Amino Acid Transport in Germinating Castor Bean Seedlings1 - NCBI

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Plant Physiol. (1981) 68, 560-566. 0032-0889/81/68/0560/07/$00.50/0. Amino Acid Transport in Germinating Castor Bean Seedlings1. Received for publication ...
Plant Physiol. (1981) 68, 560-566

0032-0889/81/68/0560/07/$00.50/0

Amino Acid Transport in Germinating Castor Bean Seedlings1 Received for publication January 6, 1981 and in revised form March 16, 1981

SIMON P. ROBINSON2 AND HARRY BEEVERS Thimann Laboratories, University of California, Santa Cruz, California 95064 ABSTRACT

During germination and early growth of the castor bean (Ricinus communis) nitrogenous constituents from the endosperm are transferred via the cotyledons to the growing embryo. Exudate collected from the cut hypocotyl of 4-day seedlings contained 120 millmlar soluble amino nitrogen and glutamine was the predominant amino acid present, comprising 35 to 40% of the total amino nitrogen. To determine the nature of nitrogen transfer, the endosperm and hypocotyl were removed and glutamine uptake by the excised cotyledons was investigated. Uptake was linear for at least 2 hours and the cotyledons actively accumulated glutamine against a concentration gradient. The uptake was sensitive to respiratory inhibitors and uncouplers and effux of glutamine from the excised cotyledons was negligible. Transport was specific for the L-isomer. Other neutral amino acids were transported at similar rates to glutamine. Except for histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids. For glutamine transport, the Km was 11 to 12 millimolar and the V was 60 to 70 micromoles per gram fresh weight per hour. Glutamine uptake was diminished in the presence of other amino acids and the extent of inhibition was greatest for those amino acids which were themselves rapidly transported into the cotyledons. The transport of amino acids, on a per seedling basis, was greatest for cotyledons from 4to 6-day seedings, when transfer of nitrogen from the endosperm is also maximal. It is concluded that the castor bean cotyledons are highly active absorptive organs transporting both sucrose and amino acids from the surrounding endosperm at high rates.

in the endosperm increases initially, reaching a peak after 5 to 6 days, then declines to zero by about day 8 when the endosperm has completely disappeared. Some of the amino acids are used for synthesis of enzymes and organelles, but eventually, all of the nitrogen originally present in the endosperm is transferred to the embryo and it seems likely that amino acids produced by hydrolysis of endosperm proteins are transported into the cotyledons. Previous studies of amino acid uptake in higher plants have dealt mainly with transport into roots (17, 21, 23), leaf slices (4, 13, 14, 16), or cells from suspension cultures (1-3, 6-9). However, in germinating cereal grains material hydrolyzed in the endosperm is transferred to the embryo by a specialized absorptive organ, the scutellum, and studies with corn (19) and barley (18) have demonstrated that excised scutella rapidly transport amino acids. The properties of an equally active system for transport of amino acids in the cotyledons of germinating castor bean seedlings are described in this paper. MATERIALS AND METHODS

Growth Conditions. Seeds of castor bean Ricinus communis cv. Hale were soaked for 24 h in cold running tap water, placed in moist vermiculite, and germinated in the dark at 30 C in a humidified growth chamber. The time of planting was taken as time zero and the seedlings were used on day 4 for most experiments. At this stage, the endosperm had an average fresh weight of 0.8 g and the pair of cotyledons weighed 55 mg and had a total surface area of 5.5 cm2. Collection of Exudate. The hypocotyl of each seedling was cut with a razor blade about 5 mm below the cotyledons which remained enclosed by the endosperm (Fig. 1). The hypocotyl stump, still attached to the cotyledons, was inserted into a narrow During germination of the castor bean, the storage reserves of tube and 20 seeds were placed in a humid chamber at 25 C. the endosperm are hydrolyzed and the metabolic products are plastic 4 h, material that had exuded from the hypocotyl stump After transferred into the growing embryo. There is a massive conver- was collected ethanol was added to a final concentration of sion of the endosperm lipid reserves into sucrose, and the cotyle- 80%o (v/v). Theand exudates were centrifuged at 40,000g for 15 min dons, buried within the endosperm, absorb the sucrose and trans- and the supernatant fraction was dried under a stream of N2. port it via the translocation stream to the growing portions of the of Soluble Nitrogen. The endosperm tissue from five Extraction seedling (Fig. 1). Kriedemann and Beevers (11) demonstrated that seedlings was added to 30 ml boiling 80%1o (v/v) ethanol, chopped if the endosperm is removed, these cotyledons retain the ability to with a razor blade, and further disrupted in a glass-glass homogabsorb sucrose at high rates. The cotyledons accumulate sucrose enizer. The extract was centrifuged and the pellet was further against a concentration gradient and the uptake is sensitive to extracted with 70% then 50%o ethanol and the supernatant metabolic inhibitors (10, 11) showing that an active process is solutions combinedethanol and dried under a stream of N2. The cotyleinvolved. dons from the same seedlings were separated from the hypocotyl This transport of sucrose provides carbon to the embryo but and extracted in ethanol in a similar fashion. In used for there must also be a transfer of nitrogen from the endosperm to amino acid analysis, the extracts were taken up insamples water and lipids the embryo during germination of the castor bean. The protein were removed with chloroform. nitrogen level of the endosperm decreases from the beginning of Amino Acid Soluble amino nitrogen was determined germination with a rapid decrease occurring after day 4 (20). As with ninhydrin Analysis. the method of Lee and Takahashi (12); following a result of hydrolysis ofthe storage protein, soluble amino nitrogen L-glycine was used as a standard. The amino acid composition was determined with a Durrum D500 amino acid analyzer. The 1 Supported by National Science Foundation Grant PCM 78 19575. amounts of glutamine and asparagine were estimated from the 2Present address: Commonwealth Scientific and Industrial Research increase in glutamate and aspartate, respectively, after hydrolysis Organization Division of Horticultural Research, GPO Box 350, Adelaide, of the samples in 6 N HCl for 24 h at 110 C in sealed tubes. SA 5001 Australia. Amino Acid Uptake. Seedlings of similar size and developmen560

AMINO ACID UPTAKE BY RICINUS SEEDLINGS

Plant Physiol. Vol. 68, 1981

tal stage were selected and the endosperm and hypocotyl were removed. The excised cotyledons were weighed, then transferred to ice-cold buffer (5 mm KH2PO4, 0.1 mm CaCl2 [pH 6.0]) for 20 to 30 min. At some developmental stages a thin film of material from the endosperm adhered to the cotyledons (11) and became slimy after soaking in ice-cold buffer. This material was wiped off and the cotyledons were lightly blotted to remove excess buffer and placed in a solution of the same buffer plus amino acid (1 ml buffer per cotyledon pair). After incubation at 25 C for 5 min, 14C-amino acid was added and the cotyledons were incubated in the dark in a shaking water bath at 25 C. Uptake of amino acids was normally determined from the loss of radioactivity in the solution with time. The quantities of material and substrates used in the individual experiments are indicated in the legends of figures and tables. To determine the extent of metabolism of the amino acids, cotyledons were removed from the incubation medium after 1 h and extracted in ethanol as described above. Amino acids were separated by TLC on silica plates using 70% ethanol as the solvent. The amino acids were localized by autoradiography and the radioactivity determined by liquid scintillation techniques. The scintillation cocktail was toluene:Triton X-100 (2:1 v/v) plus 0.4% 2,5-diphenyloxazole and 0.02% p-bis-[2-(5-phenyloxazolyl)]benzene.

Table L. Concentration of Soluble Amino Nitrogen and Individual Amino acids in Endosperm, Cotyledons, and Exudate from 4-Day Castor Bean Seedlings Soluble amino nitrogen was measured in ethanol extracts and concentrations were calculated assuming uniform distribution in each tissue, using the measured water contents of 76% (endosperm) and 58% (cotyledons). The mean ±SE for five experiments is given. The mean rate of exudation was 4.4 ± 0.3 pi/seedling.h. The concentration of each amino acid was calculated from amino acid analysis of the extracts. ND indicates the amino acid concentration was less than 0.5 mm. Concentration

LIPIDS*

SCROSE

109 ± 9

129 ± 6

119 ± 5

1.9 5.7 4.2 6.2 5.2 15.2 3.8 2.3 6.4 ND 10.6 1.9 6.8 6.2

5.7 4.7 3.6 4.5 0.6 47.1 4.3 ND 2.4 ND 8.4 0.8 5.0 4.2 ND 1.8 2.6 0.9 23.2 10.2

ND 4.7 3.2 5.0 1.5 43.6 5.1 0.9 1.3 ND 9.6 0.8 5.4 4.2 ND 2.0 2.8 4.2 15.8 9.2

Exudate

Aspartate Asparagine Threonine

Seine Glutamate Glutamine Glycine Alanine Cysteine

Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Ammonium Arginine

1.3 5.3 2.7 0.7 5.6 17.0

x

~~~~COTYLEDONSR

RCTEINPRTE--AO AciDS

.-.--

Cotyledons

ENDOSPF-ml

..e77777F-..-....*:#

{ _.,

Endosperm

mM

Soluble amino-N

Proline

RESULTS Amino Acid Levels. When the hypocotyl of 4-day seedlings was excised, the cut end attached to the cotyledons continued to exude sap for several hours and this exudate, which we presume arises principally from the phloem, contains material which is in the translocation stream (Fig. 1). The exudate was rich in amino nitrogen, but the concentration of soluble amino nitrogen was also high in the endosperm and cotyledons of 4-day seedlings (Table I). The amino acid composition of the exudate is shown in Figure 2 and it can be seen that glutamine was the major amino acid present, comprising 35 to 40%o of the total amino nitrogen. The levels of the two acidic amino acids, glutamate and aspartate, and of the neutral amino acids glycine, cysteine, methionine, alanine, and tyrosine were notably low. The amino acid composition of the exudate agrees closely with the previous results of Stewart and Beevers (20). From the levels of soluble amino nitrogen and the amino acid composition of extracts, the concentration of individual amino acids in the endosperm, cotyledons, and exudate were calculated and these data are presented in Table I. These calculations are based on the assumption of a uniform distribution

561

.

ENDosPERm

.-*-' Asp Asn Thr Ser Glu Gin Pro GSly Ala Cys Vd Met k Ls

Tr

FM

as Lp NHiA3

FIG. 2. Amino acid composition of the exudate from castor bean

seedlings. The concentration of each amino acid in the exudate is given in Table II. FIG. 1. Diagrammatic representation of the germinating castor bean in longitudinal section showing the cotyledons surrounded by the endosperm. During germination, components of the endosperm are hydrolyzed and the metabolic products are absorbed by the cotyledons and transported to the growing seedlings. Further details of the anatomy of the castor bean seedling are given in ref. 11.

throughout the water space of the tissue and any compartmentation would result in higher values. The concentration of many of the amino acids is similar in each of the three compartments but the cotyledons have lower levels of glutamate, glycine, alanine, tyrosine, phenylalanine, and arginine, but higher glutamine and aspartate than the endosperm. The levels in the exudate correspond closely to those in the cotyledons except for the decreased

562

ROBINSON AND BEEVERS

aspartate and increased lysine concentrations. These differences could arise from different rates of metabolism of the amino acids in the different tissues and/or from preferential transport of some amino acids between the tissues. Uptake of glutamine by cotyledons was further investigated since there is a large concentration gradient for glutamine from endosperm to cotyledon and the high levels of glutamine in the exudate suggest that it is the major form of nitrogen transported in castor bean seedlings. Glutamine Uptake by Cotyledons. The rate of glutamine uptake by cotyledons was not significantly changed when the hypocotyl was removed, as has been demonstrated for sucrose uptake (10), hence all experiments were done with excised cotyledons. Glutamine uptake by excised cotyledons was linear for at least 2 h and could be determined by measuring the decrease in radioactivity in the external solution (Fig. 3). When the cotyledons were incubated in radioactive glutamine for 1 h, rinsed, then extracted in ethanol, 80 to 90%o of the label lost from the external solution was recovered in the cotyledon extract. When the decrease in amino nitrogen in the external solution was measured colorimetrically with ninhydrin, there was good agreement with the rate of uptake determined by the radioactive method. Thus, the uptake by the cotyledons was accurately measured by the loss of radioactivity from the external solution. The colorimetric method allowed determination of the uptake of D-glutamine, which could not be obtained in radioactive form. When the amino acid concentration was 5 mm, the rate of uptake of D-glutamine was less than 6% of the rate of uptake of L-glutamine showing that the transport was specific for the L-isomer. Inasmuch as the hypocotyl had been removed, any exudation of material from the cotyledons would decrease the apparent rate of uptake. To check this possibility, excised cotyledons were incubated for 1 h in radioactive glutamine, quickly rinsed, and transferred to unlabeled glutamine at the same concentration. The efflux of radioactive glutamine in the following hour was very slow (Fig. 4) and was less than 5% of the rate of uptake. This shows that exudation of amino acids does not occur from the cut hypocotyl stump or the cotyledons themselves at significant rates and the measured rates of uptake reflected the true flux of glutamine into the tissue. The low rate of efflux also suggests that counterexchange of amino acids was not significant in the cotyledons. The effect of solution pH on glutamine uptake was determined in citrate-phosphate buffer to allow measurements over a wide range but similar results were obtained with Mes-Tris, MesNaOH, and Tris-HCl buffers. The rate of glutamine uptake was

Plant Physiol. Vol. 68, 1981

I

t10

a

(9lo w

Li

30

0

60

90

120

TIME (MINS)

FIG. 4. Measurements of the uptake and effiux ofglutamine by excised cotyledons. After 1 h in 5 mm [14Clglutamine, the cotyledons were rinsed and transferred to a solution containing unlabeled glutamine (5 mM). Uptake and efflux was determined from the radioactivity in the surrounding solution. Conditions as for Fig. 3.

i. £25 20 z

0

8

'U

0

5 -_

'1aU_ 0

4.0

50

6.0 pH

7.0

8.0

FIG. 5. Uptake of glutamine by excised cotyledons as a function of pH. Uptake was measured after 1 h incubation in 10 mtm KH2PO4-citric acid, 5 mM glutamine, 0.1 mM CaCl2 at the pH values indicated. The results are the mean ±SE of four separate experiments. I-a

~20 5

g10 .5 -j

0

5

30

45

60

75

90

105

120

MINUTES

FIG. 3. Glutamine uptake by excised cotyledons. Cotyledons from 5 seedlings were incubated in 5 ml of 5 mm KH2PO4, 0.1 mm CaCl2, 5 mm glutamine (0.08 Ci/mol) pH 6.0. Triplicate samples (20 Ad) were removed at intervals and uptake was calculated from the decrease in radioactivity.

not greatly changed over the pH range 4 to 8 although the highest rates were obtained at pH 6.0 to 6.5 (Fig. 5). Similar results have been reported for sucrose uptake by this tissue (10) and suggest that the uptake mechanism is insensitive to pH in this range or that the pH at the site of uptake is not altered by changing the pH of the external solution. Komor (10) measured the surface pH at the proximal side of the endosperm and on the cotyledon and found values around 5.6 to 5.7 so a pH of 6.0 was used in these experiments as it most probably reflects the pH in vivo. The rate of glutamine uptake was not significantly altered by low levels (up to 20 mM) of KCI, NaCl, MgCl2, or CaCl2 but higher levels of KCI (100 mM) were inhibitory. Accumulatio and Metabolism of Amino Acids. It was of interest to determine whether the excised cotyledons could accumulate glutamine from the surrounding solution since it is apparent that a concentration gradient occurs in vivo from the endosperm to the cotyledons (Table I). Cotyledons were incubated in 0.5, 5, or 20 mM [14CJglutamine and from the decrease in radioactivity in the

AMINO ACID UPTAKE BY RICINUS SEEDLINGS

Plant Physiol. Vol. 68, 1981

Table II. Recovery of Amino Acidsfrom Excised Cotyledons Cotyledons from five seedlings were incubated in buffer with 5 mm labeled amino acid for I h then extracted in 80% ethanol. Uptake rate (umol/g fresh wt.h) was calculated from the decrease in radioactivity in the buffer solution. The accumulation ratio (concentration in cotyledons/ concentration in external solution) for each amino acid was calculated assuming that the recovered amino acids were uniformly distributed through the water space of the tissue.

14C in exAmino Acid

Rate of Up-

take

tract as %

14C ab-

14C in amino acid

as % 14C in

sorbed

extract

72 100 83 102

56 71 87 96

14C Accu-

Valine

20.1 7.6 19.0 14.9

Table III. Effect of Various Treatments on the Rate of Glutamine Uptake by Excised Cotyledons The cotyledons from five seedlings were preincubated in 5 ml 5 mm KH2PO4, 5 mm glutamine, 0.1 mim CaCl2 (pH 6.0), with the treatments indicated for 15 min before addition of [14CJglutamine. The rate of uptake in the following 60 min during which the cotyledons were gently shaken in the respective solutions was determined. Rate of Glutamine Treatment Utk

Uptake

mulation

Ratio

pmol/g Alanine Arginine Glutamine

563

3.5 2.0 5.7 6.3

solution, the accumulation ratio (concentration in cotyledons/ concentration in external solution) for the radioactive glutamine was calculated. After 1 h, ratios of 10.5, 6.7, and 5.3, respectively, were obtained and after 6 h the values were 470, 190 and 60. The dependence of the accumulation ratio on external concentration was also reported for sucrose (10). These calculations assume that the radioactive glutamine was uniformly distributed in the tissue; any compartmentation (e.g. in the cytosol) would increase the values. The ratios would also be considerably higher if the endogenous glutamine was taken into account, while any metabolism of the labeled glutamine absorbed would decrease the accumulation ratio. The extent of this metabolism was measured by TLC of extracts from cotyledons which had been incubated in radioactive amino acids. After 1 h incubation in [14Clglutamine, 87% radioactivity in the extracts co-chromatographed with authentic glutamine (Table II) indicating relatively slow metabolism by the cotyledons. There was almost complete recovery of [14C]valine, whereas significant amounts of alanine and arginine were metabolized by the cotyledons (Table II). Thus, for glutamine and valine, at least, there was no major conversion during the relatively short incubation periods used and it is clear that these amino acids are actively accumulated against a concentration gradient by the cotyledons. Inhibitors. One currently proposed mechanism for amino acid uptake by plant tissue is an amino acid-proton co-transport across the plasmalemma driven by an ATP-dependent pump extruding protons from the cell (5). If this model is correct, treatments which depress respiration or diminish the proton gradient across the plasmalemma should inhibit amino acid transport. Glutamine uptake was not stimulated by bubbling with air, but a strong inhibition was observed when anaerobic conditions were imposed and the respiratory inhibitor sodium azide strongly inhibited uptake (Table III). Transport was also inhibited by the uncoupling agents DNP3 and FCCP, confirming the involvement of an active process. Vanadate, which is thought to inhibit the plasmalemma proton pump, inhibited uptake, by 34% but fusicoccin, which is thought to stimulate proton efflux, did not stimulate uptake of glutamine (Table III). Aminooxyacetate and methionine sulfoximine both inhibit enzyme reactions involving amino acids but neither was a strong inhibitor of glutamine transport. The strong inhibition by N-ethylmaleimide is consistent with the involvement of a protein in the transport process. Changes in Glutamine Uptake with Cotyledon Development. From the measurements of nitrogen levels during germination made by Stewart and Beevers (20), it is clear that the transfer of

N,N'-Dicyclohexylcarbodiimide, 100iLam

91

Aminooxyacetate, 5 mm Fusicoccin, I pM Methionine sulfoximine, 5 mm Sodium vanadate, I mm DNP, 500 $M Sodium azide, I mM FCCP, 50 AM8 N-Ethylmaleimide, I mM a In 0.5% ethanol.

90 91 89 66 58 45 23 23

20 -

_

16-

711 12

E

8_ 4

I

X

1

6

7

2 3 4 5 6 SEEDLING AGE (DAYS)

7

2

3

4

5

I-

12 41J .4

1.0

I ' 0.4

FIG. 6. Glutamine uptake activity of excised cotyledons at different developmental stages. Uptake was measured during 1-h incubation in 5 mM glutamine. The fresh weight of cotyledons increased from 25 to 160 mg/seedling in the period shown.

nitrogen from endosperm to embryo is maximal from about day 4 to day 7. The capacity of excised cotyledons to transport glutamine at different developmental stages is shown in Figure 6. On a fresh weight basis, the rate of uptake was constant from day 1 to day 3 then declined steadily to day 7. These changes partly reflect the continued increase in fresh weight as the cotyledons during this period. The total uptake activity, expressed per seedling, increased from day 1 to a peak at days 4 and 6 then declined sharply at day 7. This decrease may reflect the actual

grow

3Abbreviations: DNP, 2,4-dinitrophenol; FCCP, p-trifluoromethoxy carbonyl cyanide phenylhydrazone; AIB, a-aminoisobutyric acid.

% control 94 31 96 102

Bubbled with air Bubbled with N2 Ethanol, 0.5% Valinomycin, 10 ILMm

ROBINSON AND BEEVERS

564

loss of transport capacity or the development of an impermeable cuticle by the cotyledons. Komor (10) has argued that the increase in cuticular resistance is small in this period and that for sucrose uptake there is an actual decrease in the transport capacity in the later stages of development. It is clear that, on a per seedling basis, the highest rate of glutamine uptake occurs during the period when nitrogen transfer from endosperm to embryo is maximal (20). Effect of Glutamine Concentration. The rate of glutamine uptake by excised cotyledons as a function of glutamine concentration is shown in Figure 7. The transport displayed saturation kinetics with maximum rates of uptake occurring at concentrations above 20 to 30 mm. Because uptake was measured during a 1-h incubation, the concentration in the external solution was depleted by up to 22% by the end of the incubation period. In analysis of the data this was corrected for by using the mean of the initial and final concentrations. In a number of previous studies, biphasic or even multiphasic kinetics have been observed for amino acid uptake by plant tissues (6, 7, 13, 14, 17, 18). No evidence for multiphasic kinetics was observed for the uptake of glutamine by excised cotyledons over the concentration range studied (0.1 to 50 mm). In six separate experiments, the mean (±SE) values of parameters obtained by linear regression of 1 / v against 1/S (Lineweaver-Burke plot) were Vm = 59 ± 9 ,umol/g fresh weight. h and Km = 10.2 ± 1.7 mm. For the same data, the regression of V against v/ S (Eadie-Hofstee plot) yielded Vm. = 69 ± 7 ,tmol/ g fresh weight-h and Km = 11.8 ± 1.3 mm. Although the Km for glutamine is relatively high, the concentration of glutamine in the endosperm which normally surrounds the cotyledons is about 15 mm (Table I) and would thus be sufficient to support high rates of transport. The Vma. for glutamine transport compares with a Vm. of 113 ,umol/g fresh weight h for sucrose uptake in this tissue (10). Transport of Other Amino Acids. The transport of other amino acids was measured to determine whether the predominance of glutamine in the exudate (Fig. 2) resulted from its selective absorption by the cotyledons. Figure 8 shows the rate of uptake in the concentration range 0.5 to 40 mm for eight amino acids investigated. The four neutral amino acids, glutamine, alanine, tryptophan, and valine were all transported at high rates and the values for Vm. and Km obtained from Eadie-Hofstee plots were similar. Histidine and arginine, both basic amino acids, had lower Vm.x and Km values. The two acidic amino acids, glutamate and aspartate, were transported at lower rates and had a lower Vmax, but the Km values were equal to or greater than those for the neutral amino acids. The similarity in the kinetics of uptake of the four neutral amino acids suggests they might all be transported by the same mechanism, but it is not possible to determine from these

AMINO ACED CONCENTRATION mM FIG. 8. Uptake of various amino acids by excised cotyledons. Uptake was measured during a 1-h incubation. K refers to the Michaelis constant (mM) and V to the maximum velocity (jmol/g fresh wt.h) obtained by linear regression of v against v/s, where v = rate of uptake and S = substrate concentration (Eadie-Hofstee plot).

Table IV. Inhibition of Glutamine Uptake by Other Amino Acids Uptake was measured during 60 min incubation in buffer with 0.5 mm [14Clglutamine plus 10 mm of the added amino acid. The results are the mean of three experiments; standard errors were less than lO10o. Inhibition of 14C Uptake Amino Acid

Cysteine Phenylalanine Alanine Tryptophan Histidine Proline Methionine Asparagine Leucine Serine Glutamine Glycine Threonine Valine Isoleucine Glutamate

NH4Cl U..

550 40

IA

%3o S30

420 10 -

4.

bJ

0

5

1 10 GLUTAMINE

35 25 30 OD CONCENTRATION (m MOLAR)

40

FIG. 7. Concentration dependence of glutamine uptake by excised cotyledons. Uptake was measured during a 1-h incubation.

Plant Physiol. Vol. 68, 1981

75 72 62 56 55 53 51 49 47 46 43 39 37 26 24 13 12

AIB

10

Lysine Arginine D-Glutamine Aspartate

7 7 7 6

data whether the acidic and basic amino acids are transported by the same system. If all of the amino acids were transported by a common carrier, glutamine uptake should be inhibited in the presence of other amino acids. The uptake of glutamine was inhibited by other amino acids but the extent of the inhibition varied from 6% to 75% when each amino acid was present at a concentration 20-fold higher than glutamine (Table IV). The inhibition of glutamine uptake was greatest for those amino acids which were themselves rapidly transported; the acidic and basic amino acids, with the exception of histidine, were all poor inhibitors of glutamine uptake. The low inhibition by D-glutamine is also consistent with the low rate of uptake of the D-isomer.

Plant Physiol. Vol. 68, 1981

AMINO ACID UPTAKE BY RICINUS SEEDLINGS

DISCUSSION The dry castor bean seed contains about 6.2 mg (450 ,uatom) nitrogen (20) most of which must be transferred to the embryo during the first 8 days of growth. This would require transport at an average rate of 2.3 ,uatom N/seedling.h but since most of the nitrogen transfer occurs between days 4 and 7 the maximum rate would actually be about 4 ,uatom N/seedling-h. For seedlings harvested at day 4, the V.. for glutamine uptake by excised cotyledons was 69 ,umol/g fresh weight-h which is equivalent to 3.8 ,umol glutamine/seedling-h or 7.6 uatom N/seedling-h. Thus, the rate of transport measured with excised cotyledons is more than sufficient to account for the known rates of transfer but it should be noted that the in vivo rates are nevertheless quite high. From the amino acid analysis of the exudate (Fig. 2), the average ratio of carbon to amino nitrogen for the amino acids was 4.45; hence, the amino acids contribute 530 ,uatom C/ml exudate. Since the exudate also contains 70 mm sucrose (1 1) or 840 ,uatom C/ml it can be seen that the amino acids in the exudate comprise almost 40%o of the total carbon present. Taken together with the high rates of sucrose uptake previously reported for castor bean cotyledons (10, 11), the present results confirm that these cotyledons are highly effective absorptive organs which play an important role in the early nutrition of the growing seedling. The cotyledons accumulated glutamine against a concentration gradient and the transport was sensitive to inhibitors of respiration and uncoupling agents, showing that an active transport process is involved. This is supported by the fact that transport was highly specific for the L-isomer and showed saturation kinetics implying the presence of a specific carrier. Amino acids absorbed by the cotyledons later appear in the phloem exudate (20) and inasmuch as the cotyledons subsequently green and become assimilatory organs, it is tempting to speculate that the same process of phloem loading is involved at both developmental stages. If this is true, the developing cotyledons may provide a model system for studying phloem loading in leaves, but it cannot be ascertained at present whether the transport measured here directly reflects phloem loading or whether (as seems more likely) amino acids are first transported into the mesophyll tissue and subsequently into the phloem. The different relative concentrations of individual amino acids in endosperm, cotyledons, and exudate (Table I) presumably result both from different rates of metabolism in the endosperm and cotyledons and from selective uptake by the cotyledons. The high level of glutamine in the cotyledons and exudate are a consequence of the relatively high concentrations in the endosperm, high rate of transport into the cotyledon and the low rate of metabolic conversion of glutamine in both tissues (20). Indeed, previous studies have shown that amino acids, which on deamination produce intermediates of gluconeogenesis, are convened to sucrose and glutamine in the endosperm (20). It seems that the endosperm is geared toward the synthesis of glutamine for transport to the embryo. For other amino acids, notably glutamate, arginine, and aspartate, the low levels in the exudate result from metabolism of these amino acids in the endosperm and from their low rate of transport into the cotyledons. Alanine, which is transported into the cotyledons at similar rates to glutamine, is present only at low concentrations in the exudate, presumably because it is rapidly metabolized in both the endosperm and cotyledons. A number of questions about amino acid transport in plants remain unanswered. For example, it is not clear whether transport occurs via a single carrier with relatively broad specificity or if several different carriers exist, each specific for different amino acids or groups of amino acids. Many previous studies (3, 4, 6, 13, 21, 23) suggest a common carrier for two or more amino acids, but an exhaustive study of all amino acids has not been made in any tissue. In contrast, kinetic evidence for separate carriers for different amino acids has been published for roots (17) and for

565

Table V. Amino Acid Uptake Activity of Various Plant Tissues In some cases,the Km is an estimate (E) from the published data. Where more than one value is presented, biphasic or multiphasic kinetics were reported. Tissue Amino Acid Km Ref. Vm. ,umol/g mM fresh wt. h Castor bean cotyledons Glutamine 69 11.8 Barley scutella 22 3.4 Leucine 18 65 15.5 14 1 (E) Maize scutella Glutamine 19 20 1.8 Alanine 4 Pea leaf slices Barley leaf slices Glycine 3 0.4 13 27 6.2 AIB 1 Barley leaf slices 14 0.3 5 10.5 Melon root tips 36 0.02 21 Phenylalanine 7 Mustard roots Glycine 2(E) 23 Barley roots Proline 0.2 0.0016 17 6 0.061 12 0.160 35 1.7 4 Filamentous brown alga Leucine 0.14 15 2 Suspension cultured cells Leucine 1.9 1 0.4 0.017 6 Suspension cultured cells Cysteine 1 0.35 150a 0.02 Suspension cultured cells Threonine 3 36a 0.025 9 Suspension cultured cells Glutamine 0.046 7 Suspension cultured cells Alanine 5a 154a 8 a nmol/106 cells * h.

cells from suspension cultures (2, 8). Genetic evidence for specific carriers comes from the isolation of a mutant carrot cell line deficient in tryptophan transport but with unaltered leucine transport (22). In the present study, glutamine uptake was inhibited by all other amino acids which were themselves rapidly transported into the cotyledons. This is suggestive of a common carrier with a broad specificity but the presence of additional carriers cannot be ruled out. Whatever the mechanism, it is clear that most amino acids were transported into the castor bean cotyledon. With the exception of histidine, the acidic and basic amino acids were transported at lower rates than the neutral amino acids and they were also poor inhibitors of glutamine uptake. These differences may in part be explained by the fact that uptake was measured under conditions which were optimal for glutamine transport and the pH optimum for uptake of acidic amino acids may be lower as found by Lien and Rognes (13). Some published values for Km and Vm.x for amino acid transport in various plant tissues are compiled in Table V and two groups can be distinguished in this list. In the first group, the Km values are high (1-6 mM) and the maximum rates of transport, expresscd on a fresh weight basis, are also generally high. This group includes the absorptive organs (cotyledons and scutella) where transport normally occurs from one tissue or cell type to another. These organs are adapted to transport sugars, amino acids, and peptides at high rates from the relatively high substrate concentrations in intracellular spaces of the tissues that surround them. In leaf slices transport may also involve transfer between different types of cells if phloem loading is involved. Direct measurement of phloem loading and long distance transport of amino acids indicated a Km value of 3 to 50 mm for leucine (16) which is of the same order as for amino acid uptake by leaf slices (Table V). In the second group, the Km values are much lower (mostly below 1 mM) and in most cases the Vm,= values are also lower

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than for the first group. It may be significant that in all examples the second group of tissues normally transport amino acids from a dilute solution external to the plant (or cell). Whether these two groups have different types of amino acid transport systems or whether they differ only in kinetic parameters is not established. The castor bean cotyledons transport sucrose and amino acids at high rates and both systems have now been characterized. There is good evidence that sucrose uptake occurs by a proton cotransport in this tissue (10). It has been suggested that amino acid transport in plants also occurs by a proton co-transport mechanism (5) and we will provide evidence that this is indeed the case for amino acid transport in the castor bean cotyledons in a subsequent paper. LITERATURE CITED 1. BLACKMAN MS, CN McDANIEL 1978 Amino acid transport in suspension cultured plant cells. I. Methods and kinetics of L-leucine uptake. Plant Sci Lett 13: 27-34 2. BERLIN J, U MESTERT 1978 Evidence for distinct amino acid transport systems in cultured tobacco cells. Z Naturforsch 33c: 641-645 3. CHERUEL J, M JULLIEN, Y SURDIN-KERJAN 1979 Amino acid uptake into cultivated mesophyll cells from Asparagus officinalis L. Plant Physiol 63: 621626 4. CHEUNG YNS, PS NOBEL 1973 Amino acid uptake by pea leaf fragments. Specificity, energy sources and mechanism. Plant Physiol 52: 633-637 5. ETHERTON B 1980 Amino acid transport in higher plants. In RM Spanswick, WJ Lucas, J Dainty, eds, Plant Membrane Transport: Current Conceptual Issues. Elsevier/North Holland Biomedical Press. Amsterdam. pp 261-272 6. HARRINGTON HM, IK SMITH 1977 Cysteine transport into cultured tobacco cells. Plant Physiol 60: 807-811 7. KING J 1976 Uptake by soybean root cells of ['4CJalanine over a wide concentration range. Can J Bot 54: 1316-1321 8. KING J, R HIiui 1975 Amino acid transport systems of cultured soybean root

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cells. Can J Bot 53: 2088-2091 9. KING J, V KHANNA 1978 The effect of ammonium ions on uptake of glutamine and other amino compounds by cultured cells of rapeseed. Planta 139: 193197 10. KOMOR E 1977 Sucrose uptake by cotyledons of Ricinus communis L: characteristics, mechanism and regulation. Planta 137: 119-131 11. KREIDEMANN P, H BEEVERS 1967 Sugar uptake and transportation in the castor bean seedling. I. Characteristics of transfer in intact and excised seedlings. Plant Physiol 42: 161-173 12. LEE YP, T TAKAHASHI 1966 An improved colorimetric determination of amino acids with the use of ninhydrin. Anal Biochem 14: 71-77 13. LIEN R, SE RoGNES 1977 Uptake of amino acids by barley leaf slices: kinetics, specificity and energetics. Physiol Plant 41: 175-183 14. REINHOLD L, RA SHTARKSHALL, D GANOT 1970 Transport of amino acids in barley leaf tissue. II. The kinetics of uptake of an unnatural analogue. J Exp Bot 21: 926-932 15. SCHMTZ K, W RIFFARTH 1980 Carrier-mediated uptake of L-leucine by the brown alga Giffordia mitchellae. Z Pflanzenphysiol 96: 311-324 16. SERVAITES JC, LE SCHRADER, DM JuNG 1979 Energy-dependent loading of amino acids and sucrose into the phloem of soybean. Plant Physiol 64: 546550 17. SOLDAL T, P NISSEN 1978 Multiphasic uptake of amino acids by barley roots. Physiol Plant 43: 181-188 18. SOPANEN T, M UUSKALLIO, S NYMAN, J MIKOLA 1980 Characteristics and development of leucine transport activity in the scutellum of germinating barley grain. Plant Physiol 65: 249-253 19. STEWART CR 1971 Some characteristics of the uptake of glutamine by corn scutellum. Plant Physiol 47: 157-161 20. STEWART CR, H BEEVERS 1967 Gluconeogenesis from amino acids in germinating castor bean endosperm and its role in transport to the embryo. Plant Physiol 42: 1587-1595 21. WATSON R, L FOWDEN 1975 The uptake of phenylalanine and tyrosine by seedling root tips. Phytochemistry 14: 1181-1186 22. WIDHOLM JM 1974 Cultured carrot cell mutants: 5-methyltryptophan resistance trait carried from cell to plant and back. Plant Sci Lett 3: 323-330 23. WIUGHT DE 1962 Amino acid uptake by plant roots. Arch Biochem Biophys 97:

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