Plant Cell Reports (1991) 10:512-516 PlantCeil Reports �9 Springer-Verlag 1991

Avocado fruit protoplasts: a cellular model system for ripening studies

Frank W. Percival 1, Laura G. Cass, Kristin R. Bozak, and Rolf E. Christoffersen

Department of Biological Science, University of California, Santa Barbara, CA 93106, USA 1 Present address: Department of Biology, Westmont College, Montecito, CA 93108

Received June 28, 1991/Revised version received September 24, 1991 - Communicated by C. Quiros

Summary. Mesocarp protoplasts were isolated from ma- ture avocado fruits (Persea americana cv. Hass) at vary- ing stages of propylene-induced ripening. Qualitative changes in the pattern of radiolabel incorporation into polypeptides were observed in cells derived from fruit at the different stages. Many of these differences correlate with those observed during radiolabeling of polypeptides from fresh tissue slices prepared from unripe and ripe fruit. Protoplasts isolated from fruit treated with propy- lene for one day or more were shown to synthesize cellu- lase (endo-B-1,4-glucanase) antigen, similar to the intact propylene-treated fruit. These results suggest that the isolated protoplasts retain at least some biochemical char- acteristics of the parent tissue. The cells may also be used in transient gene expression assays. Protoplasts iso- lated from preclimacteric and climacteric fruit were equally competent in expressing a chimeric test gene, composed of the CaMV 35S RNA promoter fused to the bacterial chloramphenicol acetyltransferase gene, which was introduced by electroporation.

Key Words: Avocado Protoplasts - Ripening

Cellulase - Electroporation

Abbreviations: PCM, Murashige and Skoog salts and growth factors, supplemented with 3% sucrose, 0,3 % glucose, 0.3 % enzymatic casein hydrolysate, 0.5 M man- nitol, and 5 mM CaCI2; CAT, chloramphenicol acetyl- transferase.

Introduction

The ripening of avocado fruit involves a complex set of biochemical and physiological changes which lead to the development of an edible fruit. One aspect of ripening is the softening o f mesocarp tissues, a consequence of cell wall degradation (Brady 1987, Fischer and Bennett

Offprint requests to: R. Christoffersen

1991). In avocado fruit, the mRNA for cellulase (endo- 13-1,4-glucanase) has been shown to accumulate during normal ripening (Christoffersen et al. 1984) or in re- sponse to ethylene treatment of unripe fruit (Christoffersen et al. 1989, Tucker and Laties 1984). It is derived from cel l , a single member of a small cellulase gene family (Cass et al. 1990). Although ethylene is generally recognized as the primary regulatory agent of fruit ripening, the signal transduction pathway, from stimulus recognition to activation of cel l and other ripen- ing-associated genes, remains to be elucidated.

A major difficulty in studying ripening at the molecu- lar level at present is that there are no cellular model sys- tems for the process. Recently, it has been shown that protoplasts isolated from several diverse plant organs will respond to either hormonal or environmental signals in a manner analogous to the tissue from which they were de- rived (Callis et at. 1988, Hooley 1982, Howard et al. 1987, Huttly and Baulcombe 1989, Lipphardt et al. 1988). In this paper, we describe a protoplast model sys- tem from ripening avocado fruit that retains a pattern of protein synthesis that is similar to the parent fruit tissue. Furthermore, these cells are competent in transient ex- pression assays of foreign genes that have been intro- duced by electroporation. This protoplast system may prove useful in elucidating the cellular and biochemical mechanisms that are responsible for the activation of the cell gene during fruit ripening.

Materials and methods

Plant Material Mature avocado fruit (Persea americana cv, Hass) were picked from a single tree and placed individually in jars through which a flow (45-60 ml/min) of humidified air was maintained. Propylene (300 pJ/l) was introduced to the air stream of the fruits to in- duce ripening, while control fruits did not receive propylene. Ripening was monitored by measuring the rates of endogenous ethylene produc- tion during the treatment period. When treated in this manner, fruit generally begin to produce a measurable amount of autocatalytle ethy- lene after 1 day of propylene treatment and complete the climacteric

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within 3-4 days, although the specific time course varies from fruit to fruit.

Ethylene Analysis. Ethylene concentrations were determined with a Shimadzu GC6A gas chromatograph equipped with a 2.4 mm x 5 m Porapak N column and a flame ionization detector. Protoplast ethylene production was determined by sealing a protoplast suspension (0.5 ml) in a 12 x 75 mm tube with a serum stopper and shaking gently at 25~ for 1 h. Ethylene accumulation in the headspace was then determined in a 1 ml sample, withdrawn with a syringe.

Preparation of Protoplasts. Fruits were surface-sterilized in a solution containing 10% (v/v)bleach and 1% (v/v)Liqui-nox (Alconox, Inc.) for 30 rain and then rinsed several times with sterile water to remove bleach and detergent. All subsequent manipulations were performed using aseptic procedures. Cores of mesocarp tissue were taken with a No. 5 cork borer, and disks (ca. 0.5 mm thick) were cut with a scalpel, avoiding the green tissue nearer the outside of the fruit. Disks from two cores prepared in this way (approximately 1 g of tissue) were placed in 4.5 ml predigestion solution (see below) in a 60 mm petri dish for 30 min, after which the predigestion solution was removed and replaced with 4.5 ml digestion solution. The predigestion and digestion solutions both contain 0.6 M mannitol, 0.5% BSA, 0.1 mM dithiothre- itol, and mineral salts (Patnaik et al. 1981), while the digestion solution contained, in addition, 1% (w/v) Rhozyme HP-150 (Genencore), 1% (w/v) Cellulysin (Calbiochem), and 0.5 % (w/v) Macerase (Calbiochem). Prior to use, the digestion mixture was centrifuged 10 rain at 12,000g, and both solutions were sterilized by filtration (0.45 #m). After overnight digestion (at least 18 h), the tissues were teased apart with dissecting needles to release the protoplasts, and the suspen- sions were filtered through nylon mesh (150 #m openings) into 15 ml centrifuge tubes. The cell suspension was overlaid with 5 ml of medium containing Murashige and Skoog salts (Gibco BRL, Inc.), growth factors (Dodd and Roberts t985), 3% sucrose, 0.3% glucose, and 0.3 M mannitol. The protoplasts were then floated by centrifuga- tion at 50g for 10 rain. The ceils were washed once and finally sus- pended in 3 ml of the same medium. The numbers of viable cells were determined using a hemacytometer and estimated as the numbers of cells with typical morphology that also excluded phenosafranine (Widholm 1972).

In Vivo Labeling and lmmunoprecipitation of Celhdase. A 50 #1 aliquot of protoplasts (approximately 7 x 104 cells) was mixed with 5 #I of Tran35S-label (ICN; 10mCi/ml, 1100 Ci/ntMot) and incubated for 5 h at 25~ with gentle shaking. Aliquots of the labeled protoplasts were added directly to SDS sample buffer and boiled for the analysis of total labeled protein. Alternatively, the labeled protoplasts were dis- persed in immunoprecipitation buffer (10 mM Tris-HC1, pH 7.4; 2 mM EDTA; 0.15 M NaCI; 1% (w/v) NP-40; 5 #g/ml Aprotlnin) with a small mortar and pestle and insoluble material was removed in a micro- centrifuge. Control serum and formalin-fixed Staph-A cells (Pansorb, Calbiochem) were added to the sample to preadsorb nonspeciflc anti- gens. The sample was then divided in two aliquots and either control serum or antiserum to avocado cellulase (Christoffersen et al. 1984) was added. After incubation at 4~ overnight, antigen-antibody complexes were recovered by adsorption to fixed Staph-A cells. The pellet was extensively washed with immunopreclpitation buffer then boiled in a small volume of SDS sample buffer. The samples were then analyzed by SDS-PAGE and autoradiography.

Electroporation of Fruit Protoplasts. The plasmid pCaMVCAT (Fromm et al. 1985) was a gift from Michael Fromm and Virginia Walbot. The plasmid was maintained in E. coli strains HB101 or DH5ot, and purified by cesium chloride banding following normal pro- cedures (Maniatis et al. 1982).

The procedure for electroporation was essentially that of Boston et al. (1987). Protoplasts (0.5 ml, containing 0.7 - 1.0 x 10 e cells) were combined with 0.25 ml 20% PEG-8000 in F medium in sterile Gene Pulser cuvettes (Biorad). F medium consists of 140 mM NaCI, 5 mM KC1, 0.75 mM Na21-IPO4, 5 mM glucose, and 125 mM CaCI2, adjusted

to pH 7 (Krens et al. 1987). Supercoiled plasmid DNA (50 #g) was added in a volume of 50 #1 or less, and the suspension was mixed gent- ly and placed on ice for 10 rain. Eleetroporation was carried out using a Biorad Gene Pulser (120-160 V with the 250 #F capacitor), and the samples were returned to the ice bath for an additional 10 min. After this period, the cells were transferred to 15 ml disposable centrifuge tube and diluted with four 1 ml portions of sterile F medium, added at 5 rain intervals. The diluted cell suspensions were centrifuged at 25g for 3 rain to float the cells, the underlying liquid was removed, and the cells were washed 3 times with 5 ml of PCM. The cells were resus- pended in PCM to a total volume of 2 ml, poured into a 35 mm petri dish, and incubated at room temperature for 18-24 hr.

Extraction and Assay of Chtoramphenicol Acetyl Transferase. After incubation, the ceils were centrifuged at 25g for 3 min, and the under- lying liquid was removed with a syringe. Lysis was obtained by adding 1 ml 0.25 M Tris HC1, pH 7.5 to the cells, and rapidly drawing them into and out of a 1 ml micropipette tip 30 times. The lysate was centrifuged 15 min in a mlerofuge, and the supernatant fluid was re- moved and heated for 10 min at 60~ The samples were assayed for CAT activity using the HPLC assay of Young et al. 1985 and for pro- tein using the Biorad protein reagent with bovine serum albumin as a standard. S-Acetyl-coenzyme A was synthesized from eoenzyme A and acetic anhydride; the 1-O-acetyl- and 3-O-acetyl- chloramphenieol standards were synthesized from chloramphenieol and acetic anhydride (Young et al. 1985).

R ~ s u l t s a n d d i s c u s s i o n

Isolation o f Protoplasts f rom Ripening Avocado Fruit

Pro top las t s w e r e read i ly ob ta ined f r o m m a t u r e avocado

frui ts be fore , du r ing , and a f te r the c l imac te r ic r ise o f

e thy lene p roduc t ion . T h e y ie lds o f cel ls ob t a ined ranged

f rom 2 x 10 6 to 6 x 10 6 v i a b l e cel ls pe r g r a m t issue, w i th

s o m e w h a t h i g h e r y ie lds o b t a i n e d f r o m c l imac te r ic and

pos t -c l imac te r i c fruit . T h e m e s o c a r p o f avocado , exc lud-

ing vascu la r t issue, cons i s t s p r i m a r i l y o f two cell types

(P la t t -Alo ia et al. 1980, Chr i s t o f f e r s en et al. 1989). T h e

ma jo r i ty o f cel ls a re s i m p l e p a r e n c h y m a cel ls tha t are

f i l led w i th mu l t ip l e oil d rop le t s . T h e second cell type is

an id iob las t w h i c h is d i s t i n g u i s h e d f r o m the p a r e n e h y m a

cel ls in h a v i n g a s ing le la rge oi l d rop le t and secondary

wal l depos i t ion . T h e p o p u l a t i o n o f p ro top las t s re leased

f r o m the m e s o c a r p cons i s t ed o f these two cell types in

p ropo r t i ons tha t a p p r o x i m a t e those o f the pa ren t t issue,

and the re we re no o b v i o u s m o r p h o l o g i c a l d i f f e rence ob-

se rved b e t w e e n p ro top la s t s i so la ted f r o m frui t a t d i f ferent

s tages o f r i pen ing . T h e spher ica l shape o f m o s t o f the

p ro top las t s de r ived f r o m the p a r e n c h y m a cel ls suggests

tha t subs tan t ia l r emova l o f the cel l wal l had occur red ,

a l t h o u g h s t a in ing w i t h C a l c o f l u o r W h i t e (data n o t shown)

revea led the p re sence o f res idua l wal l mater ia l o n the sur-

faces o f some cel ls . Spher ica l id iob las t p ro top las t s were

n e v e r obse rved . Al l i n t ac t - appea r ing cel ls in the var ious

p repa ra t i ons exc luded p h e n o s a f r a n i n , and this t echn ique

was used rou t ine ly to e s t ima te cel l v i ab i l i ty ( W i d h o l m

1972) . T h e v iab i l i ty o f the cel ls was a lso indica ted by

the i r capac i ty to car ry ou t p ro t e in syn thes i s and to express

514

transiently a plasmid-borne chloramphenicol acetyltrans- ferase gene (see below). It proved infeasible to separate the protoplasts from lipid droplets released from broken cells during the isolation procedure, since the high lipid content of the cells caused them to float in all wash proce- dures.

Protoplast Ethylene Synthesis

Protoplasts isolated from climacteric fruit synthesized ethylene at a higher rate than cells obtained from unripe fruit--1.7 __. 0.7 nl compared to 0.34 + 0.39 nl C2H4 hr-1 per 106 cells, respectively (means + SD, P < 0.05). Since a stimulation of ethylene production during proto- plast isolation, attributable to a protein factor in Cel- lulysin preparations, has been commonly observed (Anderson et al. 1979, Fuchs et al. 1989), it is likely that some fraction of the ethylene synthesis in the avocado protoplasts is artifactual. However, the higher rates ob- served in cells isolated from climacteric fruit suggests an increased capacity for ethylene synthesis in these ceils that cannot be attributed to external factors. Addition of ACC to the protoplast suspensions derived from either ripe or unripe fruits failed to increase ethylene synthesis by the cells (data not shown), suggesting that ethylene forming enzyme may be the rate limiting step for ethylene synthesis in these cells.

Proteins from the labeling study were also analyzed by SDS-PAGE to look for qualitative changes in protoplast protein synthesis as a function of ripening of the parent tissue, and these were compared to differences in proteins synthesized by ripe and unripe fruit tissue slices (Fig. 1). In both protoplasts and fruit slices, the synthesis of pro- teins with apparent masses of 27, 41, 52, 55, and 69 kD increases as the fruit ripens. Also, several polypeptides that are actively synthesized in both protoplasts and slices from unripe fruit cease being labeled in preparations from ripe fruit (e.g., the cluster of three peptides with apparent masses of 49, 5l , and 53 kD). On the other hand, all the protoplast preparations actively synthesized a 37 kD pro- tein that was not labeled noticeably in the fruit slices.

Apparently some aspect of the protoplast preparation procedure, perhaps wounding of the tissue or some sub- stance in the digestion mixture, brings about this induc- tion. In one case, there appears to be an interaction be- tween ripening and protoplast preparation that affects the pattern of protein synthesis. A 30 kD polypeptide that is synthesized in ripe but not unripe fruit slices is not no- ticeably labeled in any of the protoplast preparations.

Protoplast Protein Synthesis

Protoplasts isolated from fruit at all stages of ripening were competent in protein synthesis, although the extent of label incorporation from 35 S-amino acids into TCA- precipitable material by the cells decreases with the stage of ripening. The average rate of label incorporation for preclimacteric protoplasts was 64.7 _+ 25.7 x 106 cpm per hr per 106 cells, while the rate for protoplasts iso- lated from fruit that had been producing autocatalytic ethylene for 1 day or longer was 14.4 + 5.0 x 106 cpm per hr per 106 cells (means + SD, P < 0.01). A simi- lar decrease in the rate of radioactive amino acid incor- poration into protein with ripening has also been ob- served in avocado fruit tissue slices (Richmond and Biale 1967), although the reasons underlying this change are unclear. Since studies of polysome profiles from ripen- ing avocados suggest that the rate of protein synthesis actually increases during the climacteric (Tucker and Laties 1984), changes in the internal pools of free amino acids may be the explanation for the decrease in label in- corporation. In the protoplasts, the CAT activities ob- tained in transient expression assays did not appear to vary with the stage of ripening from which the cells were isolated (see below). Thus, if the rate of protein synthe- sis itself is actually lower in protoplasts taken from cli- macteric fruit, it is apparently not low enough to become limiting for the expression of electroporated genes.

Fig. 1. Autoradiogram of SDS-PAGE separation of 35S-labeled pep- tides synthesized by protoplasts derived from fruit after 0, 1, 2, 3, and 4 days of propylene treatment (left) and by tissue slices taken from ripe and unripe fruits (right). Polypeptides that are ripening-induced in both fruit and protoplasts are labeled with a diamond (~); polypeptides la- beled with a open triangle ( ~ ) are discussed in the text.

The regulation of protein synthesis in the protoplasts was examined further by studying the synthesis of cellu- lase by the isolated cells. Cellulase activity (Awad and Young 1979, Pesis et al. 1978) and protein (Christoffersen et al. 1984) accumulates during the cli- macteric ripening of avocado fruit. A similar rapid accu- mulation of cellulase antigen is observed when unripe fruit are treated with propylene, an ethylene analog (Christoffersen et al. 1989). Protoplasts isolated from propylene-treated fruit also synthesize cellulase antigen (Fig. 2). Cells were incubated with 35 S-amino acids, and an anti-avocado cellulase antibody was used to immuno- precipitate cellulase protein that was synthesized during the labeling period. SDS-PAGE and autoradiography re- vealed radiolabeled material in immunoprecipitates from cells derived from fruits that had received as little as one day of propylene treatment, but not from cells from the untreated control. The electrophoresis resolved this ma- terial into two bands: one with an apparent mass of 52 kD and another at 48 kD. The larger band corresponds to the molecular weight of mature avocado cellulase (Bennett and Christoffersen 1986). Immunoblots of extracts from ripe fruit do not reveal the presence of a cross-reactive product at 48 kD (Christoffersen et al. 1989). Although this could represent a real difference between protoplasts and intact fruits, this seems unlikely, since elec- trophoretic analysis of the total labeled protoplast pep- tides did not show increased synthesis of a 48 kD product but did reveal the increase in the synthesis of the 52 kD protein. A more likely possibility is a gel artifact due the presence of a large amount of precipitated rabbit im- munoglobulin in this region of the polyacrylamide gel.

Fig. 2. Autoradiogram of SDS-PAGE separation of immunoprecipi- tates of in vivo labeled protoplast polypeptides from fig. 1 with either antiserum to avocado cellulase (A) or control serum (C).

515

c -

O

r - v

"5 <~

0

6O0

5O0

400

3O0

200

100

0 110

0

o �9

o I I i I I

120 130 140 150 160

Volts

170

Fig. 3. CAT activities in protoplasts after electropomtion with pCaMVCAT DNA at different voltages. Each point represents the ac- tivities obtained in ceils from a different individual fruit; protoplasts from unripe fruit ( (D); protoplasts from ripe fruit ( 0 ) .

Transient Expression of Electroporated Genes

The potential of the avocado fruit protoplasts as a system for studying the control of gene expression during ripen- ing was investigated by testing their ability to express a plasmid-borne CAT gene after electroporation. There is no detectable endogenous CAT activity in these cells. Thus, CAT expression after electroporation is completely DNA-dependent. Polyethylene glycol in the electropora- tion buffer was required for effective uptake and expres- sion of exogenous DNA by the cells. CAT activities in the electroporated ceils increase with increasing PEG con- centration up to 6.7 %. However, when 13.3 % PEG was tested in a preliminary experiment, cell viability de- creased markedly, even when the cells had not been shocked. Finally, although CAT activities varied in pro- toplasts isolated from different individual fruit, cells ob- tained from unripe and from ripening fruit are equally competent in expressing the electroporated CAT gene (Fig. 3).

Studies using this system are currently in progress to investigate the 5' flanking sequences of the avocado cellu- lase gene (Cass et al. 1990). This system may also prove useful for the analysis of regulatory elements from other ripening-related genes from avocado (Bozak et al. 1990), tomato (Bird et al. 1988, Deikman and Fischer 1988), or other fruit.

Acknowledgements. Supported in part by a U.S. Department of Agri- culture Competitive Research Grant to R.E.C. and a Faculty Develop- merit Grant fi'om Westmont College to F.W.P. We thank Drs. M. Fromm and V. Walbot for the gift of the pCaMVCAT. The cellulase antiserum was a gift from M. Durbin and L.N. Lewis.

516

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