isolation of intact mesophyll protoplasts from the leaves

Proc. Indian Acad. Sci. (Plant Sci.), Vol. 89, Number 2, March 1980, pp. 79-90. (~) Printed in India.

Isolation of intact mesophyll protoplasts from the leaves of higher plants for photosynthetic studies

K P CHELLAPPAN* and A G N A N A M Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021 * Present address:Department of Botany, Annamalai University, Armamalai- nagar 608101

MS received 14 May 1979; revised 4 January 1980

Abstract. From a survey of 104 herbaceous species from the Madurai Kamara.~ University Campus collected for isolating intact mesophyll protoplasts 36 species belonging to C~, C4 and CAM photosynthetic pathways were found to yield between 15-85Yo of protoplasts. Species which easily yielded intact mesophyU ceils by mild mechanical grinding were not amenable for protoplast isolation. The protoplasts of CAM species were the largest and those of C4 were the smallest in size. The isolated mesophyll protoplasts were photosynthetically active in terms of COs fixation and O3 evolution. Potassium ferricyanide was ineffective as Hill oxidant while dichlorophenolindophenol induced high Hill activity with the isolated protoplasts. The protoplasts maintained their photosynthetic stability up to 12 hr after isolation.

Keywords. Mesophyll protoplast ; photosynthesis ; enzymatic isolation.

1. Introduction

Isolated intact mesophyll celts from the leaves o f higher plants have extensively been used for various metabolic studies and particularly for photosynthetic research by several workers, chiefly because of the methodological advantages they confer as compared with intact leaf (Takebe et al 1968 ; Gnanam and Kulandaivelu 1969 ; Edwards and Black 1971 ; Jensen et al 1971). Similarly, with the successful iso- lat ion of plant protoplasts from the leaves and other parts of higher plants, they have played a vital role in opening up new vistas and at tracted the attention of plant physiologists, molecular biologists, pathologists and cytogeneticists (Bajaj 1977). Besides being used in protoplast culture and somatic hybridisation, plant protoplasts have been used to investigate various aspects in plant physiology particularly in photosynthesis (Kanai and Edwards 1973a ; Gotierrez et a11974; Ku et al 1974; Hall and Cocking 1974; Huber and Edwards 1975 ; Nishimura and Akazawa 1975; Ra thnam and Edwards 1976), virology and pathology (Aoki and Takebe 1969),

r.(a)--i 79

80 K P Chellappan and A Gnanam

cytogenetics (Carlson 1973) and cell modification and t, ptake (Davey and Cocking 1972; Giles 1974).

Several techniques are available for the enzymatic isolation of mesophyll protoplasts from the leaves of various species of higher plants and for studying the role of the two types of leaf ceils in Ca plants in photosynthetic carbon assimilation (Ruesink 1971; Takebe et al 1968; Otsuki and Takebe 1969; Cocking 1972; Kanai and Edwards 1973a, b). We report in this paper the screening of 104 herbaceous plant species of diverse taxa comprising the physiologically classified C3, C4 and CAM species from the Madurai Kamaraj University Campus for the enzymatic isolation of intact mesophyll protoplasts.

2. Materials and methods

2.1. Plant species

The plant species used for the isolation of mesophyll protoplasts were either natu- rally growing as weeds or cultivated as ornamentals or crops in the University Campus. The plants had an approximate 12 hr photoperiod and mean day temperature of 36 ~ C and mean night temperature of 27 ~ C. Fully expanded and mature leaves from healthy plants were used for protoplast isolation.

2.2. Preparatzon of leaf tissue

The method used for preparing leaf tissue for enzymatic digestion depended on the species. In dicots, the lower epidermis of the leaves was peeled off with for- ceps or the lower surface of the leaves was rubbed gently with a tooth brush to break through the epidermal tissue. The leaf tissues were thencut into small segments of 2--4era ~. In monocots, particularly Gramineae members, the 1eaves after removing the mid-vein were cut with a razor blade to give transverse sections less than 1 mm wide. The leaf segments were then vacuum-infiltrated in the enzyme digestion medium (1 g/10 ml) in a petri dish or 25 ml conical flask and incu- bated under low light at 30 ~ C. The incubation time which varied with species was usually between 1-5 hr. The enzyme digestion medium contained 0 . 5 ~ macerozyme, 2~o cellulase (Onozuka 1~-10), 0.6 M sorbitol as osmoticum, 5 mM MgCI~, and 20 mM MES buffer [2-(N-morphotino) ethanesulphonie acid]. The pH of the medium was adjusted to 5.8 with 1 N KOH.

2.3. Isolation and purification

At the end of the enzyme incubation, the leaf segments were gently shaken to libe- rate the protoplasts. This crude protoplast mixture contained the released intact and broken protoplasts, chloroplasts and undigested tissue including epidermal and vascular tissue. This crude mixture had to be purified to obtain intact mesophyll protoplasts by filtration using nylon sieves. For C3 species, the protoplast mixture was filtered successively through a 35 mesh screen, 211/z and 88/z nylon nets. For C a species, 35 mesh screen, 88/z and 44/z nets were used. For CAM species, filtration was done with 35 mesh screen and 211/z net. The filtrates were then centrifuged at 300 g for 3 rain. The pellet was resuspended and washed twice in a wns~ng medium containing 50 mM tricine-KOH buffer (pH 7.8), 0.6 M sorbitol

Isolation of intact mesophyll protoplasts 81

(concentration same as in the enzyme medium) and 5 mM MgCI~. In a few species, the pellet contained essentially pure intact spherical protoplasts which was stored in the washing medium in dark at 4 ~ C until use. However, the protoplasts o f many species had to be further purified, since the pellet contained chloroplasts and other small cellular debris mixed with the protoplasts. The protoplasts were layered on the top of 20~ sucrose solution in a test tube and centrifuged at 200 g for 5 min. The protoplasts which floated on the top of the sucrose solution were collected by a pasteur pippette. The protoplasts of sorghum, maize, paddy, wheat, etc., which did not float were purified by using aqueous liquid-liquid two phase method described by Kanai and Edwards (1973b).

2.4. Yield and size

The intact protoplasts in a given suspension were counted using the Neubaur improved double hemocytometer. The yield of intact protoplasts was calculated after knowing the total number of cells in a thin leaf section of known area under light microscope. The average size of the protoplasts was determined by measur- ing the diameter of 100 protoplasts per species using an ocular micrometer which was precalibrated with stage micrometer under microscope.

2.5. COz fixation assays

COz fixation assays were done at 30 ~ C in a total volume of 0.5 ml reaction mixture containing 50 mM tricine-KOH (pH 7.8), 1 mM MgClz, 1 mM MnCI~, 1 mM EDTA, 0.6 M sorbitol (concentration same as in the enzyme medium), 5 mN[ Nai l 1~ CO3 (1 # Ci/10/t reel) and the protoplasts containing 10-15/tg chlorophyll. Illumination was provided by a bank of cool fluorescent lamps at 15,000 lux. Fifty/zl samples were removed at various intervals and the reaction ~as stopped with 50/zl 10 ~ TCA and the incorporation of 14CO 3 into acid stable products was determined by ECIL LSS z~ liquid scintillation counter.

2.6. 03 evolution

Photosynthetic O 3 evolution was monitored polarographically at 30~ using a YS[ clark-type oxygen electrode hooked to a Heath Serve recorder model EU-20B. The reaction mixture used was the same as that of CO3 fixation assay, except that NaH14CO3 was omitted. The reaction was run in a total volume of 1 ml containing 15-30/tg chlorophyll equivalent of protoplasts. Light was supplied at 10,000 lux by a bank of cool fluorescent lamps.

2.7. Hill reaetion

Hill reaction with mesophyll protoplasts was measured spectrophotometrically by following photoreduction of Hill oxidants, protassium ferricyanide and dichlorophenolindophenol (Vernon and Shaw 1969; Anderson et al 1972).

2.8. Chlorophyll estimation

The total chlorophyll content of the protoplasts was determined according to Amen (1949).


3. Results

'Out o f 104 species screened for m e s o p h y l l pro top~as t i so l a t i on , 68 d id not y ie ld in t ac t p r o t o p l a s t s (annext,.re). F o r t he r ema in ing 36 species, several p a r a m e t e r s

such as o smot i c c o n c e n t r a t i o n o f so rb i to l , enzyme c o n c e n t r a t i o n , p H o p t i m u m

a n d i n c u b a t i o n t ime were d e t e r m i n e d for the i nd iv idua l species ( tab le 1). T h e

36 species y i e l d i n g in tac t p r o t o p l a s t s inc luded 22 Ca species, 11 C 4 species a n d

Table 1. List of plant species which yielded intact mesophyll protoplasts.

Plant species

Cone. of enzymes (%) Cone. of pH of the Incubation Yield of Average sorbitol/ Maeero- Cellulase enzyme time (hr) intact size mannitol zyme medium protoplast (t~m)

(M) (%)

1 2 3 4 5 6 7 8

C3 species Capsicum

frutescens L. 0" 7 0" 2 1- 0 5" 7 3 30 45 Catheranthus

roseus L. 0" 6 0" 5 2' 0 5" 8 3 35 46 Cucumis mela L. 0" 6 0.5 1.0 5.8 3 40 42 C. sativus L. 0"6 0.5 1.0 5.8 2 42 40 Datura metal L. 0.7 0.2 2"0 5.7 3 45 38 HelianthusannusL 0"5 0"2 2-0 5"8 5 45 42 Impatiens

balsamina L. 0" 6 0" 2 1.0 5" 8 2 50 48 L ycopersicon

lycopersicum (L.) Karst. 0"6 0"2 2"0 5"7 3 35 42

Luffa acutangula (L.) Roxb. 0-6 0"2 1-0 5-5 4 35 40

Mollugo oppositifolia L. 0-8 0"5 2"0 5"8 4 55 42

M. pentaphylla L. 0"8 0"5 2-0 5-8 5 35 40 Nicotiana

tabacum L. 0" 8 0.5 2" 0 5" 8 5 60 38

Ocimum basilicum L. 0"6 0" 5 1 "0 5" 8 3 45 36

Oryza sativa L. 0" 5 2" 0 3" 0 5" 5 5 25 37 Pedalium murex L. 0-5 0-2 2"0 5"5 3 20 38 Petunia hybrida

Hort. 0" 8 0" 5 3" 0 5" 8 5 40 42 Physalis minima L. 0.7 0.5 2"0 5" 8 4 35 40 Pisum sativum L. 0.5 2"0 3.0 5.5 3 30 38 Solanum nigrum L. 0"6 0"2 2"0 5"8 3 38 42 Trichosanthes

bracteata (Lamk.) Voigt. 0"5 0.5 2.0 5.5 4 35 40

Isolation o f intact mesophyll protoplasts

Table 1--(Contd.)

83

1 2 3 4 5 6 7 8

C 4 species

Tridax procumbens L.

Triticum aestivum L.

,Cynodon dactylon (L.) Pers. 0.5 0 .2 2" 0 5" 5 4 25 24

Eleusine coracana 0" 5 0" 2 2" 0 5" 5 5 40 28 Euphorbia hirta L. 0"6 0"5 2"0 5"7 3 38 30 MoUugo nudicaulis

Lain. 0" 8 0 .5 2" 0 6 .0 5 60 34 Pennisetum typhoi-

des (Burm) 0" 6 0" 5 2" 0 5.5 5 50 24 Portulaca grandi-

flora Lindl. 0 .45 0"5 2 .0 5 .8 4 20 28 P. oleracea L. 0"45 0 .5 2"0 5 .8 4 15 26 Saccharum

officinarum L. 0-7 1"0 4"0 5"5 5 30 24 Sorghum vulgare L. 0- 6 0 .5 2' 0 5 .5 4 30 24 Trianthema

portulacastrum L. 0 .6 0 .5 1.0 6 .0 5 35 28 Zea mays L. 0 .6 0.1 2"0 5.5 5 35 26

0 .7 0 .5 2 .0 6 .0 5 55 48

0 .5 0 .2 2 .0 5.5 5 45 37

C A M species

Kalanchoe flori- bunda W. & A. 0 .4 0"2 1 .0 5"8 3 40 75

Notonia grandi. flora DC 0"33 0"2 1"0 5 .8 2 85 100

Aloe sp. 0 .55 0" 2 1- 0 5" 5 3 30 95

3 CAM species and the yield was between 15-85~, the average size being in the range of 24--100/z. The isolated mesophyll protoplasts were morphologically intact and spherical in shape. The photomicrographs for some species are given in figures 1-4. The room temperature of 30~ was sufficient for the enzyme digestion of the leaf tissues of all the species. The incubation time ranged between 1-5 hr.

The metabolic competence of the isolated intact protoplasts was checked in certain species and the protoplasts were photosynthetically active as they showed Comparable rates (table 2). The rate of COz fixation in 6 species ranged between 5-60/J reel mg ehl -x h -x without the addition of any phosphorylated compound. ~3-onerally, the rate of CO z fixation was low in the mesophyll protoplasts of C 4


species and with the addition of various substrates, tl-~e rate increased n'any-fold (data not shown). The light-dependent O3 evolulion rate ~as in ll'_e rans o f 15-60/t mol mg ch1-1 h -1.

The intactness of the isolated protopla sts was further cl~.ecked by ff~eir photochemical activity with Hill oxidants, ferricyanide and dichlorophenolindophenol (table 3). While ferricyanide was a very poor Hill oxidant, dichlorophenolindophenol was efficient in the Hill reaction and the spectrophotometric assay for photoreduction of this dye with the mesophyll protoplasts of 6 species was between 64-180/z mol mg oh1-1 h -1.

The photosynthetic stability of protoplasts after isolation was checked with 3 species for COn fixing capacity by withdrawing at 2 hr intervals, aliquots of protoplasts stored in the washing medium in dark at 4 ~ C after isolation. The mesophyll protoplasts of Notonia grandiflora, Nicotiana tabacum and Mollugo nud icaulis retained 90~ of their original activity for 5 hr, 9 hr and 12 hr respectively after isolation.

4. Discussion

Successful enzymatic isolation of mesophyll protoplasts from many C8 species and mesophyll protoplasts and bundle sheath strands from a number of C 4 species have been previously described (Otsuki and Takebe 1969; Power and Cooking 1970 ; Evans et al 1972; Kanai and Edwards 1973a, b; Ku et al 1974; Gutierrez et al 1974; Huber and Edwards 1975 ; Okuno and Furusawa 1977). The present study aims at adding to the list a number of commonly available herbacious species which yield intact mesopkyll protoplasts, and not reported earlier. Tile species were selected mainly on the basis of their easy availability. We have also successfully isolated the mesophyll protoplasts and bundle sheath strands from the leaves of C 4 species such as Mollugo nudicau!is, Euphorbia hirta, Sorghum vulgate, Pennisetum typhoides and Eleusine coracana nat reported earlier. The results of our study do not necessarily represent the best results for individual species but might be useful to develop procedures to many other species.

The yield of intact mesophyll protoplasts depends on the age and the physiological state of plants (Takebe et al 1968 ; Otsuki and Takebe 1969) and better results than ours could be obtained if materials of proper age and physiological state are used. However, since factors like concentration of the osmoticum and the enzymes, pH of the enzyme medium and the incubation time vary with individoal species, individual species should be studied in detail to obtain maximum yield of intact protoplasts. Besides macerozyme and Onozuka R-10 used in our study, several other cell wall degrading enzymes under the commercial names meicellase, hemicellulase, rhozyme, rohament P, driceUase and different commercial grades of Onozuka are also used in protoplast isolation. Use of these enzymes in different combinations may improve the yield of protoplasts in several species. Pro- longed incubation of the leaf tissue in the enzyme medium was found to enhance the yield of protoplasts in many species. However, longer incubation invariably led to the loss of phtosynthetic function in the species studied. Also, species like Dolichos lab lab, D. biflorus, Phaseolus mungo, Thunbergia grandiflora, Arachis hypogaea, Canna indica, and several species of lpomoea which readily yielded intact

Isolation of intact mesophyll protoplasts 85

Figures 1-4. Th~-iso|ated intact mesophyll protoplasts. 1. Mollugo oppositifolia (• 600), 2. M. "nudlcaulis (X 675). 3, Sorghum vulgate ( • 300). 4. Notonia grandbqora (X 450),

Isolation o f intact mesophyll protoplasts 87

Table 2. Photosynthetic rates of intact mesophyll protoplasts of higher plants (data are the average of three independent measurements).

Plant species CO2 fixation O2 evolution t* moles CO2 t~ moles 02

flexed evolved mg ch1-1 h -i mg oh1-1 h -1

Nicotiana tabaeum 60 35 Mollugo nudicaulis 15 25 M. oppostitifolia 50 30 Impatiens balsamina 40 24 Sorghum vulgate 5 60 Notonia grandiflora 55 32

Table 3. Photochemical activity of intact mesophyll protoplasts of higher plants (data are the average of three independent measurements).

Plant species (tz reel reduced mg oh1 -x h -1)

ferricyanide dichlorophenolindophenol

Nicotiana tabacum 4 98 Mollugo nudicaulis 0 64 M. oppositifolia 5 120 Impatiens balsamina 0 70 Sorghum vulgate 0 180 Notonia grandiflora 4 76

The Hill oxidants used were potassium ferricyanide (1 mM) and dichlorophenolindophenol (0"25 raM).

mesophyU ceils upon mild mechanical grinding were not amenable to protoplast isolation even with higher concentrations o f enzyme and longer incubation. Simi- larly, species which yielded intact protoplasts did not yield intact cells by mechanical grinding. I t may be also noticed from table 1 tha t there is a marked differ- ence in the size o f the protoplasts of C3, C4 and CAM species. CAM [protoplasts are the largest in size (75-100 p), C3 protoplasts are medium sized (36-48/0 and C~ protoplasts are the smallest (24-30/~).

The isolated protoplasts were photosynthetically active with fairly high rates o f CO z fixation and 02 evolution. The intactness of the photosynthetic apparatus is evidenced by tile Hill reaction with Hill oxidants, ferricyanide and 6ichloro- phenolindopheaol. Ferricyanide was impermeable to intact protoplasts and hence


there was little photoreduction of this dye with the isolated protoplasts. Ferri- cyanide-dependent 02 evolution has been used as a measure of intactness of chloroplasts and this dye had no stimulation of Oz evolution when the chloroplasts were intact (Lilley et al 1975; Rathnam and Edwards 1976). However, Rajendrudu et al (1979) have reported high Hill activities with ferricyanide with the intact mesophyll ceils of certain plants. The Hill oxidant, dichlorophenolindophenol could easily penetrate into the protoplasts and show significant rates of photoreduction (table 3). The present results on the Hill reaction by the mesophyll protoplasts with ferricyanide and dichlorophenolindophenol as oxidants are consistent with those of Ku et al (1974) who demonstrated that ferricyanide was relatively ineffective as electron accepter for the Hill reaction by the intact mesophyll protoplasts of several other C3 and C 4 species. They also showed that dichlorophenolindophenol and p-benzoquinone induced much higher rates of Hill reaction with the mesophyll protoplasts indicating their easy penetration into tl~e protoplasts.

The mesophyll protoplasts of our preparations could maintain most of their original photosynthetic activity up to 12 hr after isolation, when kept stored in dark at 4 ~ C. With their photosynthetic stability, mesophyll protoplasts serve as a useful tool for isolating functional chloroplasts for physiological and biochemical studies (Rathnam and Edwards 1976).

Acknowledgements

The financial support to KPC from University Grants Commission in the form of a fellowship is gratefully acknowledged.

ANNEXURE

List of plant species which did not yieM intact mesophyll protoplasts.

Abelmoschus esculentus (L.) Moenich; Abutilon indicum (L.) Sweet; Aerva lanata (L.C.) Juss ; Allamania nodiflora R.Br. ; Amaranthus tricolor L. (C4) ; Arachis hypogaea W~lld. ; Basella alba L. ; Barleria cristata L. ; Borreria articularis (L.F.) F. N. Will ; Cajanus cajan (L.) ; Canna indica L. ; Cayratia moUissima Gangnep ; Cassia occidentalis L. ; Celosia argentea L. ; Centella asiatica (L.) Urban ; Citrul- lus vulgaris Schrad; Clerodendron inerme Gaertn. ; Clitoria ternatea L. ; Comme- lina bengalensis L. ; Crotalaria juncea L. ; C. laburnifoBa L. ; C. verrucosa L. ; Croton sparciflorus L. ; Cyperus rotundus L. (C4); Digerla muricata (L.) Mart; Dolichos biflorus L. ; D. lab lab L. ; Eichhornia crassipes (Mart.) Solms. ; Evolvulus alsinoides (L.) L. ; Gomphrena decumbens Jacq. (C4) ; G. globosa L. (C4) ; Helio- tropium indicum L.; H. scabrum Retz. (Ca); Ipomoea batatas (L.) Lamk. ; L carnea Jacq. ; I. longiflora R. Br. ; I. pes caprae (L.) S~,eet ; I. quamoclit L. ; Yusticia procumbens L. ; Lagenaria vulgaris Ser. ; Leueas aspera (WiUd.) Spreng; Maranta arundinacea L. ; Merremia emarginata (Burro. f.); Mollugo cerviana (L.) Ser. ; M. lotoides L. ; Momordica charantia L. ; Musa paradisiaca L. ; Ocimum sanctum L. ; Oldenlandia corymbosa L. ; O. herbacea L ; Passiflora edulis Sims ; Pavonia zeylanica Cav. ; Piper betle L. ; Phaseolus mungo L. ; P. radiatus L. ; Phyla nodiflora (L.) Greene. ; Polycarpaea corymbosa (L.) Lamk. (C4); Polygala bulbothrix Dunn. ; Sida cordifolia L. ; Solanum melongena L. ; Tagetus erectus L. ;

Isolation o f intact mesophyll protoplasts

Thunbergia grandiflora (Roxb . ex Ro t t l . ) R o x b . ; Turnera ulmifofia var. elegans L. ; Urena lobata L. ; elegans Jacq . ; Zornia diphylla (L.) Pers .

89

Tribulus terrestris L. (C4) ;

Waltheria indica L. ; Zinnia

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isolation of intact mesophyll protoplasts from the leaves

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