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Plant Physiol. (1979) 64, 88-93 0032-0889/79/64/0088/06/$00.50/0

Content and Vacuole/Extravacuole Distribution of Neutral Sugars, Free Amino , and Anthocyanin in Protoplasts1 Received for publication December 26, 1978 and in revised form March 15, 1979 GEORGE J. WAGNER Department ofBiology, Brookhaven National Laboratory, Upton, New York 11973

ABSTRACT Hybrid ( from anthesis stage ) and Tulipa cv. Red Shine for amino and other studies, and cv. Most Miles for Neutral sugar, free , and anthocyanin levels and vacuole/ sugar analysis (petals from 1- to 2-day postanthesis flowers and extravacuole distribution were determined for Hippeastum and Tulipa oldest from 5-day postanthesis ). Tulip were and Tudipa protoplasts. Glucose and fructose, the predominant obtained from K. Van Bourgardein and Sons, Inc., Babylon, N.Y., neutral monosaccharides observed, were primarily vacuolar in location. and Hippeastrum from Jackson & Perkins Co. Tulip bulbs were Glutamine, the predominant free amino acid found, was primarily extra- rooted at 9 C then held at 0.6 C as long as 10 months before vacuolar. y-Methyleneglutamate was identified as a major constituent of induction of vegetative and floral growth at 22 C. In this way Tudpa protoplasts. Qualitative characterization of Hippeastrum petal and flowers and leaves were made available for 10 months of the year. vacuole organic acids indicated the presence of oxalic, malic, citric, and Hippeastrum flowers were made available year round. For this isocitric acids. Data are presented which indicate that vacuoles obtained , postflowering vegetative growth (3 months) was terminated by gentle osmotic shock of protoplasts in dibasic phosphate have good by drying the bulbs followed by vernalization for a minimum of purity and retain their contents. 6 weeks at 1.1 C. In this way lots of bulbs were made to on schedule twice a year. Protoplasts and vacuoles were prepared and washed as previ- ously described (4, 26) except that of tissues was for 10 to 12 h at 18 C unless otherwise specified and protoplasts were washed twice with 40 ml of0.7 M mannitol, 10 to 25 mm Na-citrate Loalization of compounds in intracellular compartments is (pH 5.5). Cellulysin (Calbiochem) was purified by centrifugation crucial for understanding biochemical processing. In mature plant of a 20 g/70 ml solution at 5,000g for 5 min followed by passage cells the two major fluid compartments-with respect to volume- of the supernatant through a Sephadex G-25 coarse column (75 are the vacuolar ( sap) and the . Other fluid com- x 4.5 cm) equilibrated with and eluted with 1 mm NaCl. partments include the saps of such as and Sugar Analysis. Approximately 104 protoplasts were counted in mitochondria etc. and perhaps specialized regions of membrane a 0.2-mm-deep well-type slide and homogenized in 5 ml of 0.3 M systems such as the ER. mannitol, 10 mm Na-citrate (pH 5.5) using a glass tissue homog- Histochemical and kinetic (pulse-chase-type) analyses have enizer. An equal number of vacuoles were lysed by dilution with been the principal means for studying the distribution of metab- 1 ml of H20. Samples were passed through columns (0.9 x 20 cm) olites between the vacuolar sap and cytosol. Direct analysis of containing Dowex 50-H', X8, 200 to 400 mesh, and Dowex 1- these compartments is now possible following the isolation from formate, X8, 200 to 400 mesh and the neutral eluates lyophilized. protoplasts ofintact vacuoles and a fraction enriched in cytosol or Total neutral carbohydrate was determined according to the by quantitatively comparing the contents of vacuoles and entire method of Dubois et al. (5) with D-glucose as standard, glucose by protoplasts (3, 4, 9, 12, 15, 21, 27). the glucostat test (Worthington Biochemical Co.), and fructose by This work describes the qualitative and quantitative character- the method of Roe (20). Sucrose was assayed as fructose after ization and vacuole/extravacuole distribution of neutral sugars separation by paper chromatography in solvent 1 (see below). and free amino acids in pigmented Hippeastrum and Tulipa petal Analytical high pressure liquid chromatographic analysis of re- and Tulipa leaf protoplasts. Quantitative determination and vac- ducing sugars was performed according to D. T. Liu (in prepara- uole/extravacuole distribution of anthocyanin in petal protoplasts tion). Three solvent systems were employed for sugar analysis by and preliminary qualitative characterization oforganic acids from descending paper chromatography on Whatman No. 1: 1) ethyl petal tissue and vacuoles of Hippeastrum are also reported. Data acetate-pyridine-acetic acid-water-methanol (7:5:1:1.5:1); 2) ben- are provided in support of the contention that vacuoles isolated zene-l-butanol-pyridine-water (1:5:5:3); 3) methylethyl ketone- 1- by gentle osmotic shock ofprotoplasts in dibasic phosphate buffer butanol-acetic acid-water (3:2:2:2). Sugars were detected by spray- are relatively free from contamination and retain their contents ing with alkaline silver nitrate. and are thus suitable for direct studies. A Preliminary qualitative characterization of organic acids in preliminary report describing parts of this work has appeared Hippeastrum petal extracts and vacuoles was by paper chromatog- elsewhere (25). raphy in solvent 1 and a mixture of ethanol-NH40H-H20 (4:1:1). Soluble constituents of aqueous petal homogenates and vacuole MATERIALS AND METHODS preparations were passed through Dowex 50 and Dowex 1 as above and eluted from the latter with 4 N formic acid. Acids were Plants were grown under greenhouse conditions at approxi- analyzed by paper chromatography after evaporation of formic mately 22 C. Tissues used included Hippeastrum cv. Dutch Red acid and were detected by spraying with 0.05% bromocresol green in ethanol. 'This research was carried out at Brookhaven National Laboratory Amino Acid Analysis. Determinations were made using an under the auspices of the United States Department of Energy. automated amino acid analyzer. Asn, Gln, Thr, Ser, Glu, Asp, 88 Plant Physiol. Vol. 64, 1979 INTRACELLULAR DISTRIBUTION IN PROTOPLASTS 89 and y-methyleneglutamate were separated at 42 C on a column were recovered from the top 1 ml of the 15% zone, enriched (0.9 x 50 cm) of Aminex A6 resin- 12 ,um (Bio-Rad Laboratories). cytosol was retained in the 17% zone, and residual chloroplasts Sample buffer was 0.2 N Na, 0.3 N citrate-HCl (pH 2.12) contain- were sedimented. Addition of D-[U-'4CJglucose or '°CdCl2 to the ing 15% polyethylene glycol 400. Samples were eluted with 0.3 N 17% zone prior to centrifugation resulted in a purified vacuole Li, 0.3 N citrate-HCl (pH 2.8). Other amino acids were separated fraction containing 0.075% of the added label, indicating that at 54 C on a column (9 x 22 cm) of Durrum 6A resin-9 ,um cytosol was removed from vacuoles during flotation. Underlayer- (Durrum Chem. Co.). Sample buffer was as above. A three-step ing the 17% zone with zones of 40 and 50%1o sucrose (w/w), 10 mm elution was employed using 0.2 N Na, 0.3 N citrate-HCl at pH Hepes-NaOH (pH 8.0), 10 miM MgCl2, 1 mim DTT resulted in the 3.25, 4.25, and 4.27, successively. Tryptophan determination was simultaneous recovery of class I chloroplasts at the 40/50%o inter- not made. Extracts of protoplasts and vacuoles were prepared as face when vacuoles were released in 0.2 M Na4P207-HCI (pH 8.0) described for sugar analysis, centrifuged at 100,000g for 30 min at (see ref. 26). 4 C, and aliquots ofthe supernatant diluted with the sample buffer described above. RESULTS Contamination Assessment. Osmotic shock of protoplasts to prepare intact vacuoles results in three protoplast fractions: intact The content and vacuole/extravacuole distribution of neutral vacuoles, particulate , and "enriched" cytosol. The par- sugars in pigmented Hippeastrum and pigmented Tulipa petal and ticulate cytoplasm fraction consists of aggregated, filterable ma- Tulipa leaf protoplasts were investigated by quantitative compar- terials produced during vacuole emergence. The enriched cytosol ison of protoplast and isolated vacuolar contents. Enzymic (Glu- fraction contains soluble materials of protoplasts which remain in costat), colorimetric assay liquid and paper chromatographic anal- the phosphate buffer (used for osmotic shock) after removal of yses indicated that glucose and fructose were the primary constit- the particulate cytoplasm fraction and sedimentation of vacuoles uents and were principally localized in the vacuole (Table I). (for details see refs. 4 and 26). The three protoplast fractions as Millimolar concentrations of vacuolar glucose were 29, 159, and well as washed vacuoles were assayed for evidence of , 150 for Hippeastrum, Tulipa petal, and Tulipa leaf protoplasts, mitochondrial, microsomal, and nuclear content. Chl (chloroplast) respectively (Table II). It should be noted that protoplasts and was determined by the method of Arnon (1), Cyt c oxidase isolated vacuoles are derived from leaves previously plasmolyzed (mitochondria), and Cyt c reductase (microsomal) as described by and thus the cellular concentrations in vivo (if protoplast and in Hodges (8), the latter in the presence and absence of 12 ,iM vivo levels are the same) are lower than those computed (estimated rotenone and 1 UM Antimycin A. RNA and DNA were measured factor -0.7). Calculations were made using volumes determined using the procedure of Schmidt and Thannhauser (22). Anthocy- from average values for diameters given in the legend of Table II. anin was determined in 0.3% HCI in methanol at 25 C using the Vacuolar diameters were estimated for petal protoplasts using extinction coefficient anthocyanin as a vacuolar marker and for leaf protoplasts after equilibration with neutral red. Calculations indicated that vacu- ew = 33,000 [cm2/moll. oles occupy approximately 70 to 80%o ofprotoplast volumes (Table Osmolarity measurements were made using a model 31245 Ad- II). Sucrose was detected in all three tissues and except for Tulipa vanced Osmometer (Advanced Instruments Inc.). leaf protoplasts, it is approximately 40% vacuolar. In protoplasts Leaf vacuoles were separated from contaminating chloroplasts glucose and fructose combined accounted for 63 to 74% of the using a modification of the procedure previously described for total reducing neutral sugar and reducing sugar plus sucrose simultaneous recovery of vacuoles and chloroplasts (ref. 26, see accounted for 89 to 91% of the total neutral sugar (Table I). Data footnote 7). After release of vacuoles, the suspension in dibasic represent the mean values of three experiments, and variation was phosphate buffer was centrifuged at 5OOg for 3 min and the on the order of 10 to 15%. Only the small amounts of xylose and supernatant made 17% (w/v) with sucrose. Twenty ml of this galactose were observed by means of paper chromatography. mixture was overlayered with 10 ml of 15% (w/v) sucrose made Several major unknowns which were observed on paper chro- 20 mm with Hepes-NaOH (pH 8.0), and was centrifuged, with matograms of Tulipa petal fractions were not characterized. The slow acceleration, at 5,000g for 10 min at 20 C. Purified vacuoles content and vacuole/extravacuole distribution of free amino acids

Table I. Neutral sugar content and vacuole/extravacuole distribution in protoplasts.

Hippeastrum petal Tulipa petal Tulipa leaf Sugar protoplast vacuole % in vacuole protoplast vacuole % in vacuole protoplast vacuole % in vacuole

ng/unit percent ng/unit percent ng/unit percent glucose 2.42 1.96 81 3.65 4.0 110 5.0 5.96 120 fructose 2.04 1.73 85 0.83 0.4 49 0.98 0.99 101 sucrose 0.45 0.20 44 0.47 0.20 42 1.37 1.31 95 total reducing sugar 6.07 4.68 77 7.11 6.44 91 8.03 9.27 115 total neutral sugar 7.65 7.38 96 9.02 8.21 91 11.8 13.6 116

percent percent percent percent percent percent glucose plus fructose 73 79 -- 63 68 --- 74 75 --- as a percent of reducing sugar reducing sugar plus 91 69 -- 89 83 --- 91 87 --- sucrose (x2) as percent of total neutral sugar 90 WAGNER Plant Physiol. Vol. 64, 1979 in Hippeastrum petal, Tulipa petal and leaf protoplasts are given were observed for Hippeastrum protoplasts and vacuoles obtained in Table III. Free amino acids were substantially lower in concen- after digestion with 4% Cellulysin, 0.6 M mannitol at 34 C for 4 h. tration than neutral sugars (see Table I for comparison). Qualita- To verify the presence of glutamine and asparagine, samples of tively and quantitatively similar results to those given in Table II Hippeastrum and Tulipa petal protoplast and vacuole lysates were

Table II.

D-glucose content and vacuole/extravacuole distribution in protoplasts.

Volume determinations are based upon estimated average diameters of protoplasts and vacuoles within protoplasts of 100 An and 80 pm (Hippeastrum), 70 Ilm and 65 Am (Tulipa petal), and 80 ,m and 75 pm (Tulipa leaf). Extravacuole values were calculated.

Tissue & Compartment % of proto mM Fraction Content Volume vol. Conc.

pmoles/ x 10-3 Il % mMolarity unit Hippeastrum petal protoplast 13.4 0.523 vacuole 10.9 0.38 73 29 extravacuole 2.5 0.143 27 17.5 Tulipa petal protoplast 20.3 0.18 vacuole 22.2 0.144 80 154 extravacuole 0 0.036 20 0 Tulipa leaf protoplast 27.8 .268 vacuole 33.1 .221 82 150 extravacuole 0 0.047 18 0

Table III.

Free amino acid content and vacuole/extravacuole distribution in Hippeastrum petal, Tulipa petal, and leaf protoplasts.

Data were derived from 4 separate preparations of Hippeastrum petals (anthesis stage), 5 preparations of Tulipa petals (1 to 2 days post-anthesis stage), and two preparations of Tulipa leaves (oldest leaves from 5-day post-anthesis plants. Preparations contained 5 to 7 x 106 protoplasts and 2 to 4 x 106 vacuoles. The approximate percent of amino acids in the vacuole was computer from the means. Unknown IV is identified as ymethyleneglutamate - see results.

Amino Hippeastrum petal Tulipa leaf Tulipa leaf Acid Protoplast Vacuole % in Protoplast Vacuole % in Protoplast Vacuole % in vacuole vacuole vacuole

1015moles/unit % 10-15moles/unit % 10-15moles/unit %

UNKiU 20 + 13 8 + 3 40 123 + 40 10 + 4 8 0 0 - Asp 23 + 30 11 + 11 48 Trace Trace - 55/39 49/27 80 Glu 75 + 6 10 + 6 13 142 + 40 16 + 9 11 10/81 42/31 48 Pro 51 + 35 12 + 17 24 Trace Trace - 135/114 95/82 71 Gly 24 + 20 8 + 6 33 134 + 30 24 + 15 18 161/137 150/121 91 Ala 133 + 47 15 + 10 11 400 + 50 40 + 18 10 450/473 350/317 72 1/2 Cis Trace Trace - 42 + 10 Trace - 15/19 16/12 82 Val 59 + 60 13 + 25 31 145 + 50 32 + 25 22 135/127 78/32 42 Met 5 + 5 1 + 2 20 Trace Trace - 32/29 29/17 75 Ileu 17 + 9 8 + 7 47 73 + 20 40 + 30 55 60/51 42/31 66 Leu 35 + 20 11 + 6 31 122 + 43 27 + 16 22 152/112 101/131 90 Tyr 18 + 6 3 + 4 17 43 + 16 19 + 13 44 65/57 60/59 98 Phe 12 + 12 6 + 4 50 47 + 23 19 + 9 40 97/106 72/27 45 y amino butyric 96 + 70 5 + 3 5 60 + 15 7 + 5 12 187/162 23/31 16 Lys 50 + 6 25 + 1 50 130 + 46 19 + 16 15 124/115 40/32 30 UNK II 62 + 13 16 + 20 26 90 + 60 10 + 9 11 0/0 0/0 0 His 23 + 10 19 + 12 83 100 + 70 32 + 24 32 52/71 39/27 54 Arg 109 + 34 54 + 33 50 110 + 17 19 + 42 17 180/161 146/112 76 Thr 68 + 41 27 + 11 40 270 + 60 32 + 24 13 191/173 95/81 48 Ser 225 + 70 50 + 17 22 530 + 140 60 + 40 11 283/287 250/263 90 Asn 105 + 40 50 + 4 48 220 + 70 26 + 17 12 221/217 172/117 66 Gln 800 + 70 166 + 6 21 1140 + 260 117 + 54 10 1801/1670 146/127 7 UNK IV 0 0 - 1260 + 920 275 + 126 22 263/241 225/217 80 UNK III 0 0 - 123 + 40 10 + 4 1 Trace Trace - Plant Physiol. Vol. 64, 1979 INTRACELLULAR DISTRIBUTION IN PROTOPLASTS 91 Table IV.

Estimations of chloroplast, mitochondria, and distribution in Tulipa leaf and petal protoplast fractions.

Fractions were prepared from 5 to 10 x 106 protoplasts per experiment. Percent values are derived from the sum of the means of that recovered from the particulate cytoplasm, "enriched" cytosol and unwashed vacuole fractions combined. Petal oxidase was assayed in the presence of digitonin (8) and NADH cytochrome c reductase in the presence of Antimycin A and rotenone - see methods.

Chlorophyll Cytochrome c Oxidase NADH Cytochrome c Reductase

Fraction Leaf Leaf Petal Leaf Petal

mg/106 protoplasts pezrcent pmoles/min percent tmoles/min percent ILmoles/min percent pnoles/min percent Particulate 2.4 + .35 82 3.4 + 1.2 72 1.7 + .7 88 2.7 + .9 88 1.1 + .9 81 Cytoplasm "Enriched" Cytosol .34 + .4 12 1.3 + .5 28 .23 + .2 12 .37 + .2 1 2 .25 + .05 18

Unwashed .17 + .15 6 .002 + .001 0.04 0 0 .002 + .001 0.07 .008 + .003 .6 Vacuole

Washed .13 + .09 4.5 0.007 + 0.006 .15 0 0 0 0 0 0 Vacuole

Purified .012 + .01 0.4 Vacuole __ Number of 4 3 __ 3 Experiments ultrafiltered using an Amicon UM-5 membrane (500 mol wt cut- off) to remove protein and large peptides and the fitrates hydro- lyzed in 3 N HCI for 22 h at 100 C. Analysis of hydrolysates revealed a stoichiometric conversion of glutamine to glutamate and an 80o conversion of asparagine to aspartic acid. The com- pound observed in Tulipa fractions labeled UNK IV (Table III) has been identified as y-methyleneglutamate by co-chromatogra- phy with known substance. Unknown IV and authentic y-meth- yleneglutamate were equally insensitive to hydrolysis. Preliminary qualitative characterization of organic acids of Hippeastrum petal extracts and vacuoles by paper chromatography indicated the presence of oxalic, citric, malic, and isocitric acids in both vacuoles and petal extracts. Various markers were employed to assess the extent of contam- ination of unwashed and washed vacuoles with other cell organ- FIG. 1. Phase contrast light micrographs ofwashed Tulipa leafvacuoles elles. Particulate materials and enriched cytosol fractions re- (not further purified) 3A, and those purified by flotation 3B (see under covered during vacuole isolation were also assayed (Table IV). "Materials and Methods"). S: subprotoplasts; C: chloroplasts; D: debris Similar results were obtained for pigmented Hippeastrum petal (x 230). protoplasts (not shown). Approximately 70 to 90%o of the mito- chondrial (Cyt oxidase) and microsomal (NADH Cyt c reductase- The retention ofintegrity ofvacuoles after isolation was assessed rotenone and Antimycin A-insensitive) material was associated by comparing the pigment content of isolated vacuoles with with the particulate material in all tissues. Less than 1% of these protoplasts. In six experiments using Tulipa petal protoplasts 111 activities were associated with unwashed vacuoles. Hippeastrum to 82% of the anthocyanin per 106 protoplasts was recovered in and Tulipa preparations retained approximately 3% of the total 106 vacuoles. With Hippeastrum petal protoplasts 108 to 92% nucleic acids in unwashed vacuole preparations and 1% in washed recovery was observed in four experiments using approximately vacuole preparations. RNA was distributed as follows: 27% par- 106 protoplasts and vacuoles. The anthocyanin concentration of ticulate cytoplasm, 700%o enriched cytosol, and 3% unwashed vac- vacuoles of both tissues was calculated to be about 7.5 mM. uole; and DNA: 70o particulate cytoplasm, 27% enriched cytosol, and 3% unwashed vacuole. Approximately 300 pmol of RNA and DISCUSSION 70 pmol of DNA was found per protoplast for both tissues. Tulipa leaf cells contain a substantial number of relatively small The vacuolar saps of and terrestrial plants have a highly chloroplasts (5-,um length) about 5% of which sediment with variable composition (11, 24). Substances commonly found in unwashed vacuoles (Table IV). Purification of vacuoles reduces vacuoles of land plants include mineral salts, organic acids, amino this to less than 0.5%. Light micrographs (phase contrast) of acids, proteins, peptides, sugars, nucleic acids, alkaloids, , unwashed and purified Tulipa leaf vacuoles are shown in Figure and glycosides. Some of these have widespread distribution while 1. The difference in appearance of vacuoles is due to the presence others show high taxonomic specificity (24). The chief previous. of sucrose in purified vacuole suspensions. methods for determining, qualitatively and quantitatively, vacu- 92 WAGNER Plant Physiol. Vol. 64, 1979 olar and cytosol contents were histochemical and kinetic analyses. Variation in data is thought to be primarily due to physiological The difficulties and shortcomings of these methods have been factors not entirely under control, inaccuracies in counting, and discussed (10, 16). A third and direct approach involving the difficulties in selecting tissues in the same stage of development. examination of protoplasts and vacuoles has been used here to Protoplasts prepared from petals of late tulip buds showed gen- study the vacuolar and extravacuolar content of neutral sugars, erally lower levels of free amino acids than more mature tissues free amino acids, and anthocyanin in enzymically isolated proto- and also contained high levels of three unknowns which elute just plasts. Possible shortcomings ofthis approach are discussed below. prior to threonine. An elevated level of proline presumed to be Glucose was observed to be the predominant neutral monosac- associated with osmotic stress induced during protoplast formation charide of vacuoles and protoplasts of Hippeastrum petals and (18) was perhaps observed in Tulipa leaf protoplasts but was not Tulipa petals and leaves (Table I). Hippeastrum petal protoplasts evident in Tulipa petal protoplasts. have an equivalent amount of fructose, while tulip tissues contain To assess the level of contamination of vacuoles with other less of this sugar. In all tissues most of the glucose and fructose organelles, unwashed vacuoles obtained by sedimentation from were contained in the vacuole (Table I), and calculated concen- phosphate buffer were analyzed for several markers (Table IV). trations for vacuolar glucose were approximately 30 mm for Hip- The levels of Cyt oxidase (mitochondria), and rotenone and peastrum petal and approximately 150 mm for Tulipa tissues (Table Antimycin A-insensitive NADH Cyt c reductase (primarily asso- II). There is evidence to suggest that in sink tissues the vacuole ciated with ER but also with outer mitochondrial membrane and serves as a reservoir of hexose derived from sucrose hydrolysis (7). possible nuclear membrane) were found to be below 1% in un- Sucrose was present in lesser amounts except in tulip leaf proto- washed vacuoles. These activities were generally undetected in plasts and was observed to be about equally distributed between washed vacuole preparations. NADH Cyt c reductase has not the vacuole and extravacuolar space in petals and to be primarily been found to be associated with crude tonoplast or gradient vacuolar in Tulipa leave protoplasts. Outlaw et al. (17) have purified tonoplast prepared from Hippeastrum petals (in prepara- obtained data that are consistent with a vacuolar sucrose pool in tion). NADPH Cyt c reductase was rarely detected in tissue Viciafaba leaves. The vacuole is reported to be the site of stored extracts, protoplasts, or protoplast fractions of the tissues studied. sucrose in castor bean (15). Glucose, fructose, and When low activity was observed it was associated with the partic- sucrose were the most abundant neutral sugars in protoplasts and ulate fraction. In Hippeastrum, DNA and RNA levels in unwashed also tissue extracts of the tissues studied. and washed vacuoles were 3% and 1% of those obtained in Glutamine (800 ± 70 fm/Hippeastrum protoplast) was the most protoplast lysates. Pigmented petal cells are relatively free of abundant free amino acid in Hippeastrum petal and Tulipa petal chloroplasts and thus chloroplasts pose no contamination prob- and leaf protoplasts (Table II). It was primarily extravacuolar in lem. However, petal vacuole preparations do contain small all tissues, consistent with its formation in the cytosol (14). Cal- amounts of (Hippeastrum) and materials (Tu- culated glutamine concentrations in Hippeastrum cytosol and vac- lipa). With Tulipa leaves, a large number of small chloroplasts uole are 4.4 and 0.44 mm, respectively (data computed using results in contamination ofcrude vacuole pellets with chloroplasts. volume factors given in legend of Table II). Serine and, in Tulipa This level is reduced to 0.5% or less by floating vacuoles free from tissues y-methyleneglutamate (28) were the next most abundant chloroplasts and cytosol (see under "Materials and Methods"). A free amino acids. They both showed a distribution in petals similar approach has proved useful for recovering pea, lettuce, different from that in leaves. Arginine, threonine, asparagine, and tobacco, barley, and corn leaf vacuoles (Wagner, unpublished). alanine were also present in relatively large amounts. Wiemken The lack ofcytosol contamination ofwashed vacuoles is evidenced and Dunf (23) have reported the presence ofprimarily basic amino by the absence ofthree associated with flavonoid biosyn- acids in vacuoles of . y-Aminobutyric thesis which are readily demonstrated in enriched cytosol from acid, a common constituent in higher plants, was primarily extra- the tissues studied (9). vacuolar in all three tissues. Vacuoles isolated from Tulipa and Hippeastrum petals are shown It must be emphasized that the term extravacuolar, as used here, to contain all or essentially all of their anthocyanin pigment (this refers to the soluble contents of lysed protoplasts less the soluble work) and to have the same content as the protoplasts from contents ofvacuoles. Undoubtedly, organelles such as chloroplasts which they are derived (12) despite their exposure to large volumes and mitochondria are broken dunrng protoplast rupture and mix of solution lacking these substances during isolation and washing. with cytosol. Such organelles may be enriched in certain compo- In addition they are observed to retain amino acids, organic acids, nents while the cytosol is deficient in that component, and vice and large amounts of neutral sugars. The concentration of antho- versa. Thus, the extravacuole values may represent cytosol and or cyanin determined for Tulipa petal vacuoles (about 7.5 mM) is organellar saps. comparable to that estimated from petals of Ipomoea tricolor (2) Protoplasts isolated by digestion of Hippeastrum petals with 4% and rose (S. Asen, personal communication). These results provide Cellulysin, 0.55 M mannitol at 34 C for 4 h and those isolated by assurance that vacuoles retain their contents during isolation as digestion for 12 to 20 h at room temperature had similar amino described. acid contents. Epidermal peels (consisting primarily of pigmented The only apparent difference between isolated vacuoles and cells) of Tulipa petals prepared and washed with water to disrupt vacuoles in vivo is the increase in internal pH which occurs on pigmented cells (anthocyanin leached). Analysis of the wash for isolation in phosphate buffer (12). A similar increase has been free amino acid gave similar results to those obtained for pig- observed in petal vacuoles isolated in a solution consisting of 3.5% mented protoplasts of this tissue. A relatively constant ratio of Ficoll, 25 mM Hepes-NaOH (pH 8.0). This increase is not due to protoplast to peel extract amino acid was obtained for all amino the uptake of phosphate as evidenced by a lack of 32p04 uptake acids except that peel extracts contained more glutamine, gluta- during vacuole release in K2HPO4 containing H332P04. It is highly mate, and y-methyleneglutamate and less threonine and leucine likely that the rise in intemal pH is due to an electroneutral K+/ than did protoplast extracts. The incorporation of the protease H+ exchange such as that observed in studies of isolated inhibitors a-toluene-sulfonyl fluoride (250 i,g/ml phenylmethyl- (19). A Donnan phenomenon may mediate this pH sulfonyl fluoride) and 1 mm N-ethylmaleimide into all solutions change (organic acid as fixed internal anion) and also be respon- used for protoplast and vacuole preparation did not substantially sible for the stability of vacuoles in 0.2 M K2HPO4 (475 millios- alter free amino acid levels. Also, changes were not observed after molar) and weakly buffered 0.175 M NaCl (325 milliosmolar). storage ofsamples for several months at -20 C. These data suggest Vacuoles burst in buffered 400 milliosmolar mannitol. In plant that results obtained are not grossly different from tissue levels of tissues, K+/H+ exchange followed by vacuolar accumulation of free amino acid and that no release of amino acid due to proteo- K+ and organic anion synthesis is well known (13). lytic degradation occurs after protoplast and vacuole preparation. Protoplasts and vacuoles can be used for determining vacuolar Plant Physiol. Vol. 64, 1979 INTRACELLULAR DISTRIBUTION IN PROTOPLASTS 93 and cytosol contents directly but several difficulties with this phosphatase from plant . Methods Enzymol 32: 392-406 are 9. HRAZDINA G, GJ WAGNER, HW SIEGELMAN 1978 Subcellular localization of enzymes of approach becoming apparent. These include difficulties in anthocyanin biosynthesis in protoplasts. 17: 53-56 quantitation where fragile vacuoles of varying size and number 10. JENSEN WA 1962 Botanical Histochemistry, WH Freeman & Co, San Francisco are produced from protoplasts and possible alteration of in situ 11. KxAa PJ 1955 Physical chemistry of the vacuoles. In W Ruhland, ed, Encyclopedia of Plant cellular states as a result of mechanical disruption, Physiology, Vol 1. Springer-Verlag, Berlin, pp 649-660 12. LIN W, GJ WAGNER, HW SIEG?u&AN, G HIND 1977 Membrane-bound ATPase of intact and digestion during protoplast preparation (6, 18). The vacuoles and tonoplasts isolated from mature plant tissue. Biochim Biophys Acta 465: 110- possibility that protoplasts may take up substances from the 117 isolation medium has not been adequately studied. 13. MAcROBBIE EAC 1971 Fluxes and compartments in plant cells. Annu Rev Plant Physiol 22: The tissues studied provide useful material for examining the 75-96 distribution ofmetabolites which are contained in both the cytosol 14. MIFLIN BJ, PJ LEA 1977 Amino acid metabolism. Annu Rev Plant Physiol 28: 299-329 15. NISHIMURA M, H BEEvERs 1978 Hydrolases in vacuoles from castor bean endosperm. Plant and vacuole because vacuoles which have good purity and retain Physiol 62: 4448 contents can be rapidly prepared (10 min) from protoplasts in 16. OAIs A, RGS BIDWsLL 1970 Compartmentation of intermediary metabolites. Annu Rev Plant high yields (70%o Tulipa petals, 25% Tulipa leaves, and 15-20%o Physiol 21: 43-66 Hippeastrum petals). 17. OuTLAw WH JR, DB FISHER, AL CHRISTY 1975 Compartmentation in Viciafaba leaves. Plant Physiol 55: 7047 11 Acknowledgments-The author wishes to thank Drs. B. J. Miflin and P. J. Lea for their generous 18. PREmEcz G, T OLAH, A GULYAS, A NYITRAI, G PALFI, G FARRAS 1977 Is the increase in gift of standard amino acid derivatives, Dr. D. Liu for high pressure liquid chromatographic ribonuckase level in isolated tobacco protoplasts due to osmotic stress? Plant Sci Lett 9: analysis of sugars, and N. Alonzo for assistance with amino acid analysis. The recommendations 195-200 of Dr. A. A. De Hertogh on the culture of tulips are much appreciated. 19. REUNGOUD DJ, JM TAGER 1977 The permeability properties of the lysosomal membrane. Biochim Biophys Acta 472: 419-449 LITERATURE CMD 20. ROE JH 1934 A colorimetric method for the determination of fructose in blood and urine. J Biol Chem 107: 15-22 1. ARNON, DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in . 21. SAUNDERS JA, EE CONN 1978 Presence ofthe cyanogenic glucoside dhurrin in isolated vacuoles Plant Physiol 24: 1-15 from Sorghum. Plant Physiol 61: 154-157 2. 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