were further supported by scraping the appropriatezones from unsprayed TLE plates, and subjecting them to co-chromatography with authentic markers in one of the followingTLC solvent systems, using precoated, plastic-backed, 0.1-mmcellulose TLC plates: isopropyl alcohol-formic acid-water (80:4:20, v/v); 1-butanol-acetone-diethylamine-water (30:30:6:15, v/v);1-butanol-acetic acid-water (12:3:5, v/v). Labeled amino acidswere eluted from the TLE plates for determination of specificradioactivity as described previously (8).

Neutral (sugar) fractions from the Dowex procedure were evap-orated to dryness, redissolved in small volumes of water, andseparated by TLC on plastic-backed cellulose plates using threeascents of ethyl acetate-acetic acid-formic acid-water (18:3:1:4, v/v). Labeled sugars were detected by autoradiography and wereestimated with anthrone after eluting from the TLC plate with48% (v/v) ethanol (8); small samples of the eluates were taken forscintillation counting in order to calculate specific radioactivities.

RESULTS AND DISCUSSION

As the following calculation shows, only very small volumes ofphloem exudate can be expected from cut barley leaves-even ifthere is no reduction in phloem sap movement after cutting. Avigorous barley seedling at the three-leaf stage has a dry weight ofabout 100 mg, and a rate of dry weight increase of about 10 mg/day (ref. 7, and unpublished results). Assuming that each leafcontributes about 3.3 mg/day to the dry matter gained, a conver-sion efficiency of about 0.7 mg carbon converted to dry matterper mg carbon used in growth (15), and that phloem sap contains10%1o (w/v) dry matter (3), the volume of phloem sap produced byone leaf would be (3.3/0.7 x 0.1) = 47 ,ld/day, or 2 t,l/h. Suchsmall volumes-themselves the sums of far smaller volumes pass-ing through each vein of the leaf-clearly cannot be harvesteddirectly as droplets. In several dicotyledonous species it has provedpossible to harvest phloem exudate indirectly, by collecting thematerial escaping from the cut ends of petioles or other organs inrelatively large volumes of solutions containing chelating agents(5, 1 1). The chelating agents probably prevent or delay the sealingof cut sieve tubes (I 1). With all exudation techniques, it is possiblethat at least some of the material released by cut tissues representsgeneral leakage of solutes from the tissues, and/or the products ofwound-induced tissue autolysis, as well as continued secretion ofphloem sap.

In section A we describe the adaptation of the method of Kingand Zeevaart (I 1) to the collection of exudate from both turgidand wilted barley leaves. In section B, we summarize the evidencethat phloem sap contributes substantially to the observed exuda-tion. In section C, we compare the compositions of exudatesobtained from seedling leaves and flag leaves, in the presence andabsence of water stress.

A. DEVELOPMENT OF TECHNIQUES FOR OBTAINING EXUDATE

Because batches of plants varied in the amount of exudateproduced, all results were confirmed by repeating experimentswith at least two separate batches.

Exudation from Unstressed Shoots and Leaves. Figure IAshows the effect of EDTA concentration on exudation in darknessof "C assimilates from entire shoots of three-leaf plants. After a

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TULLY AND HANSON

0

0

IC)

w

x

ao.00

I Y

o.E-

'.0 o~.o.E

w

O aJO

:3

D xenU)

0 2 4 6HOURS OF EXUDATION

FIG. 1. A: Effect of EDTA concentration of exudation of 14C assimi-lates from entire shoots of barley seedlings at the three-leaf stage. Afterlabeling plants with "4C02 generated from Na2"4CO3, shoots were severedjust above the seed. Batches of four shoots were taken for exudation at pH7.0 into various EDTA concentrations (given in mm on the right of graph).Exudation was in darkness in air. As in all other time courses, data pointsare for a single batch of exuding organs per treatment. B: exudation of '4Cassimilates (0, 0), amino acids (A, A), and sugars (1, U) from first andsecond leaves of barley seedlings. Exudation was into water (-, A) orinto 5 mm EDTA (pH 7.0) (0, O, A). After labeling with '4CO2 generatedfrom Na2"4CO3, sheaths were cut 5 cm below the ligules. Exudation was indarkness in air, with six pairs of leaves per treatment.

2-h lag, exudation was greatly stimulated by to 30 mm EDTA,but little by 100 mm. A concentration of 5 mm EDTA was chosenfor use in all subsequent exudation experiments for two reasons.First, several trials indicated no consistent advantage of EDTAconcentrations above 5 mm in promoting exudation. Second, itwas technically desirable to keep the EDTA concentration low tosimplify analysis of the exudate. Experiments were terminated at6 h because exudation rates in the absence of EDTA tended toincrease in longer experiments, probably because of infitrationand subsequent deterioration of the submerged sheath tissue.

Entire shoots included sinks for organic translocates (meristemsand young leaves) as well as mature leaves. The technique wastherefore simplified by taking only fully expanded leaves, e.g.leaves I + 2 (three-leaf stage) or leaves 2 + 3 (four-leaf stage).The leaves were cut 5 cm down the sheaths from the ligule. Leaves2 + 3 were preferred because their sheaths were strong and easilyhandled. Figure lB shows results typical ofexudation experimentswith fully expanded leaves. Note that EDTA again enhanced 14Cexudation, and that the lag seen with entire shoots (Fig. IA) was

absent. Figure lB also illustrates another characteristic of exuda-tion: the time courses of amino acid and sugar exudation bothparalleled that of 14C exudation. Hence, chemical analyses alonewere used to follow exudation in some experiments.An attempt was made to increase the rate of exudation by

illuminating the cut leaves and subjecting them to an atmosphereenriched in CO2. Probably acting via photosynthetic productionof sucrose, light enhances the mass transfer rate of translocationin the phloem (20). Light also increases transpiration, principallyby causing stomatal opening; transpiration would draw some ofthe EDTA solution into the leaf via the xylem. An increase in theambient CO2 level would be expected to counteract this by reduc-ing stomatal aperture and thus lowering transpiration, while keep-ing the CO2 concentration inside the leaf high enough for active

net photosynthesis. Exudation of amino acids and sugars wasstimulated almost 2-fold by light alone, and an additional 2-foldby light and CO2 together (Table I). The CO2 treatment alsoreduced transpirational uptake of the EDTA solution. In the lightin air, 15 to 20%o of the solution was transpired during each 2-hinterval; in C02-enriched air only 5 to 10%o was transpired. Sub-sequent exudation experiments were therefore carried out in lightwith an atmosphere enriched with CO2.

In summary, optimal conditions for exudation with unstressedseedlings were: leaf sheaths cut 5 cm down from the ligule; 5 mmEDTA (pH 7.0) changed every 2 h for 6 h; light (above I mw.cm-2); water-saturated atmosphere enriched with CO2. These con-ditions also worked well with unstressed flag leaves (Table II).(Note that the flag leaf exudation data are expressed on a per-leafbasis, unlike data for seedling leaves, which are given per-leafpair.) By comparing Tables I and II it can be seen that rates ofexudation on a per-leaf basis from flag leaves were generally lowerthan the rates from seedling leaves. Because flag leaf blades weresmaller (area about 8 cm2) than seedling leaf blades (about 15cm2), exudation rates on a leaf area basis were comparable.

Exudation from Water-stressed Leaves. Stressing seedlings forI day caused severe wilting of the leaves, and reduced *leaf toabout -10 bars. When cut and placed with their cut sheaths in anEDTA solution, such leaves took up solution and rapidly regainedturgor. To maintain a low *'l1eaf during exudation, PEG 6000 wasincluded in the exudation medium (Is = -10 bars, 29.3 g + 100ml EDTA 6.2 mm, final pH 7.0). In Figure 2, the time course of4C exudation from stressed leaves into PEG + EDTA is compared

Table 1. Enhancement of Exudationfrom Unstressed Barley SeedlingLeaves by Light and C02

Batches of six leaf pairs (second + third leaves) were used for eachtreatment. Exudation from each batch was into 2 ml of 5 mm EDTAsolution, changed at 2 and 4 h. Light treatments were under fluorescentlamps (about I mw cm 2); CO2 treatments involved passing a stream of1% CO2 in air (flow rate about 6 liters/h) through the 20-liter tankcontaining the exuding leaves.

Amino Acid and Sugar ExudationTreatment

Glycine equivalents Sucrose equivalents

imol/leafpair- 6 h

Dark 0.195 0.138Light 0.327 0.258Light + CO2 0.683 0.590

Table II. Exudationfrom Unstressed and Water-stressed Flag Leaves ofBarley

Batches of 12 to 20 flag leaves were harvested near the end of grainfilling (expt. I) or in mid-grain-filling (expts. 2 and 3). Exudation was intoI or 2 ml of 5 mm EDTA solution, changed at 2 and 4 h; in expt. 3 theEDTA solution contained PEG, -10 bars. Exudation was in the light, inair (expt. 1), or air enriched with CO2 (expts. 2 and 3).

Cumulative Exudation Per Flag LeafExpi. Leaf Source and Time of Ex-No. Treatment udation "C assimilate Glycine Sucrose equiv-

equivalents alentsh dpm x 1o-6 Amot

I Field plot, un- 2 0.032 0.044stressed 4 0.066 0.104

6 0.123 0.191

2 Greenhouse, 2 0.73 0.028 0.053unstressed 4 2.10 0.080 0.170

6 4.06 0.165 0.295

3 Greenhouse, 2 0.026 0.025water-stressed 4 0.066 0.067

6 0.113 0.101

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AMINO ACIDS IN BARLEY PHLOEM. I

e 4.0- STRESSED LEAVES

EDTAO ~~~~~~~fL3.0

_ / ~~~~~~~/< g ~~~~~//2.0 _

J /oS

a1.0 AoIx

O 2 4 6HOURS OF EXUDATION

FIG. 2. Exudation of 14C assimilates from second and third leaves ofbarley seedlings water-stressed for I day. After labeling plants with "4CO2,batches of four leaf pairs were taken for exudation into water (-) or S mmEDTA (pH 7.0) (O), at-.= O bars ( ), or with PEG at =-10 bars(---). Leaves in PEG remained wilted throughout exudation; thosewithout PEG regained turgor. Exudation was in light in C02-enriched air.

with that obtained when PEG was omitted and the leaves rehy-drated during the experiment. Although the leaves in PEG +EDTA did not gain in fresh weight, and remained severely wtotedthroughout the experiment they exuded "C assimilates at aboutthe same rate as those allowed to recover. Stressed seedling leavesexuding into PEG + EDTA gave amounts of amino acids andsugars at least equal to those exuded by unstressed leaves intoEDTA alone (Fig. 3, A and B).With water-stressed flag leaves, addition ofPEG to the medium

again permitted maintenance of a low 'I'd, while exudationgenerally proceeded at rates somewhat lower than those fromunstressed leaves (Table II; Fig. 5, B and C).

B. EVIDENCE THAT EXUDATE IS PRINCIPALLY OF PHLOEM ORIGIN

Exudation of amino acids and sugars could be in part theconsequence either of EDTA-stimulated solute leakage from allsheath tissues, of autolysis of cells damaged by cutting or EDTA,or of both these degradative processes. The following arguments,based principally upon results from unstressed seedling leaves,indicate, however, that such degradative events contribute little toexudation. Two of the key arguments were checked and found tohold also for stressed seedling leaves.

Effect of EDTA. EDTA always became decreasingly effectivein stimulating exudation above concentrations of 10 to 30 mm (e.g.Fig. IA). King and Zeevaart (11) also found an optimal EDTAconcentration of 20 mm for exudation from Perilla leaves. Suchan optimal concentration connotes intervention by EDTA in somespecific process, and is hard to reconcile with effects on eithergeneral tissue permeability or on autolysis.

Effects of Light and CO2. Degradative processes would not beexpected to show the marked dependence on light and CO2 shownin Table I. If the exudate comes mainly from the phloem, thisdependence is readily explained as a consequence of enhancedphotosynthesis and phloem loading of photosynthate in the blade(20).Comparisons with Translocation in Intact Plants. Comparisons

of "4C assimilate translocation with exudation for unstressed andstressed seedlings showed that during a 6-h period, the amountsof 14C exuded from cut leaves were about 21 and 11%, respectively,of the amounts translocated from similar leaves into the lowersheaths and roots (data not shown). Values of 10 to 18% can bereached by comparing the rates of amino acid exudation (Figs. 1

and 3; Table I) from unstressed and stressed leaves with the

calculated rates of organic N export from such leaves (= 203 and260 ,ug N/leaf. day, respectively, ref. 23), and assuming that one-half (unstressed leaves) or one-third (stressed leaves) of the a-amino N in exudate is in the form of glutamine (Figs. 4C and5A). Although 14C exudation rates fell short of in vivo translocationrates, the data in Table III are evidence that translocation must beinvoked to account for exudation. More '4C assimilate was exudedduring a 6-h period than was present in the sheaths beforeexudation began. Further, the sheaths showed a small net gain in14C, not a loss, during the exudation. This can be explained onlyby movement of label from the blades into the sheaths, and outinto the medium.

Nitrate in Exudate. In general, NO3- is absent from phloemexudates or present only in trace amounts (ref. 17, and literaturecited therein) while it is commonly abundant in xylem sap and inleaf tissue. Absence of N03 from exudate would thus indicatethat the source of exudate is the phloem. Time courses for exu-dation ofNO, sugars, and amino acids are compared in Figure3. The rate of N03 release from both unstressed and stressed

A Amino Acids

05 _

Stressed IN0.3-

Control

0.I

B. Sugars40 4

Stressed

~J0.2-rntrol

0i C. Nitrateo 0.6 - ControlE

0.3-Stressed

D. Praline0.03 -

Stressed

001 Control

0 2Hours of Exudation

FIG. 3. Exudation from third and fourth leaves of unstressed barleyseedlings (0, control), and of seedlings water-stressed for 1 day (0).Unstressed leaves exuded into 5 mm EDTA (pH 7.0); stressed leaves into5 mM EDTA (pH 7.0) + PEG 6000, S. = -10 bars. Exudation was from24 leaf pairs per treatment, in light in COrenriched air. Amino acidexudation (A) is in glycine equivalents, sugar exudation (B) in sucroseequivalents. All data were from the same experiment.

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TULLY AND HANSON

leaves slowed considerably after 2 h; this was in contrast to therelease of amino acids and sugars which increased with time.Although N03 release between 4 and 6 h had slowed markedly,much N03 still remained in the sheath. In one experiment withunstressed leaves, 0.37 ,umol of N03 was exuded per leaf pairduring 6 h; extracts ofjust the 1-cm immersed sheath tip segmentsat 6 h still contained 0.54 ,umol NO3J/leaf pair.

In the light of the nitrate exudation results, the amino acids andsugars present in exudate (e.g. Tables IV and V; Figs. 4 and 5)were investigated only in the exudate fraction collected between4 and 6 h; This fraction is more likely to represent phloem sapthan the fraction collected between 0 and 2 h, which probablycontains solutes released by damaged cells or xylem vessels of thecut sheath.

Concentrations and Specific Radioactivities of Sugars. In manyspecies phloem sap is characterized by a high sucrose content,with glucose and fructose essentially absent (3, 24), although largefree hexose pools are often present in the parenchyma tissue whichconstitutes the bulk of the leaf. Barley seedlings fit this pattern. Insheath (translocation path) extracts of intact plants made 3 h afterexposure to 14CO2 in the light, sucrose was the sole labeled sugardetected by autoradiography (data not shown). Free glucose andfructose in sheath tissue became labeled only after longer incu-bations (Table IV), presumably through metabolism of [14CJsu-crose translocated into the sheath. The heavier labeling of glucosethan fructose in sheath extracts (Table IV) probably reflectsdifferential utilization (21).

Table IV compares chemical and radiochemical data for sugarsof sheath tissue and exudate. (Note that sheath tissue from intactplants-not from exuding leaves-was used because Table III andFig. 4 indicated that assimilates accumulated in sheaths ofexudingleaves.) Chemically, the exudate was enriched in sucrose comparedto the extract. Some of the reducing sugars in the exudate un-

Table III. Balance Sheetfor 14C Assimilates in Sheath Tissues andExudate of Unstressed Barley Seedling Leaves

Shoots of eight plants were trimmed to second and third leaves, andexposed for 30 min to 14C02 generated from Na214CO3. Thirty min afterthe end of '4CO2 exposure (= 0 h), the second and third leaves of fourplants were used for exudation into EDTA, and four plants were taken fordetermination of the '4C content of the 5-cm sheath section next to theblade. After 6-h exudation into 5 mM EDTA in light and C02-enrichedair, the 14C contents of the exudate and of the 5-cm sheath sections of theexuding leaves were determined.

Time of "C Assimilate ContentExudation 5-cm sheath Exudate

h dpm/leafpair

0 7,300" 06 8,700" 10,300

"Means of four replicates.

doubtedly arose by hydrolysis of exuded sucrose at the cut sheathends, because extensive hydrolysis of added tracer ["C]sucrosecould be demonstrated during exudation (data not shown). Thespecific radioactivity of exuded sucrose was more than twice that

2

0

r-'0

x 2

Ev 10.-

>v2

..6-

u

Vt

0 10 20Distance (cm)

FIG. 4. TLE separations and radioactivity scans for Dowex 50 fractionsof sheath extracts and exudate from unstressed barley seedling leaves.Eighteen seedlings were labeled with 14CO2 generated from Ba'4CO3.Second and third leaves were cut from 12 seedlings and used for exudationinto 5 mM EDTA in light and C02-enriched air. After 6-h exudation, thebasal I-cm sheath segments were cut off and analyzed (A). Comparable 1-cm sheath segments were taken at the same time from leaves of six plantswhich were left intact during the 6-h exudation period and kept in thesame environmental conditions as exuding leaves (B). The exudate ana-lyzed was collected between 4 and 6 h (C). The amounts of Dowex 50fractions applied to TLE plates were equivalent to extract from 2.4 sheathsegment pairs, or to exudate from 2.4 leaf pairs. The 14C activity appliedto each plate is given in parentheses. Positions of origin (0), anode (+),and cathode (-) are indicated. Broken lines in the Asp/Glu peak regionsshow the estimated contributions of aspartate.

Table IV. Sugars in Exudate and Sheath Tissue of Unstressed Barley Seedling LeavesTwelve plants were trimmed to second and third leaves, labeled with '4C02 generated from Ba4CO:s, and used for exudation. Exudation was for 6

h, in the light with C02-enriched air, into 5 mM EDTA. The exudate fraction collected between 4 and 6 h was taken for analysis. Twelve more plants,trimmed and labeled in parallel with plants used for exudation, were left intact and were kept in the same conditions as the exuding leaves. At the endof the exudation period, the intact plants were used as sources of 1-cm segments of sheath tissue which were cut out 5-cm down each second and thirdleaf sheath. Sheath segments were extracted in MCW. The Dowex neutral fractions of exudate and sheath segment extract were analyzed after TLC.

Sugar Exudate, 4- to 6-h Fraction Sheath Segment Extract

Sugar content' "C Content' Specific Radioactivity Sugar Content" "C Content" Specific Radioactivity

nmol dpm dpm/nmol nmol dpm dpm/nmol

Sucrose 27.4 (1.00)b 29.700 (1.00) 1083 64.7 (1.00) 25,600 (1.00) 396Glucose 30.6 (1.12) 8,580 (0.29) 280 128.0 (1.98) 5,610 (0.22) 44Fructose 4,600 (0.15) 2,190 (0.09)

' Expressed per leaf pair (exudate), or per sheath segment pair (extract).b Figures in parentheses express data relative to sucrose (= 1.00) within each column.

[A. !-'3 0 04dpm

.. ~~~~~~~IB.i

iN1ACTI SHEATk15

C.AC.s

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464 Plant Physiol. Vol. 64, 1979

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AMINO ACIDS IN BARLEY PHLOEM. I

of extracted sucrose. These results indicate that phloem sap con-tributes substantially to sugar exudation.There is evidence in Table IV that a small amount of sugar is

released by tissues other than phloem. First, there was moreradioactive glucose than fructose in exudate. Second, there wasabout 80%1o more chemical glucose (15 nmol) than might be ex-pected: the specific radioactivity of glucose was 280 dpm/nmolrather than the value of 542 dpm/nmol predicted if all the glucosearose by inversion of uniformly labeled sucrose of specific radio-activity 1083 dpm/nmol. Both observations indicate that someglucose of relatively low specific radioactivity leaked out of thelarge free glucose pool in the nonvascular tissue of the sheath.

Concentrations and Specific Radioactivities of Amino Acids.The a-amino nitrogen of phloem sap is often principally in theform of a small, species-dependent range of amino acids; majortransport forms include aspartic and glutamic acids and theiramides, proline, serine, and threonine (3, 24). The amino acidcomposition of phloem sap may differ quantitatively and quali-tatively from that of the free amino acid fraction extracted fromthe organs through which the phloem runs.

In Figure 4, the amino acid exuded are compared with thoseextracted from exuding sheath tissue and from sheath tissue ofattached leaves. Chemically and radiochemically, the exudatecomprised the same limited range of amino acids as the extracts,but in different proportions. Exudate contained relatively moreglutamine and [1 C]glutamine-and less glutamate and [14CJglu-tamate-than the extract (Fig. 4, B and C; Table V). The specificradioactivities of both glutamine and glutamate were at least 2-fold higher in the exudate than in the extract (Table V). Takenwith Table IV, Table V also indicates that the exudate and extractdiffered in their relative contents of amino acids and sugars; themolar ratio glutamine + glutamate/(2 x sucrose) + glucose washigher in exudates (0.69) than in extracts (0.28). With respect tothe extract, exudate was radiochemically higher in seine, alanine,and aspartate; it appeared also to be chemically higher in serineand alanine (Fig. 4, B and C).These results all indicate that the exuded amino acids come

mainly from recent photosynthate traveling through the phloem.Note that the extract of exuding sheath tissue (Fig. 4A) showedchemical and radiochemical increases in glutamine, serine andalanine relative to the extract of attached sheath tissue (Fig. 4B).This can be ascribed to a build-up of basipetally translocatedassimilate in the veins of the sheath.The ratio of amino acids to sugars in the exudate (about I

glycine equivalent per sucrose equivalent, Table I, and Figs. IB,3A and 3B) was high, corresponding to a C/N weight ratio ofabout 10. Such a composition falls well within the range of valuesin the literature for the phloem sap of various species (e.g. 24).The high amino acid level (low C/N ratio) is consistent with thehigh nitrogen requirement for production of new leaves by theseedlings; the protein concentration (N x 6.25) of leaves was about30%o of total dry matter.

Table V. Glutamate and Glutamine in Exudate and Sheath Tissue ofUnstressed Barley Seedlings

Exudate and sheath extract were from the experiment of Table IV.Dowex 50 fractions were analyzed after TLE.

Exudate, 4- to 6-h Fraction Sheath Segment Extract

Anoinoacid I af pair Specific radioac- emol per sheath Specific radioac-tivity egmentpar tivity

dpm/nmol dpm/nmol

Gluta- 19.9 (1.00)0 172 43.3 (1.00) 79.0mate

Gluta- 38.7 (1.94) 150 29.6 (0.68) 43.3mine

a Figures in parentheses express data relative to glutamate (= 1.00)within each column.

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V

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STRESSED FLAG(B.4x10 d pm)

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Distance (cm)FIG. 5. TLE separations and radioactivity scans for Dowex 50 fractions

of exudate from stressed seedling leaves, and unstressed and stressed flagleaves of barley. Symbols are as in Figure 4. Note that the runningpositions of the amino acids differed among the TLE plates. A: secondand third leaves were from seedlings water-stressed for I day. Other detailswere as for Figure 4C. B: nine flag leaves were used, from tillers whosegrains had reached an average of 37% of their final dry weight. Threeleaves were labeled with '4C02; exudation was in light with C02-enrichedair, into 5 mM EDTA. Exudate was collected for analysis between 4 and6 h; an amount equivalent to that from 1.8 leaves was applied to the TLEplate. C: 14 flag leaves were used, from plants water-stressed by withhold-ing irrigation for 2 days. Grain dry weight was 43% of the final value.Exudation was into 5 mm EDTA containing PEG, I. = -1O bars. Anamount equivalent to that from 2.8 leaves was applied to the TLE plate.Other details were as for B.

C. EFFECTS OF WATER STRESS AND DEVELOPMENTAL STAGE ONAMINO ACID COMPOSITION OF EXUDATE

Water-stressed Seeding Leaves. Water stress slightly increasedthe amino acid to sugar ratio of the exudate (Fig. 3, A and B), butdid not change the amino acid composition very markedly (com-pare Fig. 5A with Fig. 4C). Chemically and radiochemically,water-stressed seedling leaves exuded relatively more serine. Theincrease in serine may arise because more serine is produced inwater-stressed leaf blades via the glycolate pathway (12). Thescanner trace of Figure 5A shows a small but significant peak of"4C in the proline zone, which was resolved by TLC into prolineand y-aminobutyric acid. Figure 3D confirms the presence of asmall amount of proline in exudate (about 5% of total a-aminoN). This is consistent with results ofN balance studies (23) whichindicated that proline was only a minor transport form ofN fromstressed seedling leaves.

Plant Physiol. Vol. 64, 1979 465

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Plant Physiol. Vol. 64, 1979

Unstressed and Water-stressed Flag Leaves. The amino acid tosugar ratio of exudate from unstressed flag leaves was like that forseedling leaves (Table II). Unstressed flag leaves in the mid-grain-filling period exuded the same amino acids as unstressed seedlingleaves; the chemical and radiochemical proportions differed inthat there was more alanine and less serine in flag leaf exudate(Fig. 5B). The flag leaves of Figure 5B were probably mobilizinglittle of their N to grain because the plants were regularly wateredwith nutrient solution, and the flag leaves showed no visiblesenescence symptoms. In an experiment carried out close to theend of the grain-filling period, in which the flag leaves had begunto yellow and die at the tips, the amino acid exudation patternwas still very like that of Figure 5B.As with seedling leaves, flag leaf exudate did not change drast-

ically during water stress (Fig. 5C); there was an increase in therelative amount of serine, and the appearance of "'C in the prolinezone, which was shown by TLC to be contributed about equallyby proline and y-aminobutyric acid. The amino acid to sugar ratiorose (Table II); this rise, seen also with seedling leaves, is consistentwith a stress-induced decline in net photosynthesis accompaniedby precocious protein hydrolysis and N mobilization.

CONCLUSIONS

The following conclusions draw additional support from the'3N and "'C tracer results reported in the second paper in thisseries (9).

1. The five principal amino acids found in barley phloemexudate were glutamine, glutamate, serine, alanine, and aspartate.The exudate composition in the absence of water stress was quitesimilar during vegetative and reproductive growth, and probablyalso during senescence. The composition was not drastically mod-ified by water stress, which led to an increase in the relativeamount of serine, and to the appearance of small amounts of y-aminobutyric acid and proline. That the proline levels in exudatefrom stressed leaves are low supports our previous conclusion thatproline does not assume a major role in N transport during waterstress (23).

2. During water stress and senescence, there is a shift fromrecent N03 assimilation to net protein hydrolysis as the mainsource of organic N for translocation from the blade (23). Thatexudate composition is maintained fairly constant in the face ofsuch a shift means that there are defacto preferred transport formsfor a-amino N. If no such preferences existed, the exudate fromwater-stressed or senescing leaves would presumably resemble theamino acid composition of leaf protein (e.g. 10), which it clearlydoes not.

3. Following from conclusions 1 and 2, when N is remobilizedfrom leaves to grains, much of it must be channeled (via free NH3or a-amino groups) into glutamine and glutamate before export.Two related bioenergetic considerations arise from this. First,glutamine and glutamate synthesis may be driven directly by lightin leaves, eliminating a sugar requirement for the production ofthe necessary ATP and reducing power (14). The bioenergeticcosts for the synthesis of these transport amino acids from a-ketoglutarate may thus be low. Second, barley grain protein has

a very high content of amino acids of the glutamate family. If theamino acids transported to the grain are identical with (or closelyrelated to) those stored in its protein, and present in appropriaterelative amounts, the grain is spared the bioenergetic costs in-volved in metabolic conversion of the imported amino acids intothose required for the synthesis of storage proteins (4). In thewhole barley plant, N salvage from stressed or senescing leaves,organic N translocation, and grain-filling from remobilized N maybe tied closely together by shared requirements or preferences forglutamine and glutamate.

Acknowledgment-We wish to thank J. A. D. Zeevaart for his helpful suggestions.

LITERATURE CITED

1. BHATIA CR, R RABSON 1976 Bioenergetic considerations in cereal breeding for proteinimprovement. Science 194: 1418-1421

2. CANVIN DT, H BEEVERS 1961 Sucrose synthesis from acetate in the germinating castor bean:kinetics and pathway. J Biol Chem 236: 988-995

3. CRAFTS AS, CE CRISP 1971 Phloem Transport in Plants. WH Freeman & Co, San Francisco4. DUFFUS CM, R ROSIE 1978 Metabolism of ammonium ion and glutamate in relation to

nitrogen supply and utilization during gain development in barley. Plant Physiol 61: 570-574

5. FELLOWS RJ, DB EGLI. JE LEGGET 1978 A pod leakage technique for phloem translocationstudies in soybean (Glycine max [L.] Merr.). Plant Physiol 62: 812-814

6. FLINN AM, CA ATKINS, JS PATE 1977 Significance of photosynthetic and respiratory exchangesin the carbon economy of the developing pea fruit. Plant Physiol 60: 412-418

7. HANSON AD, CE NELSEN, EH EVERSON 1977 Evaluation of free proline accumulation as anindex of drought resistance using two contrasting barley cultivars. Crop Sci 17: 720-726

8. HANSON AD, RE TULLY 1979 Light stimulation of proline synthesis in water-stressed barleyleaves. Planta 145: 45-51

9. HANSON AD, RE TULLY 1979 Amino acids translocated from turgid and water-stressed barleyleaves. II. Studies with '3N and 14C. Plant Physiol 64: 467-471

10. KAWASHIMA N, SG WILDMAN 1970 Fraction I protein. Annu Rev Plant Physiol 21: 325-358II. KING RW, JAD ZEEVAART 1974 Enhancement of phloem exudation from cut petioles by

chelating agents. Plant Physiol 53: 96-10312. LAWLOR DW, H FOCK 1977 Water stress induced changes in the amounts of some photosyn-

thetic assimilation products and respiratory metabolites of sunflower leaves. I Exp Bot 28:329-337

13. LOWE RH, JL HAMILTON 1967 Rapid method for determination of nitrate in plant and soilextracts. J Agric Food Chem 15: 359-361

14. MIFLIN BJ, PJ LEA 1977 Amino acid metabolism. Annu Rev Plant Physiol 28: 299-32915. MCCREE KJ 1976 The role of dark respiration in the carbon economy of a plant. In RH Burris,

CC Black, eds, CO. Metabolism and Plant Productivity. University Park Press, Baltimore,pp 177-184

16. NELSEN CE, GR SAFIR, AD HANSON 1978 Water potential in excised leaf tissue. Comparisonof a commercial dew point hygrometer and a thermocouple psychrometer on soybean,wheat, and barley. Plant Physiol 61: 131-133

17. PATE JS, PJ SHARKEY 1974 Phloem bleeding from legume fruits-a technique for study of fruitnutrition. Planta 120: 229-243

18. PETERSON DM, TL HOUSLEY, LE SCHRADER 1977 Long distance translocation of sucrose,serine, leucine, lysine and CO. assimilates. II. Oats. Plant Physiol 59: 221-224

19. ROSEN H 1957 A modified ninhydrin colorimetric analysis for amino acids. Arch BiochemBiophys 67: 10-15

20. SERVAITES JC, DR GEIGER 1974 Effects of light intensity and oxygen on photosynthesis andtranslocation in sugar beet. Plant Physiol 54: 575-578

21. SWANSON CA, ED EL-SHISHNY 1958 Translocation of sugars in the Concord grape. PlantPhysiol 33: 33-37

22. TREVELYAN WE, JS HARRISON 1952 Studies on yeast metabolism. 1. Fractionation andmicrodetermination of all carbohydrate. Biochem J 50: 298-303

23. TULLY RE, AD HANSON, CE NELSEN 1979 Proline accumulation in water-stressed barleyleaves in relation to translocation and the nitrogen budget. Plant Physiol 63: 518-523

24. ZIEGLER H 1975 Nature of transported substances. In MH Zimmermann, JA Milburn, eds.Transport in Plants. 1. Phloem Transport. Encyclopedia of Plant Physiology, New Series VolV. Springer-Verlag, New York, pp 59-100

466 TULLY AND HANSON

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