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Plant Physiol. (1992) 99, 996-10040032-0889/92/99/0996/09/$01 .00/0

Received for publication October 23, 1991Accepted January 17, 1992

Phloem Transport of Amino Acids in Relation to theirCytosolic Levels in Barley Leaves'

Heike Winter, Gertrud Lohaus, and Hans Walter Heldt*Institut fur Biochemie der Pflanze, Universitat Gottingen, Untere Karspule 2, 3400 Gottingen, Germany

ABSTRACT

A comparison of barley (Hordeum vulgare L.) leaves was madebetween the cytosolic content of amino acids and sucrose asdetermined by subcellular fractionation and the correspondingconcentration in phloem sap, which was collected continuously forup to 6 days from severed aphid stylets. Because amino acids werefound to be almost absent from the vacuoles, and because theamino acid patterns in the stroma and cytosol are similar, wholeleaf contents could be taken as a measure of cytosolic amino acidlevels for a comparison of data during a diurnal cycle. The resultsshow that the pattern of amino acids in the phloem sap was verysimilar to the pattern in the cytosol. Therefore, we concluded thatthe overall process of transfer of amino acids from the cytosol ofthe source cells into the sieve tubes, although carrier mediated,may be a passive process and that the translocation of amino acidsvia the sieve tubes requires the mass flow of sucrose driven by theactive sucrose transport involved by the phloem loading.

In plants, the products of photosynthesis generated in theleaf mesophyll cells are exported to other parts, such as rootsor filling seeds, via the sucrose and amino acids in the phloemsap. The analysis of metabolite concentrations in the phloemsap collected from aphid stylets severed by a laser beam (2,7, 8) makes it possible to monitor the photosynthesis productsexported from the leaves. With respect to the productivity ofa plant, it is important to know which factors are governingthe partitioning of photosynthesis products between carbo-hydrates and amino acids exported via the sieve tubes. Anelucidation of the matter requires a comparison of the corre-

sponding metabolite levels in the sieve tubes and in thesubcellular compartments of the leaf cells.A refinement of the nonaqueous fractionation of lyophi-

lized leaves, reported recently (18), enabled us to determinein spinach leaves the metabolite contents in the vacuolar,chloroplastic, and cytosolic compartments simultaneously.Using this method in combination with the aphid techniquefor the collection of phloem sap, we found that in the phloemsap the concentrations of amino acids are about the same,whereas that of sucrose is one order of magnitude higherthan the corresponding concentration in the cytosol. Al-though with spinach leaves the chance of obtaining phloemsap samples upon severing an aphid stylet is very high, thephloem sap from the severed aphid stylets comes to a stop

1 This work is supported by the Bundesminister fur Forschung undTechnologie (031 9296 A).

after 10 to 15 min, producing such low quantities of sap(approximately 5 nL) that exact volume determinations arenot possible, and therefore, the concentrations of the phloemsap constituents can only be estimated (18). As shown in thispaper, this is different with barley (Hordeum vulgare L.)leaves. Although with barley leaves the chance of obtainingan exuding aphid stylet by laser beam is rather low, asuccessfully severed stylet can continuously exude phloemsap at a relatively high rate for up to 6 d. This enables thecontinuous determination of the concentrations of phloemsap constituents during several diurnal cycles with relativelyhigh accuracy and, thus, monitoring of the assimilate exportfrom a leaf.

In the present report, by adaptation of the nonaqueousfractionation technique to barley leaves, we determined thecontents of various metabolites such as sucrose and aminoacids in whole leaves and in the vacuolar, chloroplastic, andcytosolic compartments, and in the phloem sap collected fromthese leaves. A comparison of these data during a diurnalcycle reveals the dependence of metabolite export via thesieve tubes from photosynthesis metabolism in the sourcecells.

MATERIALS AND METHODS

Plant Material

Barley (Hordeum vulgare L., var Apex; KleinwanzlebenerSaatzucht, Einbeck, Germany) was grown hydroponically in5-L vessels containing fire clay particles in a climatized cham-ber (15 h light, 22°C; 9 h dark, 170C ). The nutrient mediumcontained 4 mm Ca(NO3)2, 4 mM MgCl2, 6 mm KNO3, 2 mMMgSO4, 1 mm KH2PO4 and Na2-Fe-EDTA, and trace elementsas described by Randall and Bourma (17). The illuminationwas 350 uE m-2 s-' using fluorescent lamps (Osram LumiluxL58/31; Osram, Berlin, Germany). Twenty-one-day-oldplants were used for the experiments. At the indicated times,the plant leaves were frozen in liquid nitrogen. With plantsharvested in the light, illumination was continued duringfreezing.

Plant Extracts for Assay of Metabolites

Approximately 0.5 to 1 g frozen leaf material was groundin a mortar to a fine powder while kept under liquid nitrogen.To this material, 0.6 mL buffer containing 20 mm Hepes (pH7.0), 5 mm EGTA, 10 mm NaF, and 5 mL chloro-form:methanol (1.5:3.5, v/v) were added. The sample washomogenized until completely thawed and then kept on ice

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AMINO ACID LEVELS IN PHLOEM SAP AND CYTOSOL OF BARLEY LEAVES

for 30 min. From this homogenate the water-soluble metab-olites were extracted twice with 3 mL water. The aqueousphases were combined and evaporated in a rotatory evapo-rator. The dried residue was dissolved in 2 mL H20 Li-Chrosolv, E. Merck, Darmstadt, Germany) and stored at-850C until analysis. The remaining chloroform phase wasused for the assay of Chl (1).

Determination of Subcellular Metabolite Levels byNonaqueous Fractionation

The nonaqueous fractionation of barley leaves, frozen inliquid nitrogen and lyophilized, was carried out according tothe method of Gerhardt and Heldt (9) with the followingalterations: (a) the nonaqueous fluids contained C2C4 insteadof CC14. The leaf homogenate was suspended in heptane-C2C14 (density = 1.30 g/mL). Two milliliters of this suspen-sion was added to a centrifugation tube containing, frombottom to top, 2 mL heptane-C2CI4 (density = 1.56 g/mL)and 12 mL of heptane-C2CI4 with an exponential densitygradient between 1.50 and 1.32 g/mL. (b) For assay ofmetabolites, chloroform-methanol extracts were prepared(see above). For assays of marker enzyme activities and ofmetabolites, see ref. 20. Amino acids were assayed as de-scribed previously (18).The method of Riens et al. (18) was used for the evaluation

of the distribution of metabolites among the stromal, cyto-solic, and vacuolar compartments from the assay of markerenzyme activities and of metabolite contents in seven frac-tions of the density gradient centrifugation.

Collection of Phloem Sap

For collection of phloem sap, the stylet of Rhopalosiphumpadi L. was severed by a laser beam (2, 7, 8). For details seeref. 18.

RESULTS AND DISCUSSION

Metabolite Content of Whole Barley Leaves

Barley leaves grown during a 15-h light/9-h dark cyclewere quenched in their metabolism by placing them in liquidnitrogen. Table I shows the metabolite contents of leaf sam-ples quenched after 9 h of illumination and 5 h of darkness,respectively.From the metabolites analyzed in a leaf sample, a certain

portion was contained in the sieve tube/companion cell sap.A maximum estimation for that portion was obtained fromthe assay of the total leaf sucrose content at the end of thedark period on the assumption that all of this sucrose waspresent in the sieve tubes. As shown in Figure 3A, the leafsucrose content at the end of the night was 1.5 ,umol/mg Chl.With a sucrose concentration in the phloem of 0.9 M, the partof the leaf volume representing the sieve tube/companioncell sap volume was a maximum of 1.7 gL/mg Chl. Compa-rable results have been determined by light microscopicmorphometric analysis of such barley leaves (H. Winter,unpublished results).These data indicate that the contribution of the amino acids

Table I. Total Metabolite Content of Barley Leaves Illuminated for 9h and after 5 h of Darkness

Results are mean values from three experiments with differentplants.

Metabolite Light Dark

nmol/mg Chl3-PGA 367 160Nitrate 97,300 130,000Sucrose 17,100 6,142Aspartate 1,750 750Asparagine <60 400Glutamate 5,300 3,550Glutamine 1,380 493Serine 2,170 1,160Glycine 1,370 351Threonine 770 467Alanine 883 348Valine 218 196Isoleucine 97 101Leucine 121 134Lysine 73 90Tyrosine 96 117Other 190 280

z amino acids 14,400 8,430E amino acids/sucrose 0.84 1.37

contained in the phloem sap to the total leaf contents isnegligible because they amount to only 2 to 5% of the totalamino acids in the leaves (Table IV). As shown by Pate (16),the xylem sap of barley contains a certain portion of aminoacids, mainly amides. A comparison of the vacuolar contentsof nitrate, asparagine, and glutamine (Table II) clearly dem-onstrates that the amount of amino acids in xylem sap doesnot affect our data.

Determination of the Distribution of Metabolites betweenThree Subcellular Compartments

In parallel leaf samples to those used for the measurementsshown in Table I, the distribution of the various metabolitesamong the stromal, cytosolic, and vacuolar compartmentswas determined after nonaqueous fractionation (9) accordingto the distribution of the corresponding marker enzymesNADP-glyceraldehyde phosphate dehydrogenase, phos-phoenolpyruvate carboxylase, and a-mannosidase (Tables IIand III). It should be noted that these analyses yield onlyestimations because the evaluation is based on the simplifi-cation that the leaf consists of homogenous cells. In reality,the mesophyll cells of barley leaves made up only 67 ± 3%of the total leaf cells, the vascular tissue was 6 ± 0.7%, andthe remainder consisted of epidermis and stoma cells (datafrom microscopic analysis, see above). Moreover, the contentsof the cytosolic, mitochondrial, and peroxisomal compart-ments were not differentiated. The validity of the results isdemonstrated by the finding that the vacuolar compartmentdid not contain any 3-PGA2 but it did contain practically all

2 Abbreviation: 3-PGA, 3-phosphoglycerate.

997

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Plant Physiol. Vol. 99, 1992

Table II. Subcellular Metabolite Contents in Barley Leaves Illuminated for 9 hResults are from three experiments with different plants ± SD. Values were corrected for contam-

ination by sieve tube content. n.d., Not detectable.Metabolite Stroma Cytosol Vacuole

nmol/mg Chl3-PGA 217±44 151 ±44 0Nitrate (n = 2) 0 0 97,334Sucrose 0 7,540 ± 3,080 9,590 ± 3,080Aspartate 737 ± 158 930 ± 298 105 ± 123Asparagine n.d. n.d. n.d.Glutamate 2,600 + 424 2,440 + 954 265 ± 318Glutamine 593 ± 179 759 ± 207 14 ± 28Serine 652 ± 217 1,457 + 304 43 ± 87Glycine 191 ± 150 751 ± 423 382 ± 341Threonine 231 ± 116 501 ± 162 35 ± 49Alanine 177 ± 168 680 ± 203 27 ± 44Valine 83 ± 48 124 ± 65 9 ± 17Isoleucine 37 ± 19 55 ± 27 4 ± 8Leucine 50± 30 70± 33 0Lysine 29± 18 43± 18 0Tyrosine 37 ± 19 56 ± 24 2 ± 4

Xaminoacids 5,410 + 1,550 7,860 886z amino acids/sucroce 1.04 0.09

of the leaf nitrate. It should be noted that in the nonaqueousfractionation the detection limit for each compartment was1% of the total leaf content.The results in Table II clearly demonstrate that in illumi-

nated barley leaves the amino acid contents of the vacuolarcompartment, with the exception of glycine, are very low.Even in the case of glycine, on the reasonable assumptionthat the vacuolar volume is 10-fold higher than that of thecytosol, the vacuolar concentration would be only 5% of thatin the cytosol. In previous studies with spinach leaves, theamino acid concentrations in the vacuoles were found to bemuch lower than in the cytosol, although the vacuolar con-tent of amino acids was still much higher than in barleyleaves.

Table Ill. Subcellular Metabolite Contents in BarleyResults are from three experiments with differen

Metabolite Stroma

3-PGANitrateSucroseAspartateAsparagineGlutamateGlutamineSerineGlycineThreonineAlanineValineIsoleucineLeucineLysineTyrosine

E amino acidsE amino acids/sucrose

46 ± 190

<61 ± 122300 ± 68120 ± 8

1,560 + 319290 ± 69381 ± 18563 ± 18177 ± 61101 ±4271 ± 3527 ± 531 ± 2333 ± 1327 ± 0

3,180

The virtual exclusion of amino acids from the vacuoles,especially in barley leaves, is surprising in view of the resultsof Dietz et al. (6), who found that about 50% of the totalamino acids contained in the protoplasts of barley leaveswere located in the aqueously isolated vacuoles. The totalamino acid concentration amounted to 77 mm in these vac-uoles. Moreover, these authors found that a number of aminoacids including alanine, glutamine, leucine, and methioninewere taken up specifically into isolated vacuoles. Transportof phenylalanine into barley leaf vacuoles also has beenreported recently (11).

Because most amino acids are able to penetrate membranesby diffusion (24), the virtual absence of amino acids from thevacuoles in intact illuminated barley leaves might be due to

Leaves after 5 h in the Darkt plants ± SD.

Cytosolnmol/mg Chl

109 ± 160 130,

2,150 ± 430 3,270 ± 90

01,140 ± 319128 ± 99554 ± 226172 ± 133173 ± 79191 ±8769 ± 5546 ± 2078 ± 4033 ± 3077 ± 4

2,9301.36 ± 0.57

Vacuole

5 ± 51,000 ± 20,000,,810 ± 184180 ± 53280 ± 8852 ± 17774 ± 35

219 ± 92116 ± 126112 ± 3356 ± 4557 ± 2228 ± 1525 ± 1923 ± 1713 ± 4

2,0400.53 ± 0.17

998 WINITER ET AL.




an active extrusion of amino acids from the vacuoles byspecific transport. This may explain our consistent findingthat in darkened barley leaves the vacuolar content of aminoacids is significantly higher than in illuminated leaves (TableIII). Therefore, the high concentrations of amino acids foundin vacuoles from protoplasts (6) may be the result of theincubation of the plant cells for the preparation of protoplasts.

Phloem Sap Composition

The collection of phloem sap samples from barley leavesby the aphid technique was very time consuming and re-quired much patience because the chance that phloem sapactually exuded from a severed aphid stylet was only 1 in200. This is different when working with leaves of spinachand maize, in which the corresponding success ratio was 1in 2 to 10 (G. Lohaus, unpublished results). On the otherhand, a successfully severed aphid stylet normally exudedphloem sap at a relatively high rate for such a long time thatfrom a single stylet a series of samples could be collectedwithin as many as 72 h. An example of this is shown inFigure 1. Whereas during the illumination period the phloemsap was exuded at a rate of 40 to 50 nL/h, it decreased to 20nL/h during darkness. The phloem sap was collected at 80to 90% humidity in a microcapillary tube and placed overthe severed stylet in such a way that the front edge of it camein close contact with the leaf surface. The other end of thecapillary tube was closed with a cap. In this way, any aircirculation leading to the evaporation of the phloem sapsamples was prevented very efficiently. Studies with refer-ence capillaries containing sucrose and amino acids at con-centrations imitating phloem sap demonstrated that underthe conditions of the phloem sap collection evaporation ofthe samples obtained was undetectable (G. Lohaus, unpub-lished results). Because the phloem sap from the barley leaveswas usually collected for about 4 h, yielding samples of 100to 200 nL, the volume determination from the length of theliquid column in the capillary tube could be made with anaccuracy of 100 ± 10%. The determinations of sucrose andamino acids in the samples were done with an accuracy of100 ± 5%. Therefore, the absolute concentration values de-termined in the phloem sap from barley leaves shown inTable IV can be regarded as highly reliable.

~ 60 EXUDATION RATE

B 40 \ \/VC.2 20-~

1400 200 1400 200 1400 200

time (h)Figure 1. Exudation rate of phloem sap from one single aphid styletduring a period of 60 h under a light/dark regimen.

Table IV. Concentrations of Metabolites in the Phloem Sap ofBarley Leaves Collected after 9 h of Illumination and 5 h in the Dark

Results are means ± SD.

Metabolite Light Dark

mM

Sucrose 1,030 ± 182 930 ± 162Aspartate 26 ± 6.5 51 ± 23Asparagine 2.4 ± 2.4 3.5 ± 2.7Glutamate 89 ± 24 120 ± 36Glutamine 8 ± 3.3 4.6 ± 2.6Serine 25 ± 13.5 20 ± 9.5Glycine 5.8 ± 3.8 0.8 ± 0.5Threonine 7.3 ± 2.4 10 ± 6.2Alanine 11.3 ± 2.5 12.6 ± 4.2Valine 3.7 ± 1.4 5.8 ± 3.4Isoleucine 1.1 ± 1.0 2.6 ± 1.8Leucine 1.1 ± 1.0 3.1 ± 2.3Lysine 1.7 ± 1.1 3.9 ± 2.9Tyrosine 0.5 ±0.5 1.6 ± 1.3

2 amino acids 186.0 ± 63.4 244.0 ± 96.4E amino acids/sucrose 0.18 0.26

In the phloem sap from illuminated barley leaves, theconcentration of sucrose was found to be about 1 M (TableIV). The most abundant amino acids are glutamate, aspartate,and serine. Similar results were reported earlier (22). Atpresent, it is not possible to make an exact quantitativecomparison of the metabolite concentrations in the phloemand in the cytosol because the volumes of the subcellularcompartments of barley leaf mesophyll cells are not preciselyknown, and therefore, the contents of the subcellular com-partments listed in Tables II and III cannot be translated intoconcentrations. A preliminary estimation by morphometricanalysis of EM shows that about 7% of the total cell volumein barley leaves corresponds to the cytosolic compartment(H. Winter, unpublished results). Because these leaves containan aqueous space of 700 ,L/mg Chl, a cytosolic volume ofapproximately 50 ,uL/mg Chl can be evaluated. As a result,the data shown in Table II would yield 150, 50, and 20 mmfor sucrose, glutamate, and aspartate cytosolic concentrations,respectively. Compared with these values, the concentrationof sucrose in the phloem sap would be about one order ofmagnitude higher, whereas the cytosolic concentrations ofthe above listed (and all other) amino acids were very similarto the corresponding concentration in the phloem sap. Thepreferential extraction of sucrose from the cytosol of thesource cells into the sieve tubes over the extraction of aminoacids is illustrated by the total amino acids to sucrose ratiosamounting to 1.0 in the cytosol and to 0.18 in the phloemsap.The pattems of amino acids in the phloem sap of illumi-

nated and darkened barley leaves resemble to some extentthe corresponding patterns in the cytosol, as shown in TablesV and VI, although there are significant differences apparentwhen the percentages in the phloem sap and the cytosol aredivided by each other. With illuminated and darkened leavesfor aspartate, glutamate, valine, and lysine, ratios of >1 arefound, indicating that these amino acids are transferred into

999




Table V. Percentage of Each Amino Acid from Total Amino AcidContent in the Whole Leaf, Cytosol, and Phloem after 9 h ofIllumination

n.d., Not detectable.Metabolite Total Leaf Cytosol Phloem Phloem/Cytosol

Aspartate 12.2 11.7 14 1.2Asparagine 0.4 n.d. 1.3Glutamate 37.0 30.0 48.0 1.6Glutamine 9.6 10.1 4.3 0.4Serine 15.0 18.8 13.4 0.7Glycine 9.5 10.0 3.1 0.3Threonine 5.3 6.5 3.9 0.6Alanine 6.1 8.8 6.1 0.7Valine 1.5 1.5 2.0 1.3Isoleucine 0.7 0.7 0.6 0.9Leucine 0.8 0.9 0.6 0.7Lysine 0.5 0.5 0.9 1.8Tyrosine 0.7 0.7 0.3 0.4Other 1.3 1.7

the sieve tubes with some preference over the other aminoacids. With glutamine, on the other hand, this ratio is partic-ularly low, which suggests that the release of this substancefrom the source cells into the sieve tubes is restricted inrelation to the other amino acids.

Previous comparative studies of amino acid levels in leavesand in the phloem sap obtained from these leaves showedthat in illuminated spinach leaves under growth conditions,the amino nitrogen was mainly exported as glutamine, as-

partate, and glutamate, representing 28, 26, and 24% of thetotal amino acids in the phloem sap (18). A comparison ofthe relative distribution of these amino acids in the leavesand in the phloem sap indicated that glutamate was trans-ferred preferentially over the other amino acids, whereas thetransfer of glutamine, despite its high content in the phloem,appeared to be restricted with regard to its very high cytosoliclevel. Variable results have been obtained with maize leaves(15, 23). In experiments from our laboratory, the main con-

stituents of the phloem sap were glutamine, alanine, gluta-mate, and asparagine (23). Of these amino acids amountingto 28, 28, 13, and 13% of the total amino acids in the phloem,respectively, the amides appeared to be transferred prefer-entially from the source cells into the sieve tubes. It may benoted that with spinach and barley leaves the relative aspar-agine content in the phloem sap was also much higher thanin the leaves. Apparently, there exists a mechanism for a

preferential transport of asparagine in the three differentplants investigated, although in spinach and barley it is notmuch utilized because in the phloem sap obtained from theseleaves asparagine made up only 2 and 1%, respectively, ofthe total amino acids.

Comparison of the Diurnal Changes of Amino Acid Levelsin Leaves and in the Phloem Sap Obtained from TheseLeaves

As shown in Tables V and VI, the pattem of amino acidsin whole barley leaves is not very different from the corre-

sponding pattern in the cytosol. This observation and similarresults recently obtained with spinach leaves (18) reveal thatfor studying the dependence of the amino acid compositionof the phloem sap on the metabolism of the source cell, it isnot necessary to determine cytosolic metabolite levels. In-stead, it seems sufficient to determine the whole leaf contentas a reasonable measure of the cytosolic amino acid levels.Based on this finding, we compared the amino acid pattemsin barley leaves and in the phloem sap obtained from theseleaves in a diumal cycle. In three experimental series, leavesof barley plants were quenched in their metabolism for sixdifferent times during a day/night cycle for assay of metab-olites, and under identical conditions, phloem sap was col-lected for several cycles from successfully severed stylets.Results of the continuous measurement of the concentrationsof glutamate and glycine in phloem sap samples obtainedfrom the exudation of a single aphid stylet are shown inFigure 2. From the exudation of four stylets, each measuredover a period of 20 to 60 h, mean values that were calculatedfor the composition of the phloem sap in a diumal cycle are

presented in Figure 3 with mean values of the correspondingleaf contents.

For many amino acids the diumal changes of the concen-

trations in the phloem sap reflect changes of the leaf content.Of glycine, serine, and glutamine, which are substancesformed during photosynthetic metabolism, the decrease inthe leaf content in the dark period is accompanied by a

decrease in the corresponding phloem sap concentrations.Whereas in the case of glycine the correlation between leafand sieve tube content is very high, with serine being one ofthe three major phloem sap constituents and with glutamine,the relative decrease in the phloem sap concentration in thenight is less than the decrease in the leaf content. The markedincrease in valine, leucine, isoleucine, lysine, phenylalanine,and tyrosine in the leaves during the dark period is closelyreflected by a corresponding increase in the phloem sap. Itmay be noted, however, that with most of these amino acidsthe relative concentration increase in the phloem sap in the

Table VI. Percentage of Each Amino Acid from Total Amino AcidContent in the Whole Leaf, Cytosol, and Phloem Sap after 5 h ofDarkness

n.d., Not detectable.Metabolite Total Leaf Cytosol Phloem Phloem/Cytosol

Aspartate 8.9 6.4 20.9 3.3Asparagine 4.7 n.d. 1.4Glutamate 42.1 36.3 49.2 1.4Glutamine 5.8 5.0 1.9 0.4Serine 13.7 21.2 8.2 0.4Glycine 4.1 7.2 0.3 0.05Threonine 5.5 6.4 4.1 0.6Alanine 4.1 6.8 5.2 0.8Valine 2.3 2.4 2.4 1.0Isoleucine 1.2 1.7 1.1 0.6Leucine 1.6 2.9 1.3 0.4Lysine 1.1 1.0 1.6 1.6Tyrosine 1.4 3.1 0.7 0.2Other 3.3 1.8

1 000 WINTER ET AL.




.2

Ec0._

a,-

0u

_R

0

1400 200 1400 200

time (h)1400 200

Figure 2. Diurnal variations of glutamate and glycine concentrationsin the phloem sap from one single aphid stylet during a period of60 h under a light/dark regimen. (Same experiment as in Fig. 1.)

night is higher than the corresponding increase in leaf con-

tent.This reflects a general increase in amino acid concentrations

in the phloem sap during the night. Similarly, in the case ofglutamate, the phloem sap concentration increases consider-ably during the night, whereas the leaf content remainsalmost unaltered. With aspartate, the phloem concentrationis largely increased during the night, although the leaf contentdecreases significantly. Apparently during darkness, aspar-tate is preferentially extracted from the source cells into thesieve tubes over other amino acids, which is also demon-strated from the results in Table VI.

Relationship between Phloem Loading of Sucrose andAmino Acids

Under the conditions of our experiments, the barley leavesshowed a net photosynthesis rate of about 80 ,umol C02/mgChl.h. If 80% of the fixed carbon were exported as sucrose

via the sieve tubes, this would require a translocation rate of5.3 ,umol sucrose/mg Chl.h. In the phloem sap collectedduring the illumination period (Table IV), the ratio of aminoacids to sucrose is 0.18, which related to the translocationrate of sucrose assumed above yields a translocation rate fortotal amino acids of about 1 Amol/mg Chl - h.The sucrose accumulated in the leaves by the end of the

illumination period is only about 3 times the amount presum-

ably translocated in 1 h during the light period. Because ofthe low content of starch in barley leaves (10 jtmol/mg Chlend of illumination period), the leaf sucrose may be requiredfor the dark respiration of the leaf mesophyll cells. For thesereasons, the export of sucrose from the source cells will belargely decreased upon darkening. From the decrease in theleaf sucrose content during the second half of the night period

(Fig. 3A), it can be calculated that during this time the rateof sucrose translocation is <0.6 ,umol/mg Chlh, which isonly 10% of the full rate assumed during the illuminationperiod.

In contrast to sucrose, amino acids are present in leaves insuch high amounts after the end of the illumination periodthat an export from the source cells via the sieve tubes at thefull rate of 1 ,umol/mg Chl *h during the whole night wouldconsume only about 60% of the leaf amino acid pool. But, infact, the translocation of amino acids appears to be restrictedduring the dark period. This is obvious from the amino acidto sucrose ratio in the phloem sap obtained in the dark. If inthe dark the amino acids were loaded into the phloem at fullrate, the ratio of amino acids to sucrose in the phloem sapamounting to 0.18 in the light period should increase 10-foldto 1.8, but this is not the case. Although there is a certainincrease of the amino acid to sucrose ratio observed in thephloem sap upon darkening of the leaves, this ratio is only0.3, even at the end of the illumination period. That theincrease of this ratio between the illumination and darkperiod is only twofold indicates that during the second halfof the dark period the translocation of amino acids is de-creased to <20% of the rate evaluated for the light period.

This decrease in amino acid translocation during darknessmay also explain the increase in the leaf contents of certainamino acids such as leucine, isoleucine, valine, tyrosine, andphenylalanine, which is observed during the dark period.The increase may be due to continuation of the biosynthesisof these amino acids while their export is inhibited.The question arises as to which mechanism achieves the

apparent decrease in amino acid translocation during thedark period. Because of the high amino acid contents in theleaf at the end of the dark period, it seems unlikely that thetransfer of amino acids from the cytosol of the source leavesto the sieve tubes is limited to any major extent by thecytosolic amino acid concentrations. As shown earlier withspinach leaves (18) and also in the present report with barleyleaves, the sucrose is concentrated during transfer from thecytosol of the source cells into the sieve tubes by a factor ofabout 10, whereas amino acids seem to remain in the sievetubes at a similar concentration as in the cytosol. Althoughthe phloem loading probably involves a passage through theapoplast (21), the overall process of the transfer of aminoacids from the cytosol of the source leaf into the sieve tubesmay be a passive process.The translocation of amino acids via the sieve tubes may

require the mass flow of sucrose driven by the active sucrosetransport (3, 5, 10, 19) involved in phloem loading. Thephloem transport of xenobiotica can be regarded as a modelfor such a proposed dependence of amino acid phloemtransport on the mass flow of sucrose. Substances like 2,4-D, which because of their lipid solubility are able to diffusefrom leaves exposed to these substances into the sieve tubes,are only translocated via the sieve tubes when the leaves aresynthesizing sucrose (12). There are many indications in theliterature that CO2 and NO3- assimilation are closely linkedto each other (4, 13). One factor in this may be the adjustmentof sucrose and amino acid export from the source cells viathe sieve tubes. In the phloem-loading process, the transferof amino acids from the cytosol of the source cells will require

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_ o

,0 Q

0.3

0.2 {

00

0.1 0&Il..

time (h)

Figure 3. Diurnal variations of the content of sucrose (A) and amino acids (B-G) in mature barley leaves and in the phloem sap obtainedfrom such leaves under identical conditions. The leaf contents (0) are mean values from three series of measurements, and the phloem sapconcentrations (O) are mean values from the exudation of four aphid stylets (20-60 h each).

0

E

v

E

-E

0

E

4--

0

0

1~E

4--a

0U

Q0

0

E0

F TOTAL AMINO ACIDS0

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time (h)

G AMINO ACIDS/SUCROSE0

_ , %%

,0 --_

I0




specific membrane translocators because of the apoplasticmechanism most probably involving the passage of twomembranes.

In plasma membrane vesicles, four amino acid transportsystems have been found: two for transport of neutral aminoacids, one for acidic amino acids, and one for basic aminoacids (14). Although it is not clear whether and how thesecharacterized membrane translocators are involved in phloemtransport, it seems most likely that these or similar translo-cators participate in the phloem-loading process. Because ofthe wide spectrum of amino acids translocated via the sievetubes, a regulation of the phloem loading with amino acidsat the site of membrane transport would involve a synchron-ous regulation of the various specific amino acid membranetranslocators. Although an individual regulation of the var-

ious translocators may occur, the coupling of amino acidtranslocation to the mass flow of sucrose, as proposed above,may be a very simple way for a general adjustment of theleaf export of amino acids to the export of carbohydrates.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Dieter Heineke, Dr. SieglindeBorchert, and Burgi Riens for valuable discussions.

LITERATURE CITED

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1 004 WINTER ET AL.



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