Physiological Aspects Sugar Exchange between the ...tained300,AL oftheenzymeextract, 400,uL of0.1 Msuccinic acid, pH 5.0 (with 0.8 mg of BSA and 1.75 ,uL of f-1-mercaptoethanol), and

Plant Physiol. (1992) 100, 1815-18220032-0889/92/100/181 5/08/$01 .00/0

Received for publication January 15, 1992Accepted July 16, 1992

Physiological Aspects of Sugar Exchange between theGametophyte and the Sporophyte of Polytrichum formosum

Sylvie Renault, Jean Louis Bonnemain*, Loic Faye, and Jean Pierre Gaudillere

Laboratoire de Physiologie et Biochimie Vegetales (Centre National de la Recherche Scientifique, Unite deRecherche Associee 574), 25 rue du faubourg St Cyprien, 86000 Poitiers, France (S.R., JIL.B.); Laboratoire desTransports Intracellulaires (Centre National de la Recherche Scientifique, Unite de Recherche Associee 203),

Universite de Rouen, F 76130 Mont St. Aignan, France (L.F.); and Institut National de la Recherche Agronomique,Centre de Bordeaux, Station de Physiologie Vegetale, 33140 Villenave d'Ornon, France U.P.G.)

ABSTRACT

The sporophyte of bryophytes is dependent on the gametophytefor its carbon nutrition. This is especially true of the sporophytesof Polytrichum species, and it was generally thought that sucrosewas the main form of sugar for long distance transport in theleptom. In Polytrichum formosum, sucrose was the main solublesugar of the sporophyte and gametophyte tissues, and the highestconcentration (about 230 mM) was found in the haustorium. Incontrast, sugars collected from the vaginula apoplast were mainlyhexoses, with traces of sucrose and trehalose. p-Chloromercuri-benzene sulfonate, a nonpermeant inhibitor of the cell wall inver-tase, strongly reduced the hexose to sucrose ratio. The highest cellwall invertase activity (pH 4.5) was located in the vaginula, whereasthe highest activity of a soluble invertase (pH 7.0) was found inboth the vaginula and the haustorium. Glucose uptake was carrier-mediated but only weakly dependent on the external pH and thetransmembrane electrical gradient, in contrast to amino acid up-take (S. Renault, C. Despeghel-Caussin, J.L. Bonnemain, S. Delrot[1989] Plant Physiol 90: 913-920). Furthermore, addition of 5 or50 mm glucose to the incubation medium induced a marginaldepolarization of the transmembrane potential difference of thetransfer cells and had no effect on the pH of this medium. Glucosewas converted to sucrose after its absorption into the haustorium.These results demonstrate the noncontinuity of sucrose at thegametophyte/sporophyte interface. They suggest that its conver-sion to glucose and fructose at this interface, and the subsequentreconversion to sucrose after hexose absorption by haustoriumcells, mainly governs sugar accumulation in this latter organ.

the presence of chloroplasts throughout the young sporo-phyte (1). This dependence varies in importance, dependingon the genus, but is dramatic in various species of Polytrichum(in which photosynthesis only balances respiration) (26, 28).The study of sugar transport in mosses began in the 1960s.

Two major results have been shown: (a) the transport ofassimilates is localized in leptoid cells of Polytrichum, with avelocity similar to that in higher plants (9, 10), and thisvelocity is markedly reduced to a few millimeters per hour inFunaria hygrometrica, in which conducting tissues are poorlydifferentiated (4); (b) in Funaria, transfer cells of the hausto-rium play a key role in sugar absorption (sucrose, glucose) bythe sporophyte (3, 4). Uptake is inhibited by uncouplers(CCCP1), metabolic inhibitors (KCN and NaNO3), and pho-tosynthesis inhibitors (DCMU) (4).On the other hand, several important aspects of this subject

are still unknown, particularly the kind of sugars present atthe gametophyte/sporophyte interface and, consequently,the mechanism of sugar uptake by the haustorium. Therefore,the aim of this research was to identify the sugars of thevaginula apoplast and to study their metabolic fate fromgametophyte to sporophyte at the vaginula/haustorium in-terface and their uptake properties. This work is part of acomparative study of the mechanisms of nutrient exchangebetween generations in plants.

MATERIALS AND METHODS

In mosses, as in angiosperms, there are no symplasticconnections at the interface between the two generations,and the epidermis of the organism emerging from the zygotemay be modified into transfer cells. In Polytrichum formosum,as in many mosses, these epidermal transfer cells border thehaustorium, i.e. the part of the sporophyte that is embeddedin the cavity of the upper part (vaginula) of the gametophyte.It was shown recently that the plasmalemma of epidermaltransfer cells of P. formosum haustorium creates a protonmotive force much larger than that of other cells of thesporophyte (30). This proton motive force is used to energizethe absorption of amino acids released into the apoplast ofthe gametophyte (5, 6, 27, 30). The sporophyte is also de-pendent on the gametophyte for its carbon nutrition, despite

1815

Plant Material

Gametophytes and sporophytes (during their develop-ment) of Polytrichum formosum were used as the experimentalmaterial. Sporophytes were isolated as previously described(30). The haustorium was placed in a standard medium (0.125mM CaCl2, 0.125 mM MgCl2, and 0.1 mm KCl). The temper-ature was 20 ± 10C, and unless otherwise stated, lightintensity was 48 umol.m-2 s-' (Sylvania fluorescent tubes).Sporophytes 3.0 ± 0.5 cm long were used.

1 Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydra-zone; a-AIB, a-aminoisobutyric acid; DES, diethylstilbestrol; 3-0-MeG, 3-0-methyl glucose; PCMBS, p-chloromercuribenzene sulfo-nate; PD, potential difference.

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Plant Physiol. Vol. 100, 1992

"CO2 Assimilation

Two techniques have been used. Carbon flux between thegametophyte and the sporophyte was measured after "4CO2labeling (1110 Bq ,mol -', 400 Aimol' photons.m-2 s-, 350AMmol- CO2 in air) by mosses during 30 min. Carbon parti-tioning was measured after a 0-, 6-, 24-, and 48-h chase indarkness, in the gametophyte, the haustorium, the seta, andthe capsula. Cellular metabolites were extracted by N,N-dimethylformamide. Starch was solubilized by amylogluco-sidase (1 h, 450C, pH 4.6). Radioactivity was measured byliquid scintillation counting.

In another set of experiments, 2 1iL of phosphate buffer(pH 8) containing ["4C]Na2CO3 (74 kBq) were placed on 20gametophyte leaves. After 6 or 12 h, the seta of the sporo-phyte was cut, frozen, and lyophilized. Sugars were extractedand analyzed by the method described in the followingsection.

Uptake of Labeled Compounds

Isolated sporophytes were prepared as previously de-scribed (30). After 1 h of preincubation in standard medium(buffered at pH 5 with 10 mm citrate and 20 mm disodiumphosphate), sporophytes were incubated in 2 mL of the samemedium containing one of the following labeled sugars: 1mM [3H]sucrose (18.5 kBq), 1 mm [3H]glucose (18.5 kBq), 1mM [3H]3-O-MeG (18.5 kBq), 1 mM [14C]sucrose (148 kBq), 1mM [14C-fructosyl]sucrose (185 kBq), or 1 mM [14C]glucose(111 kBq). After uptake, the sporophytes were rinsed in abath (1 min) of unlabeled medium. The tissues that hadabsorbed 3H-sugars were digested for 24 h (600C) in thepresence of 100 IAL of perchloric acid and 200 AlL of H202,and their radioactivity was counted by liquid scintillationspectrometry (Intertechnique SL 33) after addition of 5 mLof scintillant (PCS II, Amersham). The tissues that had ab-sorbed 14C-sugars were rapidly frozen and lyophilized beforeextraction of sugars according to the method of Dickson (8).Lyophilized tissues were homogenized in metha-nol:chloroform:water (12:5:3). The water-alcohol fractioncontained the carbohydrates. The sugars were concentratedby drying and were redissolved in 80% (v/v) ethanol. Asym-metrically labeled sucrose was hydrolyzed (230 units of in-vertase/mL of 100 mm acetate buffer) and then dissolved in80% ethanol. Sugars were analyzed by paper chromatogra-phy in a solvent consisting of ethyl acetate:acetic acid:water(3:3:1). The radioactivity on the chromatograms was countedby a low background gas flow counter (Manu 16, Numelec,France).

Endogenous Sugars

For determination of the soluble sugars of the main organs,10 mg of P. formosum tissues (leaves, axis, haustorium, andseta) were incubated for 15 min at 600C in 1 mL of 80%ethanol. The liquid fraction was frozen before analysis byGC.

For the extraction of the sugars from the apoplastic cavityof the vaginula, 1 IAL of standard medium (pH 6) was insertedin each vaginula of 50 Polytrichum. After the vaginula apo-plast was rinsed for 30 min at 40C, the solution was collected,

boiled at 1000C for 10 min (in the presence of polyvinylpo-lypyrrolidone), and then rapidly frozen (-250C). Sugars wereanalyzed by GC and HPLC.

Invertase Extraction and Assay

Polytrichum tissues (250 mg; axis, vaginula, haustorium,and seta) were homogenized in 20 mm citrate and disodiumphosphate (pH 6.5) and centrifuged for 20 min at 9000g, andthe supernatant (representative of the intracellular com-partment) was frozen. The pellet was then incubated for 3 hin the same medium with 1 M NaCl. After centrifugation (20min at 9000g), the supematant was frozen (this fraction isconsidered representative of the extracellular compartment,containing cell wall-bound enzymes). All of these procedures(11) were carried out at 4 + 10C. For the determination ofinvertase activity, 0.1 mL of the enzyme preparation plus 0.9mL of substrate (0.1 M sucrose or trehalose in a 0.1 M citratedisodium phosphate buffer, pH 4.5 or 7) was incubated at37 ± 10C (temperature of maximal activity of invertase) for3 h. The reaction was stopped by incubating the mixture for3 min in boiling water. The reducing sugars formed weremeasured according to the method of Nelson (24).

a-1-Mannosidase Assay

a-i-Mannosidase was used as a vacuolar marker (2). Theenzyme activity was assayed by measuring the liberation ofp-nitrophenyl-a-mannopyranoside. The assay mixture con-tained 300 ,AL of the enzyme extract, 400 ,uL of 0.1 M succinicacid, pH 5.0 (with 0.8 mg of BSA and 1.75 ,uL of f-1-mercaptoethanol), and 100 ,AL of 8 mm p-nitrophenyl-a-mannopyranoside (2). The reaction was started by additionof substrate. It was stopped after 30 to 240 min by additionof 400 ALL of 1 M Na2CO3, and absorbance was determinedat 405 nm.

pH Measurements

One hundred sporophytes of P. formosum were isolatedand affixed vertically to the wall of a 50-mL beaker withterostat (inert paste), their haustoria dipping into 10 mL ofstandard medium (pH 5.0). Experiments were conducted aspreviously described (30).

Electrophysiological Measurements

Sporophytes were secured with terostat in a cuvette, con-taining standard medium buffered at pH 5.0. Electrophysio-logical measurements were made in a Faraday cage, with theequipment previously described (30). After 1 h of preincu-bation, the reference electrode was dipped into the bathingmedium, and the glass micropipette was inserted into atransfer cell with a mechanical micromanipulator.

RESULTS AND DISCUSSION

Carbon Exchange between Generations and TransportForm of Sugar in the Sporophyte

Photosynthesis was extremely low in the sporophyte be-cause only 1% of the labeled carbon assimilated by the moss

RENAULT ET AL.1816



SUGAR EXCHANGE BETWEEN THE GAMETOPHYTE AND SPOROPHYTE OF MOSSES

18 Endogeneous Soluble Sugars of theGametophyte and Sporophyte

15 - Subjacent organs (axis and leaves of the gametophyte) andorgans above (haustorium, seta; i.e. the sporophyte axis) the

> / / . * generation interface were studied. Soluble sugars extracted_1211t1<- | z haustorium from these organs consisted mainly of sucrose (Table I). Theset0 0 data are in agreement with previous results conceming var-

o 9T ,h ious species of mosses (4, 10, 17, 22). Hexoses were detected

i3- / /only in small quantities. The latter result is in contradiction6 - to the data of Holligan and Drew (19), who reported that the

sugars extracted from Polytrichum junipericum leaves were

// - mainly hexoses.

Little trehalose was present in the gametophyte and the

capsula sporophyte. This sugar, previously identified in the calyptra0 T and the leaves of P. junipericum (19), is more particularly

0 10 20 30 40 50 present in liverworts (23). Some other sugars were present in

TIME (h) trace amounts in Polytrichum, including, probably, maltoseand a sugar alcohol.

Figure 1. Percentage of radioactivity found in the sporophyte of Sugar content was higher in the sporophyte, more pafic-mosses labeled for 30 min in the light by 14CO2. Inset, The in vivo ularly in the haustorium (77.6 mg g-' fresh weight, i.e. aboutcarbon transport between generations. Each point is the mean of 230 mM). This result shows the important role played by thenine mosses ± SE. Mosses with sporophytes 3.5 cm high were used. haustorium in the exchange of sugar between the gameto-

phyte and the sporophyte.

was found in this part of the moss immediately after "4CO2labeling (Fig. 1). Forty-eight hours later, 17% of the label wasfound in the sporophyte (Fig. 1). The in vivo carbon transportbetween generations could be calculated from specific radio-activity of the soluble sugars (mainly sucrose) present in thegametophyte. It was estimated at 64 nmol of carbon h-1.moss-' (i.e. 10.6 nmol equivalent hexose h-l moss-') andappeared to be constant throughout the experiment (Fig. 1,

inset).Twelve hours following [14C]Na2CO3 uptake by gameto-

phyte leaves, about 95% of the soluble radioactivity in thegametophyte and the sporophyte was found in the sucrose

fraction (data not shown). Only 6 h following this uptake,when the labeled sugars began to penetrate the seta via theleptom, 90% of the soluble radioactivity of these tissues wasassociated with sucrose, and the majority of the remainingradioactivity was associated with hexoses (data not shown).This dominance of sucrose is in agreement with the beliefthat sucrose is the main form of long distance transport ofsugars in bryophytes (4, 29) but does not exclude the possi-bility of hexose transport as a minor phenomenon.

Endogeneous Sugars in the Vaginula Apoplast

Sugars collected from the vaginula apoplast were primarilyhexoses (Table II), with only traces of sucrose and trehalose.Except for the trehalose, these results are completely oppositeto those concerning the soluble sugars of the gametophytepart subjacent to the vaginula and the basal part of thesporophyte (Table I). The hexose to sucrose ratio was mark-edly reduced by 1 mm PCMBS, an invertase inhibitor (25).Sugar efflux into the vaginula apoplast was also inhibited(6.1 nmol equivalent hexoses for control, 3.3 nmol equivalenthexoses for treated tissues). Because PCMBS is a nonper-

meant thiol reagent, these data suggest that sucrose hydrol-ysis takes place, at least in part, in the vaginula apoplast.

Invertase Activity

Cell wall invertase activity (pH 4.5, moderately sensitiveto 50 mm Tris) was detected primarily in the vaginula (Fig.2). Much lower activities were detected in the axis of thegametophyte and in the seta. This invertase is referred to as

an acid invertase; it is widely distributed in various tissues ofhigher plants (12, 15, 20, 33) and is generally localized in the

Table I. Soluble Sugars Extracted from Different Tissues of PolytrichumResults are in milligrams per gram fresh weight (mean ± SE, n = 3).

Tissue Sucrose Trehalose Glucose Fructose

GametophyteAxis 9.25 ± 2.2 0.78 ± 0.10 0.39 ± 0.13 0.14 ± 0.01Leaves 21.7 ± 4.9 0.50 ± 0.20 0.67 ± 0.12 0.67 ± 0.11

SporophyteHaustorium 77.6 ± 9.6 1.10 ± 0.20 1.23 ± 0.56 0.73 ± 0.38Seta 18.9 ± 6.2 0.36 ± 0.16 1.18 ± 0.50 0.81 ± 0.34

1817




Table II. Sugars Collected 30 min after Insertion of 1 ,uL of Standard Medium (oH 6) in Each of 50Vaginula (mean ± SE, n = 3)

Sugar ConcentrationTreatment--

Sucrose Trehalose Glucose Fructose

mM

Control 0.17 ± 0.05 0.05 ± 0.01 4.3 ± 2.3 1.4 ± 0.6PCMBS (1 mM) 0.50 ± 0.07 0.04 ± 0.01 1.6 ± 0.7 0.6 ± 0.1

cell wall. The levels of acid invertase activity found in thevaginula and the haustorium were similar to those detectedin some higher plant tissues (13, 20).a-Mannosidase activity was found in the intracellular frac-

tion, whereas no a-mannosidase activity was detected in thecell wall fraction after 4 h of incubation. This suggests thatthe cell wall fractions were not contaminated with solubleenzymes. Furthermore, previous results (7) have shown thatthe cells of the haustorium after transfer were not damagedwhen the sporophytes were separated from the gametophyteand that the tissues of the vaginula were also undamaged.Without homogenization of tissues, acid cell wall invertase

activity was also detected in the vaginula and the haustorium(8 and 13 Amol g-1 fresh weight.h-1, respectively). Thisresult suggests that the invertase was relatively accessibleand, therefore, present in the cell wall of the epidermaltransfer cells of the haustorium and in the vaginula epidermalcells.

Soluble invertase activity (optimum pH 7.0, highly sensi-tive to 50 mm Tris) was detected, in the same way, in thevaginula and the haustorium (Fig. 2). This invertase activitywas low in the gametophyte axis and in the sporophyte seta.This invertase, referred to as a neutral invertase, has been

100

>

_c

w o, 60In P

>0

z E40

20

0A V H S A V H S

Cell wall invertase (pH 4.5) Soluble invertase (pH 7)

Figure 2. Soluble and cell wall invertase activity of various tissuesof P. formosum (gametophyte axis, vaginula, haustorium, and seta).The effect of 50 mm Tris on invertase activity (mean ± SE, n = 3) isshown. wh, Weight.

detected in various higher plants (15, 18, 21, 33) and operatesin the cytoplasm. Inhibition of the neutral invertase activitywith 50 mm Tris and the weak inhibition of acid invertase(Fig. 2) have been observed in various higher plants (18, 21).This difference is probably due to the effect of pH on theTris-enzyme interaction (18).

Soluble invertase may initiate sucrose hydrolysis in thesymplastic compartment of the vaginula. Therefore, the hex-oses found in the vaginula apoplast may be in part effluxedfrom the symplasm. Paradoxically, this enzyme was alsopresent in haustorium tissues (Fig. 2) where sucrose synthesisoccurred (Table III, compare Tables I and II). Consequently,this raises some questions about the compartmentalizationand function of the soluble invertase in this organ.

Substrate Specificity of the Invertases

Sucrose (a-D-glucopyranosyl-f-D-fructofuranoside) may behydrolyzed by either a-D-glucosidase or B3-fructosidase. Thecleavage is based on the nature of enzymic attack on thesucrose terminal residue (pyranosyl or furanosyl). Amongthese two kinds of enzymes, ,B-fructosidase is widespreadand particularly studied in higher plants (11). To determinewhether invertase in Polytrichum was an a-glucosidase ora fl-fructosidase, we compared the metabolic fate of twosubstrates, sucrose and trehalose (a-D-glucopyranosyl-a-D-glucopyranosyl).At pH 4.5, trehalose and sucrose were hydrolyzed in the

same way, at least in the vaginula (Fig. 3A). Therefore, theacid invertase is an a-glucosidase nonspecific for sucrose and

Table IlIl. Distribution of 14C after Incubation of the Sporophyte for2 h in the Presence of 1 mM "4C-Sugar

After preincubation (1 h), each group of 25 sporophytes wastransferred to 2 mL of incubation medium containing '4C-labeledsugars. After 2 h of uptake, the haustoria were cut, rapidly frozen,and then lyophilized. Experiments were repeated three times withsimilar results. After extraction, [14C](fructosyl)sucrose was hydro-lyzed with C. utilis invertase (230 units/mL of 100 mM acetatebuffer) for 3 h.

S4C-Sugar DistributionSubstrate

Sucrose Glucose Fructose

[14C]Glucosea 88 4 3[14C]Sucrose 85 5[14C](Fructosyl)sucrose 2 37 52

(hydrolyzed)a Nonradiolabeled 1 mm fructose added.

1818 RENAULT ET AL.




Cell wall SolubleVAGINULA

Cell wall SolubleHAUSTORIUM

Vaginula Haustorium Vaginula HaustoriumCELL WALL SOLUBLE

Figure 3. Substrate specificity of soluble and cell wall invertases(mean ± SE, n = 3). One hundred percent represented 76, 19, 28,and 20 ALmol-g-1 fresh wth-' for cell wall and soluble invertases ofvaginula and haustorium, respectively, at pH 4.5 (A), and 0.4, 0.1,41, and 34 ,umol-g-1 fresh wt-h-1 for vaginula and haustoriumsoluble and cell wall invertases at pH 7 (B), respectively.

could react with sucrose and trehalose. However, in thehaustorium, the affinity of the cell wall enzyme appeared tobe higher for trehalose than for sucrose.

At pH 7, in the vaginula and the haustorium, the twosubstrates were not hydrolyzed in the same way. Therefore,it seems that a-glucosidase is not the only enzyme to hydro-lyze sucrose and trehalose in these tissues (Fig. 3B). Solubleinvertase, at this pH, may be composed of an a-glucosidaseand a fl-fructosidase, which could explain the lower percent-age recorded for trehalose hydrolysis. On the other hand, at

pH 7, the cell wall invertase hydrolyzed 10 to 60 times moretrehalose than sucrose. This result suggests the existence of acell wall trehalase, active at pH 7. Because the apoplastic pHof the haustorium transfer cells was found to be about 4.6(30), this enzyme is probably not active in vivo. In P. formo-sum, trehalose is detected only in very small amounts. Thissugar is found mainly in insects, fungi, and algae (16).However, trehalase has been detected in various higherplants, especially sugarcane (14), although its substrate isabsent. Data from Cuscuta reflexa (32) have shown the toxiceffect of trehalose when trehalase activity is low. Higherplants could encounter trehalose during their symbiotic orparasitic association with bacteria, fungi, or insects. Undersuch conditions, trehalase would serve to relieve the plantfrom the toxic effects of trehalose (32). Because there is anincreasing number of reports in the literature of microorga-nisms (fungi, blue-green algae, bacteria) associated withmosses, including Polytrichum (31), the origin of the smallamounts of trehalose found in our material is unclear.

Metabolism of Sugars in the Haustorium Tissues

Tris buffer (an inhibitor of invertase activity, see Fig. 2induced a 36.1% inhibition of sucrose uptake but had amarginal but not significant effect on glucose uptake (TableIV). This result agrees with the hypothesis that sucrosewas, at least in part, hydrolyzed before its uptake by thehaustorium.

Furthermore, 33% of the label originating from ['4C](fruc-tosyl)sucrose was associated with the glucose moiety after 2h of absorption into the haustorium (Table III), indicatingthat at least a portion of the absorbed sugar was cleaved. Onthe other hand, ["4C]glucose taken up from the incubationmedium was massively converted to sucrose in the hausto-rium tissues after its absorption (Table III).

Characterization of Glucose and 3-0-MeG Uptake

As for amino acids (30), the rate of glucose uptake from a1 mm solution changed during the growth of the sporophyte;it reached 1.5 nmol h-1 sporophyte' for 2.5-cm-high spo-rophytes (Table V) and 5.7 nmol-h-1.sporophyte-1 for 3.5-

Table IV. Effect of 50 mm Tris (pH 7.8) on the Rate of 1 mm3H-Sugar Uptake

The control was incubated in the presence of 50 mm Hepes (pH7.8). After preincubation (1 h), each group of 12 sporophytes wastransferred to medium containing 3H-labeled sugar (18.5 kBq). After1 h, the sporophytes were prepared as described in "Materials andMethods" for radioactivity measurements. Each value is the meanof 24 sporophytes ± SE. Mosses with sporophytes 3.5 cm high wereused.

Sugar UptakeSubstrate

Control Tris-HCI Inhibition

nmol*h- *sporophyte` %

1 mm sucrose 0.36 ± 0.05 0.23 ± 0.02 36.1a1 mM glucose 5.68 ± 0.49 4.60 ± 0.44 19.0 (NS)

a Significant (0.05) by Student's t test.

200

1501.

IPH45_ Ii|OUrSe|7pH4.Treharose

I4W I

ioo l

50p.

I-

I-

UI-

u

z

I.-

~-.

LuLU

z

u- -1 1.

1819

ni




Table V. Effect of Various Effectors on 1 mM [3H]Glucose and 1 mM [3H]3-O-MeG UptakeThe tissue was preincubated for 1 h in a solution buffered at pH 5 with effectors (CCCP, DES, PCMBS) or without effectors (KCI). The

sporophytes were then incubated (1 h) in a solution containing effectors and 1 mm [3H]glucose (18.5 kBq) or 1 mM [3H]3-O-MeG (18.5 kBq).Each value is the mean of 24 sporophytes ± SE. Mosses with sporophytes 2.5 cm high were used.

Glucose 3-0-MeGEffector

Control Treated Control Treatednmol-h'- sporophyte-1 % inhibition nmol-h'- sporophyte-1 % inhibition

PCMBS (1 mM) 1.50 ± 0.20 0.39 ± 0.20 74a 0.76 ± 0.05 0.45 ± 0.04 42aCCCP (5 iAM) 1.58 ± 0.20 0.41 ± 0.05 74a 0.61 ± 0.03 0.46 ± 0.05 25aDES (0.2 mM) 1.50 ± 0.05 0.76 ± 0.03 49a 0.59 ± 0.06 0.56 ± 0.05 5 (NS)KCI (100 mM) 1.50 ± 0.03 1.12 ± 0.02 25a 0.62 ± 0.06 0.60 ± 0.03 3 (NS)

a Significant (0.05); Student's t test.

cm-high sporophytes (Table IV). This last value was not verydifferent from the rate of carbon transport measured in vivobetween generations (10.6 nmol equivalent hexose h-'.moss-) at the same stage of growth (Fig. 1, inset).

Glucose and 3-0-MeG uptake in the sporophyte exhibitedtwo phases over time: uptake approached saturation after 3.5h for glucose and 2 h for 3-0-MeG. After that time,uptakewas linear for at least several hours (Fig. 4). On the otherhand, glucose and 3-0-MeG uptake by isolated haustoriashowed saturation after 6 or 8 h (Fig. 4). Sugar concentration

(9 ~~~~~intact15 sporophytO

lo10 ~~~~~~haustorium0 T

1- /

0

GlucoseE 0c~ (#) | intact

! 2 sporophyte

haustorium

0 1

O 2 4 6 8 10 12TIME (h)

Figure 4. Time dependence of glucose (A) and 3-0-MeG (B) uptakeby P. formosum intact sporophytes (0) and haustoria (U). Thesporophytes were incubated (1 h) as described in 'Materials andMethods." After preincubation (1 h), the isolated haustoria weretransferred to 10 mL of incubation medium buffered at pH 5containing [3H]glucose (185 kBq) or [3H]3-O-MeG (296 kBq). Afterincubation (1 h), the sporophytes and the haustoria were rinsed ina bath (1 min) of unlabeled medium, and then the tissues weredigested as described above. Each data point represents the meanof 18 sporophytes ± SE (-) or 12 sporophytes ± SE (U).

in the isolated haustorium was calculated after the hausto-rium volume (0.79 mm3) was estimated. After 12 h of trans-port, the apparent glucose concentration in the haustorium(8.08 mm) was higher than the external glucose concentration(1 mM). In the case of 3-0-MeG, the concentration in thehaustorium (1.24 mM) was similar to its external concentration(1 mm). Because there was no accumulation, uptake of 3-0-MeG may be simply by diffusion.Uptake of glucose and 3-0-MeG was weakly dependent

on the external pH, with a slight increase at pH 5 (Fig. 5A).Glucose uptake at neutral and alkaline pH was the same asat pH 4. In contrast, amino acid uptake that occurs by H+cotransport is maximum at pH 4 and is strongly inhibited atneutral and alkaline pH (30). The apparent sucrose uptake,much less important than glucose uptake, was more depend-ent on the external pH than was hexose uptake (Fig. 5B).Sucrose uptake was maximal from pH 4 to 5, which is theoptimal pH for acid invertase activity.Uptake of glucose exhibited saturable kinetics (Fig. 6A).

Lineweaver-Burk (Fig. 6B) and Eadie-Hofstee plots showed asingle phase with an apparent Km of 46.6 mm and an apparentV.,ax of 2.8 nmol min-' sporophyte-1. These data suggestthat a carrier that becomes saturated at high concentrationscould be involved in the uptake of glucose by the transfercells. These results differ from those obtained on the sameplant material concerning amino acid uptake (5, 30), wherethe Km was much lower (about 1 mm). Uptake of 3-0-MeGwas linear with concentrations up to 150 mm.

Effect of Various Effectors on Uptake

PCMBS, a nonpermeant thiol reagent, strongly inhibitedglucose uptake, and 3-0-MeG uptake to a lesser extent (TableV). These results confirm the involvement of a carrier inglucose uptake. The protonophore CCCP and the ATPaseinhibitor DES strongly inhibited glucose uptake (74.2 and49% inhibition, respectively), but they had only a slight effecton 3-0-MeG uptake (Table V). KCl (100 mm), which inducesa dramatic decrease in the PD of transfer cells (about 100mV) (30), had a slight effect on glucose uptake and no effecton 3-0-MeG uptake. These results are in contrast with thoseconcerning amino acids; uptake of glycine (5) and a-AIB (30)is strongly inhibited by 100 mi KCl (50-80%, depending onthe experimental conditions and the substrate).The effect of CCCP and DES on glucose influx may be

1 820 RENAULT ET AL.




04;1.0

0

Ec

LU 3.0-MeG

=0.4 Sucrose

0.2 -

4 5 6 7 8pH

Figure 5. pH dependence of [3H]glucose and [3H]3-O-MeG (A)and [3H]sucrose (B) uptake by P. formosum sporophytes. Threedifferent buffers were used: 10 mm citrate, 20 mm disodium phos-phate for pH 3.5 to 7.0 (0), 20 mm Mes/NaOH for pH 5.0 (0), and20 mm Hepes for pH 7.0 to 8.0 (0). The sporophytes were preparedas previously described. Each point is the mean of 24 sporophytes± SE.

either the result of inhibition of an active uptake process or

the consequence of the inhibition of the metabolic conversionof glucose to sucrose inside the haustorium tissues. Because3-0-MeG uptake was inhibited to a lesser extent than glucoseuptake, and because uptake of the latter is weakly dependenton the transmembrane electrical gradient (slight inhibitoryeffect of 100 mm KCl), intracellular metabolism may be themajor process driving glucose uptake.

Effect of Addition of Sugars on the PD of TransferCells and on the pH of the Incubation Medium

The addition of 5 mm glucose to the incubation mediuminduced a marginal effect on the PD of the transfer cells (nodepolarization or less than 5 mV), whereas 5 mm 3-0-MeGor sucrose had no effect (data not shown). Neither sugar hadany effect on the pH of the incubation medium. These resultsare in contrast with previous data concerning amino aciduptake (27, 30); 5 mm a-AIB, leucine, threonine, methionine,or glycine induced a depolarization of about 50 mV and an

alkalinization of the incubation medium. The difference can-

not be explained in terms of rate of uptake because the influxof 5 mm glucose (Fig. 6A) and 5 mm a-AIB (figure 7 in ref.30) are nearly the same.

- o .

0750J 0.6

Hl 0.4oj

7 50-

3 ~~~~~~~~0.2-E

25.0 1 2

V/S00 25 50 75 100 125 150

GLUCOSE (mM)

Figure 6. Concentration dependence of [3H]glucose uptake bysporophytes. A, Michaelis-Menten plot; B, Lineweaver-Burk plot.After preincubation (1 h), each group of 12 sporophytes was trans-ferred to 2 mL of incubation medium buffered at pH 5 containing[3H]glucose (18.5 kBq) in concentrations ranging from 0.5 to 20mm. Sugar concentrations from 25 to 150 mm~(37 kBq of 3H) wereused. After 1 h of incubation, the sporophytes were prepared aspreviously described. Each point is the mean of 72 sporophytes± SE.

The addition of 50 mm sugar to the incubation mediumhad no effect on the PD of the transfer cells, except forglucose, which induced a slow and slight depolarization(about 25 mV) (Fig. 7). This gradual change in the transmem-brane PD is in contrast with the fast depolarization obtainedwith 5 mM or even 1 mm neutral amino acid (figure 3 in ref.27, figure 11 in ref. 30). Furthermore, as for 5 mm sugar, thisaddition had no effect on the pH of the incubation medium(Fig. 7).

In conclusion, our results show that, in agreement withprevious results concerning various bryophytes (4, 10, 17,22), sucrose is the main form of storage of soluble sugars inP. formosum. They also suggest that sucrose is the main formof carbon for long distance transport, which also confirmsprevious results (4, 29). The results presented here demon-

20 min-100

> Glucose 3-0-MeG Sucrose

0-200 1Glucose

30Me Surs4.8 - 3.0MeG Sucrose

CL4.6 -.4.4 -20mmi

Figure 7. Effect of addition of 50 mm sugar (final concentration) onthe PD of P. formosum transfer cells and on the pH of a mediumcontaining 100 sporophytes (2.5-3 cm long). These experimentswere conducted four times with similar results.

1821




strate the noncontinuity of sucrose at the gametophyte/sporophyte interface, hexoses being the main sugars foundat this interface. Sucrose hydrolysis is at least in part achievedby a cell wall invertase, which is an a-glucosidase. Thissucrose hydrolysis, and the subsequent reconversion to su-crose after hexose absorption into the haustorium, could helpto drive hexose absorption and lead to sucrose accumulation.Furthermore, our results suggest that the proton motive forceestablished at the plasma membrane level of transfer cells inthe haustorium does not play a major role in sugar uptake,contrary to what has been found for amino acid uptake (30).Our results support the hypothesis that this system of meta-bolic conversion of sugars governs sugar exchange betweenthe gametophyte and the sporophyte of P. formosum, at leastat the stage studied. This raises the question of whether ornot the absence of direct competition between some organicnutrients (e.g. amino acids and sugars) in dissipating theproton motive force may also be the case at the interfacebetween the maternal organism and embryo in angiosperms.This subject is now under investigation.

ACKNOWLEDGMENT

The authors thank Dr. M.D. Devine for correcting the English inthe manuscript and for stimulating discussions.

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1 822 RENAULT ET AL.



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