Flow cytometry and sorting of plant protoplasts

Pkysiol. Veg.• 1984. 22 (5). 541·55 4

r

Flow cytometry and sorting of plant protoplasts: problems and physiological results from a study o alkalo ids in Catharanthus roseus

Spencer BROWN·, Jean-Pierre REN AU DIN"', Charles PREVQTu and Jean GU ER

Laboraroire de Ph)'siologie ceilulaire r:egerale" , C.N .R.S.-l.N.R .A .• 91190 Gif -sur-Yc Service commun de ~yrofluorom etrie analytique er separanve..• • C.N.R.S .• B.P . 8. 94800

(Received for publicatio n May 4. 1984; accept

Abstract. Technical details are given for a genera l approac h to flow cyto metry with v Sorting rates of 1000 prot cplasts per second can be maint ained, with about 70 % re is th us possible to sort distinct classes fro m a sam ple for subseq uent reanalysis or fo Protoplasts from cell suspension cultures of Catharamhus roseus fluoresce in the indicative of their serpentine content. The intensity of fluorescence has been measured try and now cytometry (or cell by cell descrip tion of serpe ntine levels. The pH prob has been used to describe the distribution of individual values for vacuolar pH in these Both serpentine and 9·aminoacridine were conti nuou sly distributed abo ut a single skew toward s high levels. From cell sorting, a positive correlation was shown betw pH and serpentine content. The effect of ionophores upon the two indicates that se over 2 h is only par tly depe ndent upo n vacuolar pH. Key words: flo w c)'tomerr)', a pH , 9-aminoacridine. Catharanthus roseus.

Resume. Des renseignemems techniques d ordre general sane presences pour f urilisarion cy tometrie en f lux. AL'ec une erresse normale de tri de 1000 protoplaues par seconde, env/ron sonr recuperes intacts. Il est done possible de trier un echantillon sur un caracre soumettre ulterieuremem d d'aurres analyses. Les protoplasres de cellules de Catharanthu en suspension cer un spectre de fl uorescence dans le bteu dli .i leur reneur en serpenti cerre fl uorescence a ere mesuree par microfluorimem e er cysomi trte en fl ux pour dicrire teneurs individuelles en serpentine dans une population. La sonde de pH 9-flminoacridin utilisee pour apprecier fa dispersion des p H vacuolaires dans ces prot oplastes. Les teneur en 9-aminoacridine sone aistribuees d une maniere continue autour d'un mode, avec une tr du core des teneurs eievees. L'a nalyse des sous-classes triees monrre une correlation po uacuolaire et la teneur en serpentine. L'erude de reffe t des ionopnores sur ces deux fac r accumu lation de la serpentine dans les cellules pendant 2 h ne depend que parciellemem Mots des : cytometrie en nux. alcalofdes. pH vacuolaire, 9-amin oacridine, Camaranth

Abbreviations. 9AA, 9-aminoacridine; CCCP. carbonyl cyan ide m-chloropheny lhydra cient of variation; Hepes, N~ 2- h yd roxye t hyl pi perazi n e- N' -2-ethanesulphonic acid: HP mance liquid chromato graphy; Mes, 2-{N-morpho lino)et hanesulphonic acid; pH e• pH buffer, pH;. intrace llular pH. being essentially vacuolar pH with the 9AA probe; SO. st SE, standard error,

INTROD UCTION

Flow cy to m etry an d cell so rtin g has been used wi th h igh er plan t ma few instances : fo r cell cycle and D N A analysis. to so rt ch ro m os o m es a protoplast fus ion. to stu d y protoplast regeneratio n a nd vacuo la r propert Physiologie Vegetale 0031-9368/84/05 54 1 t4/$ 3.40/ © CN RS·G authier-Villars

542 S . Brown, J.-P. Re na udin, C. P revot a nd J. Guern

review of these reports (Brown, 1984) it was proposed tha t the tec stud y the physiology and biochemistry of plant cells from tissue cultures. We have set out to establish th e gene ra l working conditions fo sorting of viable protoplasts. This study addressed aspect s such a intactness which are important for subsequent manipulation of sor ted by other workers (Galbrai th and Har kins, 1982; Redenbaugh er al., al., 1983 ). Analyse s by flo w cyto metry an d by microfl uorimetry a instance, cell suspension cultures of Catharanthus roseus are studied a intrin sic fluor escence attribu ted to their serpentine con tent, and for vac with a fluorescent p robe. The presen t article develops earlier commun 1983; Brown, 1984), by addi ng physiol ogical data and much techni may be inst ructive for avoiding hazards and artefacts in fut ure wo obscu re the observ ation that flow cytornetr y can be simple, efficie Ga lbraith er al., 1983; an d fo r a review, Kruth, 1982). Since this G albraith (198 4) have described sorting of leaf proto plasts from N regeneration of the same back to plants, and have th erefore shown instru mentation for use with viable m aterial. A general scheme for compa rtme ntation of indole alkaloids betwe of suspe nsion culture s of C. roseus has been proposed (Renaudin and G et al., 1983 ). In this scheme , compartmentation is a function of the pK of the pH gradient between the suspension medium and th e vacuole. copy of such cell sus pensions indi cates marked differe nces bet ween ce with respect bo th to the intrinsic fluo rescence due to serpe ntine (Bro also to vacuolar pH (Manigault et aI., 1983; Kurkdjian er aI., 1984). N recently argued tha t these par ticularly fluorescent " alkaloid cells" for of vacuolar p H, being around pH 3 in contrast to the " normal" val cells of the same suspe nsion. Taking serpentine as one indicato r of alka loid-accumulat ing capac as a contributing factor, we have sought to describe the var iabilit alkaloid-producing cell line during the exponen tial growth pha se. Th of defined types enables further analysis, with yields sufficient for su by microfluorimetr y and flow cyto metry. Here we have used protoplast cells, as necessary for flow cytometry, could not be obtai ned from the

MATERIALS AND METHO DS

Protoplast isolation. The cell line C20 of Catharant hus roseus (L. ) G . D on was days in Gamborg medi um containing 1 ~ M 2,4-dichlorophenoxy-acetic acid a growth cha racterist ics, see Courtois and Guern (1980). Cells at five days were glass filter (no . 2) under partial vacuum and rinsed five times with 0.55 M so obtained by adding 20 ml of 0.5 % (wiv ) cellulase Onoz uka RS (Yakult Honsh ( w/v) pectolyase Y23 (Seishin Pharmaceutical, Tokyo ) in 0.55 M sorbitol adj u pH 5.5. Incubation was in an 11 cm Petri dish during 2 h at 28°C on a recip The protoplast medium consisted of 10 mM Hepes (pH 7.40 at 6°C, adjusted 4.4 mM K '") in 0.55 M sorbit ol. The digested cells were filtered thro ugh 104 40 m! tube , centr ifuged at 350 g for 1.5 min; resuspended in 40 ml fresh mediu supernat ant discarded. Protopla sts were then resuspended in 10 ml fresh m 54 urn nylon , sedimented at 200 g for 2 min, resuspended in 10 ml of 0.7 M sorb stored over ice. An aliquo t was diluted 2Q.fold for counting in a 0.2 mm deep Ma

Flow cytomerry, All media were passed through a sintered glass filter and the were filtered through 0.45 um. Solutions were not stored with any matter likely Phys iotogie Vegeta te

Alkaloids and pH by now

material. namely cotton or cellulose fibre stoppers. An 80 % (v/v) Percoll stock w 10 mM Hepes (pH 7.4 with 0.1 N HO ): this was Filtered on the da y as it tends to d of tubes. and the resulting flakes can break prot oplas ts during flow through ca pilla concentratio n was 0.5.2 106 protoplasts mI- t (internal volume of 106 prot oplasts mlsuspension was refiltered through 52 pm nylon gauze just prior to use. The flow FACS UO ( Becto n Dickinscn) with a 5 W arg on laser (Spectra- Physics). Technical given under Results, but a general configuration to study the blue fluorescence of 25 mW excitation at 351+364 nm, emission collected thro ugh a 415 LP filter, photo fluorescence gain 2 x I, scatter gain 1 x 0.5. For pr otoplaste stained with 9AA the was 1 x 0.5. A low pressure sett ing (17/30 units on the cyto meter gauge) was adopted capillaries and flow resistors.

Fluorescence. Fluo rescence was studied with a Perkin-Elmer LS5 luminescence spectro ting a rhodamine reference cell for qua ntum correction of spectra. Microfl uorimetry rescence with a Leitz MPVtI microscope and pho to m ultiplier. The intr acellular pH {pH {Mani gau lt et al., 1983) fro m the distribution of the fluorescent probe 9-aminoacri dine 10 IlM and incubated over ice for at least I h, giving intracellu lar concentrations be that self-quenching does not occur. The method assumes that the calculated pH, of indiv is essentially that of the vacuole in sit u, as this organelle occupies a bout 90 % of cell v blue fluorescence in unstained protoplasts was measured by microfluori met ry, using excitation 1/ 1) with a K430 cut-off on the epifluo rescence. lntracellular serpentine c calculated from the intensities of the protoplast and medium relative to standard soluti and applying the measured protoplast diameter.

Analysis. All chemicals were from Sigma, excepting CCCP (Boehringer No. 567110) hydr ochloride monohydrate (Fl uka 06650) and Percoll (Pnarm acia j. Serpentine c assa yed in cells by reversed-phase high performance liquid chromato graphy after Ren prot oplasts. the fo llowing extrac tio n was used: a kno wn number of prot cpl asts w sulphuric acid. stored at 4~C for convenience. and then sonicated. centrifuged at 2 700 supernatant was collected and the pellet again extracted three times with 5 ~~ sulphur extract was part itioned with I volume diethyl ether. which was subsequently rewashe of acid. The acid fractions were pooled. adjusted 10 pH 10 and extracted three li volume of chloroform. giving a crud e alkaloid extract which was then fractionated silica column as already described (Renaud in. 1984). Cells were counted after acid disso an d G uern, 1980).

RES ULTS AND DISCUSSION .Technieal aspects

From 8 g fresh weight cells, and after the subseq uent puri 5-7 10' protoplas ts were obtained; I g fresb weight contained 9.09 106 cells ( The cytometer did nOI have a sample-handling system capable of maintai suspension of protoplasts du ring analysis. The protop lasts were of differing sizes. and some settled or floated even if the medium was adjusted to be gene The result was that the ratio of pro toplasts to small contaminants could cha tube : the cell flow rate changed: and the sample at the inlet was not consta ntly of the bulk population. Where analysis required less than ten minu tes. initi the sample tube was sufficient. Herein lies one advanta ge of sorbitol rathe osmo ticum: the sugar alcohol at 0.7 M is more viscous and is almost iso pyc anal ysis. PercolJ can be added to 15 % (v/v) to approach an average isopy this favou rs weak associa tion of protoplasts (witho ut blocking flow) and so w Eventu ally, an agitator needs to be designed for prolonged sorting with pro 5 105 prot oplasts ml- 1 the flow pressure had to be increased in order t adeq uate rate of analysis. and proro piasts tended to break .

vol

544 S. Brown. J.-P. Renaudin, C. Prevot and J . Guern

Even though light scatter analysis will clearly distinguish protoplast (plastids, starch, mitochondria. etc.], well purified protoplast suspensio least in initial studies as any breakage of protoplasts (as with an instudy, treatment with 5 mM propionic acid) can then be immediately

scatter histogram.

Narrow-angle light scatter was also used to gate out debr is from ana cannot be strictly taken as proportional to d 2 (Salzman et al., 1979), of similar cells it indicates relative size. For protoplasts having a mea by microscopy , the mode of light-scatter corresponded to 34 urn (calib 15 urn]. But in another experiment the mode of light-scatter gave pr 25 pm, compared to microscopic measurement of 32 urn.

For sorting, the sheath fluid must be electrolytic; sorbitol osm unstable droplet deflection. In earlier experiments the sheath fluid wa 14 mM CaC12 (pH 7.4). When it became evident that the C. roseus pr in sorbitol, tbe sheath fluid was modified to 0.5 M sorbitol, 54 mM (pH 7.4), giving about 120 meq ions 1- 1 . Thi s more viscous fluid did But if the protoplast suspension was itself highly viscous, such as with high flow pressure was required and this was deleteriou s. Mannitol wa as a solution of the same osmotic concentration is more viscous, and a prob lem. If mannitol was not promptly rinsed small crystals formed these broke cells during now , and were subsequently very difficult to w

The 80 um nozzle caused most protoplasts to break , whilst the 10 recoveries of up to 88 % intact. A modified routine was necessary to sorting mechanism. Three droplets were deflected, the central one idea The crystal was vibrated at 15 kHz. The droplet delay could not b simplicity as for smaller nozzles since the droplets were not so readily count exceeded 15.75 droplets, as was possible with a 100 urn nozzle, t to be adjusted and new values of amplitude and oscillation-frequency t

The optimal setting was found by trial and error. The droplet del a trial sort started. A microscope slide was held for several seconds in and the number of protoplasts assessed on a micro scope. The droplet and by trial and error an optimum was found: a droplet delay of 13. adjustment of the droplet forming proces s (varying pressure, freque phase) can also raise the proportion of sorted cells intact, as if the she minimized. Changing the position of droplet formation imposes reasses delay value. If the sort was set up with microspheres, this optirnisatio to be repeated with the protoplast sampl e as the optimal setting was being modified by the larger pa rticle size, dens ities. etc.

With these procedures, protoplasts of mean diameter 40 um and 65 pm (these can pass a 52 J.U1l filter) could be sorted at 500 to 1000 pe up to several hours , with consistently more than 70 % being recovered i Whilst the cell sorter analyses cells, the sorting mechanism al (vernpty"] droplets into the sort tubes . What error does this introduce is assayed? The following example with 9AA gives the order of this extracellular source . Given a cell population of mean pH i 6.1 in subpopulations differing by 0.3 unit in pl-l, will have a two-fold differe [9AAJ. But once sorted, [9AA] in the receiving tube s will differ by 1.88 because of the error introduced by extracellular 9AA. Thi s problem ca one drople t (that containing the cell) is deflected, but recovery ma y be Phy siotog ie Vegetate

Alkaloids and pH by no w

The only UV ba nd ava ilable on the argon laser of the cell sorter is a mul of 35 1 + 364 nm. On a fluorimeter. exciting 9AA at 364 nm rather tha n at 399 nm. led to a 67 % loss of emission intensity; similarly, exciting serpe nt rather than at 305 nm lowered emission by 7 6 ~~. Therefore the mola r emissio the now cytometer was only a qua rter of tha t possible at the optimal excitatio Furthermore, the argon laser has a nominal power of only 0.2 W at 351 + 364 to 1.5 W at 488 nrn (where for instance fluorescein is excited). Despite these the fluorescence signal on the now cytometer was adequa te. The rela tive fluore sce nce intensities of micromolar aq ueous solutions (for the excita tion and emis sio n optima shown usin g 25 and 5 nm bandpas in a semimicro cuvette): 9AA 1.00 (excita tion 399 nm, emission 430 nm ); s (excita tion 305 nm. emission 443 nm); l1uo rescein 0.67 (excita tio n 478 nm, emi Because of the optica l configura tio n of the flow cytom eter, with tine excita emission measuremen t. the signals from 9AA and serpen tine are finally of si on a molar basis. (We have recentl y imp ro ved the selectivity for 9AA in th serp entine by inserting a U V int erference filter Oriel 70706, peak 452 nm, ba nd The cell sa p do es not hav e significa nt absorbance in the blue emission w 9AA and serp entine. Nor do serpe ntine a nd 9AA quench one anothers' emis emission spectra of serpe n tine and 9AA overla p to such a degree tha t it was using one excitation bea m, to simultaneously assess these two compo unds b emission s at differen t wavelengths. Yet in a two laser system it sho uld be p the 407 + 415 nm band of krypton (e.g. 100 mW on Spectra-Physics 2025-11 excite 9AA and so pefonn biparametric analysis of pH agai nst other a ttr serpentine or H oechst 3334 2 (for vital staining of DN A) excited at 351 + 364

1

Pb ysiology Serpentine content of protoplasts Th e overall method of serp entine extrac tio n and HP LC analys is has a 6.2 % for cells (three experiments each with three independent extrac tions SE % = 9.2.[6.2) and of 1.8 % for protoplasts (three values of SE % = 3.1, 2.2, 0 varia bility with cells is att ributed pa rtly .to errors in measurement of fresh w the pr otop last number is more pr ecise.

Table 1. Serpentine content of cells and protoplasts. Cells fro m a 5 da y old suspensio protoplast preparatio n. Prot oplasts were fixed immedia tely after purification (total tim 2.5 h). In experiments '; and 5 they were also washed and analysed after storage at 4 6 h. Serpent ine was assa yed by HPLC. Serpentine content

Experiment Cells

I 2 3 4

Protoplasts (nmol per 106 objects)

0.46 0.46 0.27

0. 17 0.16 0.15

0.16

0.063 0.065 0.064 0.080 0.089 0.071

,=3 h t= 6 h

5 t =3 h t =6 b mean (SE %)

Ptw siotogie Veghale

0.13

0.30 (24%)

Prot o

0.14(25 %)

4

vol.

546 S. Brown. J. -P. Renaudin, C. Prevot and J. Goern

Serpentine was assayed by H PLC both in cells and in protoplast (tab. 1). Th e serpentine con tent per protoplast was only 49 % of tha This was not due to serpentine degradation by enzymes used for pr nor to serpentine catabolism: serpentine lost from cells d uring cellu recovered in the medium (data not shown). Other alkaloid s such as ajm acids too. were massively lost during protoplast preparation (data not sh non is currently being studied. The serpentine content of protoplasts re at least 6 h (tab . 1), namely during cytomet ry.

Relation becween serp entine and fluorescence Figure 1 presents excitation and emission spectra of serpentine, protoplasts and cell sap obtained by freeze-thaw after Kurkdjian and of C. roseus C20 appear blue when illuminated by UV. This alread y kn attri buted to serpen tine; bot h th e protoplast and cell sap spectra are ty serpentine (excitation peak 305 nm, broad emission at 443 nm ) with ajm (excitation aro und 290 nm with a Stoke s shift of 30~50 nm]. Addit ion

SERPENT

9 AA Ex

Em

em 49 0

>>-

Ul

Z

ex360

-: 300

w >-

Z

Ex

Ex

em4 4 3

em443

PROTO PLASTS 300

400

500 300 (nm) WAVELENGTH

400

Figure 1. Fluorescence spectra. Excit ation spectra (Ex) are shown from 280 nm. on the excita tion monochromato r and 5 nm on the emission rnono chr omato r (se spectra (Em) are shown to 550 nm, using 5 nm bandpa ss on the excitation mo i. nml and 2..5 nm on the emission monochr omat or. 9AA 0.5 1l M aque ou s. aqu eous serpent ine tart rate. PROTOPLASTS, 20 000 prot oplasts ml - l . SAP, sap in 0.25 % H 2S0 •. Th e scan ning rate was 30 nm min - I; response factor 4; us Th e gain was different for each spectrum : the relativ e intensiries are discussed in

Alkal oids and pH by flow

cuvette reduced these peak s as with serpentine and pheno Iics. At excitation ajmalicine and pheno lics are no t significant. This cell line contains only pho ropigments (e.g. one fluorescent at excita tio n 458 nrn, emission 685 nm). Is it reasona ble to relate fluorescence and serpe ntine levels? The cel (below) aTC affirmative. Secondly, the intrinsic blue fluorescence of proto p compared by microf'luori merry to that of standard solutio ns. and to the inte fro m the serpe ntine content after extraction and HPLC. In two independen intensities by rnicrofluorimetr y on living proto plasts were only 61 and 32 % of fro m their chemical levels. This would be due to intracellular q uenching Fluore scence, as obser ved with other fluoroc hromes. So while we attribu te rescence of pro to plasts to serpentine. it is not ed that this fluorescence repres 30 % of th at predic ted from the initial serpentine content of the cells, serpentine and quen ching. The cell walls remaining after alco holic extraction also ha ve 3. blu equivalent to 20..25 % of the total cell fluorescence. This is likewise observed of, for inst ance, Ace r pseudoplaranus after the same trea tment, even though A. pro toplasts do not show blue fluo rescence. Thi s pigment canno t be ext sulp huric acid, nor by 0. 1 N NaOH, so it is not serpentine.

Histograms of serpentine and pH The scatter plot (fig. 2 A) rep resents dia meter an d blue fluorescenc 125 protoplasts meas ured by microfl uori metry. Th ere is no clear relationsh size and total fluo rescence. When fluoresce nce was divided by pro toplas calibrated agai nst serpentine stand ards, the ran ge was 1 to 25 and the d skewed rather than gaussian (fig . 2 B]. A major part of the po pulation seem to a log normal distribut ion (fig . 2 B). -...- LOG [S] - 0.6 w U Z

w

®

,,

"

Cl

0:

o

..J u,

>-