Changes in esterase and superoxide dismutase isozymes during in vitro morphogenesis in Plantago ovata Forssk

Plant Cell, l~ssueand Organ Culture 44: 123-127, 1996. © 1996 KluwerAcademic Publishers. Printed in the Netherlands.

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Changes in esterase and superoxide dismutase isozymes during in vitro morphogenesis in Plantago ovata Forssk S. Pramanik, SarmisthaSen Raychaudhuri& SubhraChakraborty Department of Biophysics, Molecular Biology & Genetics, University College of Science, University of Calcutta, 92, A. P.. C. Road, Calcutta 700 009, India Received 16 November 1994;acceptedin revisedform26 October1995 Key words: Callus, esterase, in vitro regeneration, isozyme, Plantago ovata, shoot tip multiplication, superoxide dismutase

Abstract

Callus cultures were established from hypocotyl explants of Plantago ovata in Murashige and Skoog's medium supplemented with 2,4-D/Kinetin and NAA/BA combinations. Calluses growing on NAA/BA (0.4 mg 1-I each) regenerated into plantlets after the second subculture when transferred to media containing IAA (0.2 mg 1- l ) and BA (5 mg 1-1). Shoot tip multiplication was carried out in the same media with IAA and BA. Tissue samples from calluses, regenerating plantlets and multiplying shoot tips grown in vitro were extracted with protein extraction buffer and subjected to esterase and superoxide dismutase isozyme analysis. The calluses however, showed a uniform banding in esterase even when grown on different hormone combinations. The multiplying shootlets showed two new bands which were not found in either the control or the regenerating plants. A new band was also found in the multiplying shootlets when analysed for superoxide dismutase. It is postulated that those new enzyme forms which arise in esterase as well as in superoxide dismutase may either arise de novo or due to post-transcriptional modification of the genes and are essential for shoot tip multiplication of Plantago ovata. Abbreviations: 2,4-D- 2,4-dichlorophenoxyacetic acid; Kinetin- 6-furfuryl amino purine; N A A - naphthalene acetic acid; B A - 6-benzyl amino purine; IAA-indole-3-acetic acid Introduction

Polyacrylamide gel electrophoresis of isozymes of plant tissues is a very useful biochemical index reflecting changes in metabolic activity during growth, development and differentiation. During tissue culture, a series of metabolic changes take place affecting various enzyme systems leading to callus formation and differentiation into root and shoot. This also happens during multiplication of shoot tips. An attempt at determining such changes during in vitro studies of Plantago ovata Forssk was undertaken using esterase (EC 3.1.1.1; EST) and superoxide- dismutase (EC 1.15.1.1; SOD) isozyme banding patterns. Esterase includes a group of enzymes consisting of a host of ester hydrolases. Cubadda & Quattrucci (1974) reported that esterase isozymes that act upon c~ and

/~-naphthyl acetate are carboxyl esterases. Superoxide dismutase comprises of some metal conjugating proteins whose function is to remove superoxide radicals (0 2). Since SODs protect cells from oxidative damage they are known as 'antioxidant enzymes' (Scandalios, 1969; Tanksley, 1983). Plantago ovata is an important medicinal plant. Husks of the seeds of this plant are used as laxative. In an earlier paper by our group (Pramanik et al., 1994) nuclear DNA content and chromosome variation has been studied in relation to plant regeneration. In the present paper plant regeneration and shoot tip multiplication is being correlated with esterase and superoxide dismutase isozymes as biochemical markers.

124 Materials and methods

Callus cultures were established from hypocotyl (segment of 4-6 mm in length between root and shoot) of aseptically grown seedlings (10 days old) of Plantago ovata Forssk. To Murashige & Skoog's (1962) medium, various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, a-naphthalene acetic acid (NAA) and 6benzylaminopurine (BA) were added in different combinations. The pH of the media was adjusted to 5.8, solidified with 0.8% (w/v) agar agar (Difco) and sterilized for 15 min at 1.50 kg cm -2 pressure. The cultures were grown at 2 2 - 2 5 ° C with a relative humidity of 55-60% and under 32 x 108 mol s -1 m -2 for 16/8 h light/dark period. The calluses were subsequently maintained on the same medium by subculturing every 4 weeks. For shoot tip multiplication, shoot tips were inoculated onto MS medium supplemented with different concentrations of indole acetic acid (IAA) and BA. The pH of the media was adjusted to 5.8, solidified with 0.8% (w/v) agar agar, sterilized for 15 min at same pressure and maintained under the same external environment (Pramanik et al., 1994). For isoenzyme analysis, tissue samples (Calluses, Regenerated shootlets and Multiplied shoot tips) were weighed and homogenized in ice-cold protein extraction buffer containing 0.1 (M) Tris-HCl, pH 6.8, 0.25 (M) sucrose, 1.0% PVP, 1.0 mM DTT, 0.1% ascorbic acid, 0.1% cysteine hydrochloride, 1.0 mM EDTA, 1.0 mM MgC12 at pH 6.8 in a cold room at 4 °C. The homogenates were centrifuged in a Sorvall ultracentrifuge using 50.1 S rotor at 85,000 g for one hour at 4 °C. The supernatants were decanted and the volumes adjusted to obtain equal protein amounts following Lowry et al., (1951) and preserved at - 7 0 °C for isoenzyme analysis. Vertical polyacrylamide gel electrophoresis was performed in 16 cm x 14 cm slab gels of 1.5 mm thickness in a vertical gel electrophoresis unit. Approximately 150 I.tg of protein was loaded onto each well of the gel. The electrode buffer was Tris-glycine at pH 8.3. The gel electrophoresis was performed at 4 °C for 4 h at 35 mA at constant current provided from a LKB power supply unit. Esterase activity was localized on the gels according to Shaw & Prasad (1970) with minor modifications. 40 mg of a-naphthyl acetate was dissolved in 1 ml of acetone and the volume was made up to 100 ml with 100 mM phosphate buffer (pH 6.0). Then 76 mg of

Fast Blue RR salt was dissolved by vigorous stirring. Gels were incubated at 35 °C for 20 min, then washed with distilled water. Brown bands of esterase appeared almost immediately on a clear background. Superoxide dismutase activity was localized on the gels according to Geburek & Wang (1990) with minor modifications. The gels were immersed in 40 ml 0.2 (M) Tris-HC1, pH 8.0, 5 mM EDTA buffer containing 0.2 ml of 0.5 (M) MgC12, 1.0 ml of 1% aqueous nitro blue tetrazolium and 0.5 ml of 1% aqueous phenazonium methosulphate. The gels were exposed to strong fluorescent light for 15 min followed by one hour incubation in the dark. Superoxide dismutase appeared as light bands (negatively stained) on a dark blue background.

Results and discussion

Soft, friable calluses were obtained from 0.5 mg 1-1 each of 2,4-D and kinetin and 0.4 mg 1-1 each of NAA and BA (Pramanik et al., 1994). Shoot buds were differentiated from the latter when transferred to media with 0.2 mg 1-1 of IAA and 5 mg 1-1 of BA (Pramanik et al., 1994). Clusters of 2-4 shoots were excised from the callus and rooted in hormone free basal medium. The morphology and chromosome number of these regenerants were the same as that of the control (normal seedlings). This study was carded out with a view to determine isozyme changes in spite of constancy of chromosome number in regenerants and control seedlings. The ideal medium for shoot tip multiplication was MS medium containing 0.2 mg 1-l of IAA and 5 mg 1-l of BA (Pramanik et al., 1994 ). Shoot organogenesis was measured as number of shoots regenerated from each explant of 4-6 mm in length. Each experiments were repeated ten times. Esterase

Esterase isozyme banding patterns were photographed (Fig. la). The control set (A) showed one faint band at the top, one dark band at the middle and a very faint band at the lower part. This banding pattern of the control set was compared with the tissue grown in vitro. The callus tissue grown on 2,4-D/Kin (0.5 mg 1- l each) from subculture 1 and subculture 2 (Lane B and C in Fig. la) and calluses grooving vigorously on NAA/BA subculture 2 (Lane D in Fig. la) showed the same banding patterns. These calluses revealed

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Fig. 1. Isozyme banding patterns ofF,st (a) and SOD (b) in calluses, regenerating plants and multiplied shoot tips of P. ovata in vitro [Lane A -

Control, B - 2,4 D/Kin callus (subculture 1), C - 2,4 D/Kin callus (subculture 2), D - NAA/BA callus (Subculture 2), E - Regenerated plant in IAA/BA, F - Multiplied shootiets on IAA/BA]. Very dark II, Moderately dark III, Faint III.

two bands in the middle part o f Rf values 0.37 and 0.43 respectively. The upper band was thick and dark, while the lower band was faint. The plant regenerated in I A A / B A (E), like the control set showed a band at the top, two bands at the middle. But the faint band present in the control set at the lower region (Rf value 0.75) was absent in the regenerants. The next lane in the Fig. l a was that o f rapidly multiplying shootlets; (F). Interestingly, the bands in this lane were similar to that o f the control but two new bands with Rf values 0.50 and 0.53 appeared. It seems that these two

new bands marked the appearance of two new isozyme types during shootlet micropropagation. Reports on esterase isozyme p o l y m o r p h i s m in relation to in vitro regeneration and morphogenesis are lacking in Plantago. In a previous paper o f our group (Pramanik et al., 1994) esterase p o l y m o r p h i s m was reported in different species o f P l a n t a g o in vivo. It was found that esterase banding pattern could be used as a biochemical marker for the identification o f species in Plantago.

126 Esterase isozymes in calluses of Plantago ovata growing on different hormone combinations showed similar banding patterns. This has also been reported by Nehra et al. (1991) in esterase patterns for callus tissue of strawberry. A similar record has been noted by Mangolin et al. (1994) in Cereus peruvianus. The regenerating plants of P ovata showed the same pattern as that of the control but excepting the lower band. This may be due to inactivation of genes involved in the synthesis of these isozymes during regeneration. Shootlets multiplied on IAA/BA showed two new bands which were absent either from the control or regenerating plants. It seems that during shootlet multiplication these two new molecular forms of the enzyme are essential and may indicate de novo synthesis of genes during multiplication process. Superoxide dismutase

Superoxide dismutase isozyme banding patterns are shown in Fig. lb. Depending on the Rf values the bands have been classified as follows: (a) Rf values ranging from 0 to 0.30 represent SOD-l, (b) Rf values from 0.30 to 0.50 represent SOD-2 and (c) Rf values from 0.50 to 1 indicate SOD-3. The control set (A) showed three bands of which the one at the lower part was very thick (Rf value 0.58). The next three lanes (B, C and D respectively) represent calluses growing on 2,4-D/Kin (0.5 mg 1-1 each) (B-initiation of callus and C-vigorous growth of the callus) and calluses growing vigorously NAA/BA (D). All these calluses showed a common band at the upper zone with Re value 0.27. Calluses growing on 2,4-D/Kin of subculture 1 (Lane B in Fig. lb) showed a single band in the middle zone of Rf value 0.48. In addition to this band as in (B), another band of Rf value 0.59 appeared at the lower part in C (calluses on 2,4-D/Kin (subculture 2). In the next lane (D) calluses growing vigorously on NAA/BA (Subculture 2) showed only one band with Rf value 0.27. Plantlets regenerated on IAA/BA (E) showed one band of Rf value 0.27 in the upper part. The multiplying shootlets (F) showed four bands - one at the upper region of Rf value 0.09, one at the middle (Rf value 0.30) and two at the lower region (Rf values 0.46 and 0.50). However, the band of Rf value 0.46 is unique to the multiplying shootlets only. This new band represent a new molecular form of SOD which is essential for shootlet multiplication. Perl-Treves & Galun (1991) have reported that SOD genes of tomato are developmentally regulated and respond to light and stress. Shoot tips, flower buds,

seedlings and young leaves of tomato contained high levels of mRNA transcripts of SOD. Developmental control of Cu/Zn SOD has also been recorded by Herouart et al. (1994) in transgenic tobacco. According to White et al. (1990) Cu/Zn SOD mRNA levels remain constant, but SOD 3 (Mn-SOD) mRNA level increases throughout postgerminative scutellar development. In maize total SOD activity increases in kernel extracts after 10 days post pollination. SOD 3 and Cu/Zn SOD activity increase during kernel development. According to these authors (White et al., 1990) there is a post transcriptional event that regulates the transcriptional output or possibly the half life of the SOD protein. In our investigation the new molecular form of SOD that appears in multiplying shoot tips is developmentally regulated and may arise due to de novo synthesis or may represent post transcriptional modification of genes.

Acknowledgements The authors are grateful to Professors R. K. Poddar, B. B. Biswas, C. K. Dasgupta and U. Chaudhuri of the Department of BMB, C.U. for facilities. They are also grateful to Prof. S. C. Roy and Dr. T. K. Bera of the Department of Botany, C. U. for important suggestions. The authors are thankful to CSIR for financial help.

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