Plant Physiol. (1984) 76, 680-6840032-0889/84/76/0680/05/$0 1.00/0

Extensor and Flexor Protoplasts from Samanea Pulvini'I. ISOLATION AND INITIAL CHARACTERIZATION

Received for publication March 15, 1984 and in revised form June 1, 1984

HOLLY L. GORTON* AND RUTH L. SATTERBiological Sciences Group U-42, University ofConnecticut, Storrs, Connecticut 06268

ABSTRACT

Protoplasts were isolated from extensor and flexor regions of openpulvini of the nycftnastic tree Samanea saman. Both types of protoplastsundergo many changes during isolation. Extensor protoplasts are univa-cuolate in vivo, but some become multivacuolate. All flexor protoplastsare univacoolate. In an open pulvinus, extensor cells have a higher osmoticpressure than flexor cells. However, both types of protoplasts can beisolated with optimal yield using the same osmoticum (0.5 molar sorbitol)in the digestion medium. This suggests that some leakage of osmoticumoccurs during harvest or digestion, especially from extensor tissue. De-spite these changes, both types of protoplasts extrude protons in responseto 10 micromolar fusicoccin (1.6-1.8 nanoequivalent/lO' protoplasts/minute), demonstrating that the protoplasts are metabolically active andthat proton transport mechanisms must be at least partially functional.The changes in vacuolar structue and osmotic pressure are what onemight expect if the protoplasts, which are isolated from open pulvini,take on characteristics of cells in a closed pulvinus.

Nyctinastic plants like Samanea saman2 usually spread theirpaired leaflets horizontally during the daytime and fold theminto a vertical orientation at night. The movements continueunder constant (free-running) conditions, since they are con-trolled by an internal circadian clock as well as by light anddarkness. Leaflet movement is accomplished by changes in thecurvature of a cylindrical motor organ, or pulvinus, at the baseof each leaflet. In Samanea, the terminal secondary pulvini (seeFigs. 1 and 2 in Satter et al. [21]) have been studied mostthoroughly because they are large enough for dissection (about5-7 mm long and 2-3 mm in diameter) and yet less woody thenthe larger primary pulvini. Cortical cells on opposing sides ofthepulvinus swell and shrink to generate the changes in the relativelengths of the two sides that are required for bending. Extensorcells swell as the pulvinus opens in the daytime, and the oppo-sitely positioned flexor cells swell as the pulvinus closes at night.The turgor pressure changes responsible for the swelling andshrinking are caused by uptake and release of large amounts ofsolutes, chiefly K and Cl (Samanea [21]; Albizzia julibrissin[22]; Phaseolus [11, 14]). These ion fluxes may be driven by anoutwardly directed H+ pump (10).Whole pulvini or strips or sections ofpulvinar tissue have been

used for all experiments to date. However, the presence of thecell wall often complicates interpretation of the data. For in-

' Supported by grants from the National Science Foundation toR. L. S.2Samanea saman has been reassigned to the genus Pithecolobium.

We retain the name Samanea for continuity with earlier literature.

stance, x-ray microanalysis has been used extensively to deter-mine ion distribution in pulvini. However, even with high reso-lution techniques that distinglish between elements in the walland the protoplasm (4, 20), there is a danger of ion migrationduring specimen preparation. In experiments that measure ionextrusion or uptake by excised strips of tissue (10), the wall canact as a buffer that affects the magnitude and kinetics ofobservedchanges.Although protoplasts undergo severe stresses during isolation,

in many systems they retain physiological responses observed inintact cells (9, 12, 15, 19). Protoplasts from stomatal guard cells,for example, swell in response to light (24) and shrink in responseto ABA (23) as do intact guard cells. Experiments with guardcell protoplasts are especially pertinent to our work becausestomatal guard cells are similar to pulvinar motor cells in manyrespects. Thus, protoplasts isolated from the extensor and flexorsides ofthe Samanea pulvinus might aid in addressing questionsabout pulvinar physiology and ion transport that are difficult toapproach with intact cells. They should also enable us to ap-proach a basic question about circadian rhythmicity, i.e. dorhythms persist in individual cells isolated from a multicellularrhythmic system? Unicells have well known circadian rhythms,but rhythmic organization in multicellular systems may be en-tirely different, perhaps requiring interaction among differentcells. Here we describe the isolation and initial characterizationof extensor and flexor protoplasts from Samanea pulvini.

MATERIALS AND METHODSPlant Materials. Samanea saman (Jacq.) Merrill trees were

grown at 27°C and 70% RH on a 16-h light:8-h dark regime.Illumination was provided by cool-white fluorescent bulbs (West-inghouse F96T12/CW/HO) with a photon flux density (PAR)of270 Amol m-2 s-'. Open, terminal, secondary pulvini harvestedin hours 2 to 4 of the light period from the 4th through 10thfully expanded leaves (counted from the top) on at least 10 treeswere used in most experiments.Pasmolysis Experiments. We performed simple plasmolysis

experiments to estimate the osmotic pressure within extensorand flexor cells ofopen pulvini in vivo and, hence, estimate whatosmotic pressure might be required in the cell wall digestionmedium. Sections of pulvinar tissue about 50 ;&m thick were cutusing a vibratome (Lancers series 1000), stained with neutral red,and incubated in solutions of sorbitol of varying concentrations.Qualitative estimates of the degree of plasmolysis established byeach sorbitol concentration were made using light microscopy.

Solutions. Gamborg's B-5 medium (with sucrose, withouthormones; supplied in powdered form by Grand Island Biologi-cal Company, Grand Island, NY) was used as a base in manysolutions (for composition of this medium, see [7]). Unlessotherwise noted, all other chemicals were supplied by Sigma.Predigestion solution: Gamborg's B-5 with 0.3 M sorbitol, 0.2%(w/v) BSA, 50 mM Mes, and 8 mm CaCl2 (pH 5.5). Osmotic

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ISOLATION OF PROTOPLASTS FROM SAMANEA PULVINI

0-

.C

a)3:

~210.

-o

0)

4

I I I--rl

0

S

0

d 0

I

0oI/

I I I0.50.4

* 0

0.6

FIG. 1. Photomicrographs of flexor (A) and extensor (B) cells froman open Samanea pulvinus after staining with neutral red and incubationin 0.6 M sorbitol. The stain is taken up into the vacuole and is concen-trated as the protoplast shrinks away from the wall during plasmolysis,evident in the flexor cells. The light areas in A are the spaces betweenthe shrunken protoplasts and the wall. Stain was also taken up into thevacuoles of extensor cells, but little plasmolysis was observed. Bar rep-resents 50 Am.

adjustment solution: same as predigestion solution except with4.0 M sorbitol. Ficoll cushion: 20% (w/v) Ficoll 400 in Gamborg'sB-5 with 0.5 M sorbitol and 4 mM CaCl2 (pH 5.8). B-5 washsolution: Gamborg's B-5 with 0.5 M sorbitol, 4.0 mM CaCl2, and0.5 mM DTT (pH 6.2). Digestion enzymes: 6% (w/v) each ofdriselase (Plenum Scientific Research, Inc., Hackensack, NJ),Onozuka cellulose (Gallard Schlesinger Chemical Corporation,Carle Place, NY) and pectinol AC (gift from Corning Biosystems,Corning, NY) were dissolved in Gamborg's B-5, centrifuged toremove nonsoluble debris, and desalted over Bio-Gel P6. Afterdesalting, the enzyme solution contained 7.0 to 8.0 mg/mlprotein (determined by the methods in [2]; dye reagent fromBio-Rad), and was stored frozen until use. No loss of activitywas noticed over a 2-month storage period. Before use, theenzyme solution was brought to the desired osmotic pressure(0.5 M sorbitol, except where noted), DTT was added to 1 mm,and pH was adjusted to 5.5.

Protoplast Isolation. A procedure for isolation of protoplasts(extensor and flexor combined) from Samanea pulvini has al-ready been reported (1). We modified this procedure in severalways and also worked out methods for obtaining extensor andflexor protoplasts separately. Strips of pulvinar tissue were ex-cised from the extensor and flexor sides of about 120 pulvini (see

Sorbitol, MFIG. 2. Effect ofthe concentration ofsorbitol in the digestion medium

on yield of protoplasts from extensor (@-4) and flexor (0- - -0)cortical tissue.

Fig. 2 in Iglesias and Satter [10]), finely chopped, and placed inpredigestion solution. The osmotic pressure of the predigestionsolution was then raised to the desired level (0.5 M sorbitol,except where noted) in two steps, over 20 min, with osmoticadjustment solution. Generally, at this time about 0.4 g ofextensor tissue and about 0.6 g of flexor tissue were each incu-bating in 5 ml of solution. Five ml enzyme solution was added,and the tissue was vacuum infiltrated for 1 min. The finaldigestion medium consisted ofGamborg's B-5 with 0.5 M sorbitol(except where noted), 0.1% BSA, 25 mM Mes, 0.5 mm DTT, 4mm CaCl2, and 3% (w/v) each of driselase, Onozuka ceilulase,and pectinol AC, desalted as described above. The pH duringdigestion remained between 5.1 and 5.4. Digestion proceeded for3 h at 30C with gentle agitation (about one cycle per second ona reciprocating shaker bath). Protoplasts were then filteredthrough a stainless steel screen with 63 ,m pore size, layered ona 20% Ficoll cushion, and centrifuged at 700g for 7 min. Proto-plasts were collected from the interface, resuspended in B-5 washsolution, pelleted by centrifugation at 50g for 5 min, and resus-pended in an appropriate solution for analysis. Protoplasts werecounted using a hemacytometer.

Proton Extrusion by Protoplasts. The pH of 25 to 35 Asamples containing about 104 protoplasts was measured in mi-crofuge tubes (400 1l capacity, Beckman) using micro-combi-nation pH electrodes (model MI-410, Microelectrodes, Inc., Lon-donderry, NH). The pH of extensor and flexor protoplast sus-pensions was monitored simultaneously using two replicate set-ups. Agitation was necessary during measurements to preventthe protoplasts from settling and establishing a vertical pH gra-dient in the droplet. To accomplish this, extensor and flexorsamples were placed on a platform that was raised and loweredabout 30 to 40 times per min by a motor-driven cam and leverarrangement. The amplitude of the motion (about 1 mm) wasadjusted to suit the size of the droplet.

RESULTS

Plasmolysis Experiments. In general, flexor cells from openpulvini were strongly plasmolyzed in 0.5 to 0.6 M sorbitol (Fig.IA). Few extensor cells plasmolyzed at this osmotic pressure(Fig. IB), but most plasmolyzed in 0.7 to 0.8 M sorbitol. However,there was much variability between sections cut from differentpulvini and between cells within one section.

Protoplast Isolation. Digestion and B-S wash media containing

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GORTON AND SATTER

FIG. 3. Protoplasts from flexor (A) and extensor(B) cortical cells. Vacuoles are stained with neutralred. Bars represent 25 Mum.

0.5 M sorbitol gave optimal yields for both extensor and flexorprotoplasts (Fig. 2). Increasing the sorbitol concentration to 0.6M (Fig. 2) or even to 1.0 M (data not shown) had little effect onprotoplast yield. Consequently, 0.5 M sorbitol was used for allremaining experiments.The yields shown in Figure 2 are lower than we normally

obtained because the protoplasts had to be handled in smallbatches to compare several different osmotic treatments in oneexperiment. When larger batches were used (as described in"Materials and Methods"), yields were generally between 0.5 x106 and 106 protoplasts/g fresh weight. Extensor yields were oftensomewhat higher than flexor yields, perhaps because flexor cellshave thicker cell walls (16). Leaf age also affected protoplastyield. Yields were 20 to 50% lower for pulvini from old leaves

(numbers 10 to 15, counting from the youngest fully expandedleaf) rather than young leaves (numbers 4 to 7). Wheneverpossible we excised pulvini from leaves 4 to 7, but older materialfrom leaves 8 to 10 was also used when the amount of startingmaterial was limited.

Protoplasts were examined by light microscopy to look for anystructural changes that occurred during isolation (other than lossof the cell wall, of course), and for any differences betweenextensor and flexor protoplasts. Like pulvinar cortical cells invivo (16), pulvinar protoplasts were variable in size, 10 to 35 Mmin diameter. There was no consistent difference in the meandiameter of populations of extensor and flexor protoplasts, gen-erally 22 to 28Mm. Flexor protoplasts contained one large, centralvacuole (Fig. 3A). Most extensor protoplasts appeared identical

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ISOLATION OF PROTOPLASTS FROM SAMANEA PULVINI

Flexor

5.8 -

5.7-

0 2 4 6 8Time, min.

FIG. 4. Acidification of the external medium (B-5 wash solution) byextensor and flexor protoplasts in response to 10 uM FC added at time0. See text.

I

FIG. 5. pH of the external medium (B-5 wash) from suspensions ofextensor and flexor protoplasts. Initial (i, within 5 min of adding FC)and final (f, 100 min after adding FC) measurements taken on differentaliquots from the same population of protoplasts are shown in each case.Measurements were made on the supernatant after protoplasts were

removed by centrifugation at the indicated times.

to the flexor protoplasts, but about 3% of the population weremultivacuolate (Fig. 3B). Greater than 98% of both populationsof protoplasts were viable as shown by uptake of neutral red andexclusion of trypan blue and Evans blue stains. No residual wallmaterial was seen with calcofluor white staining.Response to Fusicoccin. As an additional test of protoplast

viability, we measured H+ extrusion to the external medium inresponse to the fungal toxin, FC,3 which causes rapid energy-dependent H+ extrusion from many diverse plant systems (13).Both extensor and flexor protoplasts acidified the medium, start-ing as soon after addition of FC as measurement could beresumed (Fig. 4). While these kinetic data are useful to demon-strate the rapidity of the response to FC, they do not give a validcomparison between H+-extrusion from extensor and flexor pro-toplasts. The samples had different numbers of protoplasts andprobably somewhat different buffering capacities. More impor-tantly, extensive protoplast breakage occurred during the meas-urements because of the necessity for agitation.To avoid the problem of protoplast breakage, we used a

method for measuring H+ extrusion that did not require agitationof protoplast suspensions. Protoplasts from each treatment (ex-tensor -FC, extensor +FC, flexor -FC, flexor +FC) were dividedinto two aliquots. At the beginning of the experimental period,protoplasts were removed from one aliquot of each treatment bycentrifugation, and the pH of the supernatant was measured.The same procedure was repeated on the remaining aliquot 100min later (Fig. 5). Buffering capacity was determined for eachsample by back titration. The average rate of acidification overthe 100-min time period could then be calculated. This experi-ment was repeated three to four times; all values, for both

'Abbreviations: FC, fusicoccin.

extensor and flexor protoplasts, were within 20%. Mean acidifi-cation rates for the two populations were not significantly differ-ent: extensor, 1.6 neq!106 protoplasts/min; flexor, 1.8 neq/106protoplasts/min. Proton extrusion in response to FC was drasti-cally reduced or eliminated by 0.5 mm KCN, indicating thatmetabolic energy was required for the response. Little H+ extru-sion was observed in the absence of FC.

DISCUSSIONThe pulvinus contains a central vascular core surrounded by

several layers of cortical tissue and the epidermis. The pieces oftissue being digested contained only cortical cells and epidermalcells. Examination of the undigested debris revealed that the vastmajority of protoplasts must have come from the cortical tissue,because strips of epidermal tissue were nearly intact after the 3-h digestion period.

Plasmolysis experiments indicated that extensor and flexorcells in open pulvini have osmotic pressures equivalent to 0.7 to0.8 and 0.5 to 0.6 M sorbitol, respectively. We therefore exploredthe possibility that digestion media with different concentrationsof sorbitol, the major source of osmoticum, might be requiredfor optimum protoplast yields from the two cell types. Foroptimum yields, the digestion medium must have sufficientosmotic pressure to plasmolyze the cell, relieving turgor pressureso that the protoplast will not burst when the cell wall is removed,but it should not dehydrate the cell unnecessarily. Both extensorand flexor tissues from open pulvini gave optimal protoplastyields in 0.5 M sorbitol, suggesting that some leakage of osmoti-cum, especially from extensor cells, must be occurring duringharvest or digestion.The vacuolar structure of the protoplasts also appears to

change during isolation. In vivo, extensor and flexor cells differin the morphology of the vacuolar system, which changes as cellsgain and lose turgor. Electron microscope studies have shownthat both types of cells are multivacuolate in their low-turgorstate, but the small vacuoles merge to form a large, centralvacuole as the cells gain turgor (3, 16). The multivacuolate stageis generally less pronounced in flexor than in extensor cells.Thus, in an open pulvinus, flexor cells may be multivacuolatewhile extensor cells contain only one large, central vacuole.However, all isolated flexor protoplasts were univacuolate, andsome extensor protoplasts were multivacuolate.

In all systems, many structural and biochemical changes occurduring the transition from cell to protoplast (reviewed in Galun[6]). The changes in osmotic content and vacuolar structureobserved in our system are what one might expect of protoplaststaking on the characteristics of cells in a closed pulvinus, ratherthan the open pulvinus from which they were isolated. Suchchanges are not surprising; open Samanea pulvini will close ifthe light intensity is reduced (17), and we could not maintainconstant light conditions during harvest and isolation. Pulvinialso open and close rhythmically, and some rhythmic changeswould be expected during the 6 to 7 h required for harvest andisolation. Furthermore, the stresses of harvest and digestionmight in themselves cause such changes.

Protoplasts were viable, as shown by vital staining and by theirresponse to FC. The response to FC does not tell whether themembranes and pumps are functioning as they do in the intacttissues. However, in order for there to be a net flux of H+ to theexternal medium in response to FC, the protoplasts must have aplasma membrane capable of sustaining a pH gradient, theymust be metabolically active, and H+-pumping mechanismsmust be at least partially intact. Because H+ extrusion in responseto FC is so widespread in both whole tissues (5, 12) and proto-plasts (5, 12, 18, 19), a lack of responsiveness to FC wouldsuggest that the protoplasts were damaged during isolation, andfurther work with them would be unlikely to be productive.

Co- EXTENSOR FLEXOR

-FC *FC -FC .FCi f fI,- r~~~r

Lf f

ss_~~~~~~~~~~~~~~

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684 GORTON AND SATTER

Pulvinar protoplasts from Samanea extrude HI in response toFC slightly more rapidly than corn root protoplasts (12), butonly about one-third as fast as leaf protoplasts from oat (19),tobacco (18), or spinach (18). In the experiments presented here,however, acidification was allowed to continue for 100 mnbefore the final aliquot was measured in order to obtain a largepH change. If a shorter time period had been used, the actual,measurable pH change would have been smaller, but the calcu-lated rate of acidification undoubtedly would have been higher.Although we have probably not measured the maximal rate ofH+ extrusion in response to FC, clearly extensor and flexorprotoplasts are metabolically active and capable ofH+ extrusionto the external medium at a reasonable rate.

Protoplasts isolated from extensor and flexor cells ofSamaneapulvini should be useful to answer questions about pulvinarphysiology and circadian rhythmicity. The chief problem is thatthe amount of starting material is limited. Protoplast yields arereasonable on a fresh weight basis, but the limited availability ofstarting material resulted in low absolute numbers. Too fewprotoplasts are produced for conventional types of analysis suchas radioactive tracer techniques to follow ion fluxes or flamephotometry to measure ion concentrations. New analytic tech-niques are therefore needed. In a companion paper, we describea method for estimating elemental concentrations in individualprotoplasts (8).

Acknowledgments-We thank Dr. Philip Applewhite for his help in developingprotoplast isolation techniques, Dr. E. Marre for his generous gift of FC, AnaIglesias and Youngsook Lee for assistance harvesting pulvini, and Mark Dodd andSubir Chakrborty for careful technical assistance. The engineering skills of RobertGorton were greatly appreciated.

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2. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein using the principle of protein-dye binding.Anal Biochem 72: 248-254

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4. CAMPBELL NA, RL SATTER, RC GARBER 1981 Apophlstic transport of ions inthe motor organ ofSamanea. Proc Natl Acad Sci USA 78: 2981-2984

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14. MAYER W 1977 Kalium-und Chloridverteilung in Laminargelenk von Phas-eolus coccineus L. wihrend der circadianen Blattbewegung im tagesperiod-ischen Licht-Dunkelwechsel. Z Pflanzenphysiol 83: 127-135

15. MErrLER IF, RT LEONARD 1979 Ion transport in isolated protoplasts fromtobacco suspension cells. I. General characteristics. Plant Physiol 63: 183-190

16. MORSE MJ, RL SArrER 1979 Relationships between motor cell ultrastructureand leaf movement in Samanea saman. Physiol Plant 46: 338-346

17. PALMER JH, GF AsPREm 1958 Studies in the nyctinastic movement of the leafpinnae of Samanea saman (Jacq.) Merrill I. A general description of theeffect of light on the nyctinastic rhythm. Planta 51: 757-769

18. ROLLO F, E NIELEN, F SALA, R CELLA 1977 Effect of fusicoccin on plant cellcultures and protoplasts. Planta 135: 199-201

19. RUBINSTEIN B, TA TATTAR 1980 Regulation of amino acid uptake into oatmesophyll cells: A comparison between protoplasts and leaf segments. J ExpBot 31: 269-279

20. SATrER RL, RC GARBER, L KHAIRALLAH, YS CHENG 1982 Elemental analysisof freez-dried thin sections of Samanea motor organs: Barriers to iondiffusion through the apoplast. J Cell Biol 95: 893-902

21. SATTER RL, GT GEBALLE, PB APPLEwHrTE, AW GALSTON 1974 Potassiumflux and leafmovement in Samanea saman. I. Rhythmic movement. J GenPhysiol 64: 413-430

22. SATTER RL, P MARINOFF, AW GALsTON 1970 Phytochrome controlled nycti-nasty in Albizziajulibrissin. II. Potassium flux as a basis for leaf movement.Am J Bot 57: 916-926

23. SCHNABL H, CH BORNMAN, H ZIEGLER 1978 Studies on starch-containing(Viciafaba) and starch-deficient (Allium cepa) guard cell protoplasts. Planta143: 33-39

24. ZEIGER E, PK HEPLER 1977 Light and stomatal function: blue light stimulatesswelling of guard cell protoplasts. Science 196: 887-889

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