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Changes in Indoleacetic Acid Oxidase and Peroxidase Activities During Cotton Fibre Development
Changes in Indoleacetic Acid Oxidase and Peroxidase Activities During Cotton Fibre Development N. RAMA RAO, S. C. NAITHANI, R. T. JASDANWALA and Y. D. SINGH Department of Biosciences, Saurashtra University, Rajkot - 360005, India Received August 5, 1981 . Accepted March 3, 1982
Summary Growth analysis, IAA oxidase and both cytoplasmic and ionic ally bound wall peroxidase activities were investigated during the entire period of cotton fibre development. IAA oxidase and peroxidase activities recorded low levels during the elongation phase while during the secondary thickening phase these activities increased significantly. It is suggested that IAA oxidase in vivo may regulate the levels of IAA. The close correlation between cessation of elongation growth and increase in wall peroxidase points to an important role of this enzyme in the termination of the elongation phase. Its role as a wall rigidifying factor is discussed. Electrophoresis data supported the view that both IAA oxidase and peroxidase activities are associated with a single protein molecule.
Key words: Gossypium barbadense, cotton fibre, peroxidase, fAA oxidase, wall rigidifying/actor.
Introduction Cotton lint fibres are initiated from epidermal cells of the seed coat from a day before to a day after anthesis (Lang, 1938; Joshi et aI., 1967). The growth of the fibre involves cell elongation and secondary wall thickening (Flint, 1950). It has been shown (Hawkins and Serviss, 1930) that the initial period of fibre growth involves elongation and that the secondary wall thickening does not start till the elongation phase is completed. However, the mechanisms regulating the development of cotton fibre are poorly understood. Importance of auxin in cotton fibre development has been demonstrated (Beasley, 1973; Beasley and Ting, 1974; Beasley et aI., 1974) and it has also been suggested Qasdanwala et aI., 1977, 1980) that auxin levels may play an important role in the termination of the elongation phase. Levels of IAA in plants are regulated via synthesis, binding, derivatisation and enzymic degradation, which is catalysed by a complex of enzymes termed the IAA oxidase system. The major constituent of IAA oxidising system is peroxidase, an enzyme which is capable of utilizing hydrogen peroxide to oxidise a wide variety of hydrogen donors including phenolic compounds and IAA (Saunders et aI., 1964). Further, this enzyme is often found in significant association with cell wall and particulate fractions of plant tissues (Fielding and Hall, 1978; Choy et aI., 1979; Mader et aI., 1980). Despite the fact that many researches have been devoted to the characterization of peroxidase activity, its function still remains obscure. Its activity has been Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
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implicated in hydroxylation of proline (Ridge and Osborne, 1971), lignification (Helper et aI., 1972; Siegel, 1955) and IAA oxidation (Galston and Davies, 1969). Therefore, in the present investigation, we have assayed IAA oxidase and peroxidase activities during the entire period of cotton fibre development, with a view to examine their role in the fibre development.
Material and Methods Cotton (Gossypium barbadense L. Cv. ERB 4492) plants were grown in the field. The cultural practices including irrigation, fertilizer, insecticides etc., were conducted to maximize the lint yield. On the day of anthesis, each individual flower was tagged and the bolls were harvested for analysis after the required periods.
Fibre length and dry weight measurements Fibre length was determined by the method of Gipson and Ray (1969). A locule from a boll was placed in boiling water to allow the seeds to separate from each other and each such separated seed was placed on the convex side of a watch glass and the fibre was streamed out with a jet of water. The length of the fibre was measured from the rounded side of the seed adjacent to the chalazal end. All the seeds from the three locules were measured from three bolls and an average was calculated for the fibre length. The fibre was removed from the seed with a scalpel without removing the seed coat and the dry weight was determined after drying in an oven at 80°C for 2 days. Each result represents an average of three bolls harvested randomly at a given age. The data (plotted versus boll age) were fitted to an appropriate curve by computer curvilinear regression analysis and the rate curves were obtained by differentiating the best fit equation (Schubert et aI., 1973).
Preparation 0/enzyme extract Freshly harvested bolls were opened with a sharp knife and hairs were immediately separated from the seed and frozen. The frozen material was crushed in a cooled mortar, with borate buffer (0.2 M pH 7.6) as recommended by King (1971) to yield maximum proteins and centrifuged. The crude extract was mixed with chilled acetone (1: 2 v/v) at O°C to precipitate soluble proteins. The precipitate was separated by centrifuging in the cold at 15,000 g and stored at -5°C. Till 5 days of postanthesis, it was difficult to separate fibres from the seed. Hence, young ovules were taken for analysis, while at 8 day and in subsequent periods only fibres were used. The acetone-precipitated proteins were dissolved in 0.02M phosphate buffer (pH 6.4) and the purified extract was used for measurement of IAA oxidase and cytoplasmic peroxidase activities.
fAA oxidase assay IAA oxidase activity was determined by the modified method of Gordon and Weber (1951). The reaction mixture (5 ml) consisting of 1 ml extract, 198 J.1g MnCh, 162 J.1g 2-4 dichlorophenol, 200 J.1g IAA and 1 ml 0.02 M phosphate buffer (pH 6.4), was incubated for 30 min at 28 ± 2 °C in dark. After incubation, 2 ml of the mixture was added to 4 ml of Salkowski reagent and the colour was allowed to develop for 20 min. The absorbance of the {'ink solution was measured at 530 nm. The enzyme activity is expressed as mg IAA oxidised h - mg -I protein.
Cytoplasmic peroxidase activity The activity of cytoplasmic peroxidase was measured by recording the change in absorbance at 470nm (~~70) to the oxidation of guaiacol in the presence of H202. The activity is expressed as ~~70 min- I mg- I protein.
Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
IAA oxidase and peroxidase activities in cotton fibre
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Wall peroxidase activity (ionically bound) The wall fraction was separated from the initial borate buffer crushing by centrifuging the homogenate at 1,500 g and washed thrice or till the washing was free of peroxidase reaction with guaiacol. The wall pellet was then resuspended with 5 ml of NaCI (1 M) solution (Ridge and Osborne, 1970) for 1 h and centrifuged at 12,000 g for 10 min. The supernatant served as the source of wall peroxidase. The activity of wall peroxidase was measured as described in cytoplasmic peroxidase. The activity is expressed as ~~70 min-I mg- I protein. The protein content of the extract was estimated using Folin reagent (Lowry et al., 1951). Three replicates were taken for biochemical analysis and the mean values with standard deviation are presented.
Gel electrophoresis Electrophoretic separations of purified cytoplasmic peroxidase and IAA oxidase were performed using the system of Ornstein and Davis (1962), using 7.5 % polyacrylamide gel (in cold). The applied current was 2 m Amp per tube for 10 min, followed by 4 Amp per tube until the dye marker (0.01 % bromophenol blue) had reached the ends of the gels. Ten samples from the entire period of cotton fibre development were run simultaneously. Peroxidase isoenzymes were detected with benzidine (Sahulka, 1970) while IAA oxidase isoenzymes were visualized by the method of Hoyle (1977). The results of electrophoretic separation have been presented schematically with mobilities calculated as the relative distance traversed by the band with respect to the distance travelled by bromophenol dye.
Results
The data for fibre length and dry weight versus boll age X (days) were fitted to a best-fit polynomial with the equation: Y
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Fig. 1 A: Best-fit curves for fibre length and dry ~ weight per seed against boll age. The regression u: 1.50 coefficients are 0.998, 0.979, and the multiple ~ correlation coefficients 0.998 and 0.989 for fibre ~ 0.75 length and dry weight respectively. - B: The 0: rate curves for fibre elongation and dry weight. Fibre length, continuous lines: fibre dry weight, broken lines.
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Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
160
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N. RAMA RAo, S.
NAITHANI,
R. T. ]ASDANWALA and Y. D. SINGH
The best-fit curve for fibre length (Fig. 1 A) showed that the elongation starts 3 days after anthesis and continues up to 30 days. No significant increase was discernible in the subsequent period. The dry weight on the other hand showed a lag phase up to 20 days after which it increased exponentially till 43 days. The differential curve (Fig. 1 B) for fibre length indicated that fibre elongation stopped at 33 days after anthesis. The rate of fibre elongation was highest at 10 days. Dry weight increase was initiated around 15 days and maximum rate occurred at 30 to 40 days after anthesis or about the time elongation ceased. Low IAA oxidase activity was recorded on the day of anthesis while at day 3 it increased appreciably. Up to 10 days postanthesis a fall in its activity was discernible and it is interesting to note that the low level of IAA oxidase activity corresponded with maximum rate of fibre elongation (Fig. 1 Band 2). After day 10, IAA oxidase activity recorded an increasing trend, the increase being enormous after 25 days postanthesis. High level of activity was maintained till maturity (Fig. 2).
~~
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AFTER ANTHESIS
Fig. 2: IAA oxidase activity against boll age.
On the day of anthesis a very low level of cytoplasmic peroxidase activity was recorded. By day 5 it had increased significantly (Fig. 3 A). It was again low at 8 days postanthesis after which it showed a gradual rise up to 20 days, when a second peak was observed. The activity fell again at 25 days and recorded a sharp rise at maturity. Maximum cytoplasmic peroxidase was recorded in fully mature fibre. Substantial wall bound (ionically) peroxidase activity was observed which rose slightly at 3 days. Up to 15 days postanthesis it decreased, and the low level of this activity corresponded with maximum rate of fibre elongation (Fig. 1 Band 3 B). After 15 days wall peroxidase increased significantly and maximum activity was recorded on 40 days postanthesis. Again, it is interesting to note here that the increase in wall peroxidase corresponded with the rate of dry weight accumulation (Fig. 1 Band 3 B). In all, eleven isoenzymes of peroxidase and IAA oxidase were detected during the entire period of fibre development. A number of stage specific isoenzymes were detected e.g., band E4 which showed both IAA oxidase and peroxidase activity was present during elongation phase. Band E9 and EIO showed only peroxidase activity Z. Pjlanzenphysiol. Bd. 106. 5.157-165.1982.
IAA oxidase and peroxidase activities in cotton fibre
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o
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DAYS AFTER ANTHESIS
I·Or----------------. E I • 9 K:l K:l K:l K:l K:l K:l K:l K:l K:l K:l E2 c:::::J K:l c:::::J K:l K:l K:l K:l K:l K:l K:l
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'4 _ _ _ _
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DAYS AFTER
ANTHESIS
and were present during early differentiation stages. Isoenzyme E7 with exclusive peroxidase activity was present during differentiation and elongation phases. While, band E6 , a peroxidative, and band E 8 , with both peroxidative and IAA oxidative activity, were detected only during dry matter accumulation stages. Isoenzyme E3 , an exclusive IAA oxidase band, was also detected during dry matter accumulation stages (Fig. 4). Isoenzymes E 1, E2 and E5 were present throughout and showed both IAA oxidase and peroxidase activity. Z. Pjlanzenphysiol. Ed. 106. S. 157-165. 1982.
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Discussion From the growth data presented here, cotton fibre development can be divided into four distinct phases; (I) initiation (0-3 days), (II) elongation (5-20 days), (III) secondary thickening (25-40 days), and (IV) maturation (40-45 days). The differential curve (Fig. 1 B) for fibre elongation showed that a maximum rate of fibre elongation was achieved on 10 days after anthesis and it ceased completely on 33 days. Before the elongation was completed the secondary thickening phase started around 15 days after anthesis and the maximum rate was observed from 30 to 40 days postanthesis indicating clearly that these rather distinct phases are continuous processes which can not be delimited from one another. IAA oxidase activity recorded low levels during the elongation phase (Fig. 2) while during the secondary thickening and maturation phases it increased sharply, confirming thereby the earlier suggestion (Thimann, 1972) that this enzyme may regulate plant growth by limiting the concentration of IAA. In contrast to IAA oxidase, cytoplasmic peroxidase activity recorded significant level during elongation phase while, during secondary thickening and maturation phases it almost paralleled the trends of IAA oxidase (Fig. 2 and 3 A). Peroxidases have been implicated in IAA oxidation. However, whether oxidation of IAA by peroxidase is a general phenomenon, is not well understood. There are three hypotheses regarding the relationship between IAA oxidase and peroxidase, (i) peroxidase and IAA oxidase are different enzymes (Sequeira and Mineo, 1966; Rubery, 1972), (ii) peroxidase and IAA oxidase activities are associated with the same protein molecule but have different active sites (Siegel and Galston, 1967, Laurema, 1974; Srivastava and Van Huystee, 1977) and (iii) only a few of several existing peroxidase isoenzymes possess IAA oxidase activity (Stafford and Bravinder-Bree, 1972). Our electrophoretic studies showed that isoenzymes of peroxidase and IAA oxidase can be classified into three categories, (I) with exclusively IAA oxidase or peroxidase activity, (II) with both IAA oxidase and peroxidase activity, and (III) initially showed peroxidase activity and later IAA oxidase activity. Recent studies on etiolated pea have also revealed the existence of exclusive peroxidase and IAA oxidase isoenzymes and isoenzymes with both peroxidative and IAA oxidative activities (Brynt and Lane, 1979). It is therefore, logical to believe that both IAA oxidase and peroxidase activities are associated with the same protein and that at a given time, depending upon the allosteric modifier, either one or both active sites may function. Reports in this direction indicated the allosteric (homotropic) nature of IAA oxidase and peroxidase (Srivastava and Van Huystee, 1977; Raa, 1971). In our studies with different hydrogen donors (Unpublished data) it was observed that the patterns of changes in peroxidase level and isoenzymes in developing cotton fibre differed with different hydrogen donors. It is therefore possible that exclusive IAA oxidase isoenzymes reported in the present study may turn out to be a peroxidase with another substrate. Studies in this direction are in progress.
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IAA oxidase and peroxidase activities in cotton fibre
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Changes in wall bound peroxidase, in the present investigation, showed that during the elongation phase its levels were low while, during the secondary thickening phase it increased significantly (Fig. 3 B). Similar correlation between wall peroxidase and elongation growth has been reported by Ridge and Osborne (1971), Osborne et ai. (1972). It has been suggested that wall peroxidase may playa causative role in cessation of elongation growth (Gardiner and Cleland, 1974). Further, it has been pointed out by these authors that cell elongation may not be under the control of a single factor, but may be influenced by a series of factors including peroxidase induced lignification, auxin destruction and «extensin» induced wall stiffening. Since a similar correlation between the cell wall bound hydroxyproline containing protein «extensin» and growth has also been reported (Sadava et aI., 1973). Evidences have been presented to link ethylene induced growth inhibition to «extensin» synthesis (Sadava and Chrispeels, 1973). Lamport (1965) proposed that the cross-liking of «extensin» by means of disulphide bridges may restrict cell expansion. That peroxidase can oxidise -SH groups has also been demonstrated (Stonier and Yang, 1973). Thus, it is possible that the increased levels of wall peroxidase during the secondary thickening phase may oxidise -SH groups of «extensin» which in turn may restrict extension growth. However, little evidence supporting the -SH oxidation theory of «extensin» growth control has appeared in the last fifteen years since Lamport's suggestion, and a putative «extensin» precursor purified by Stuart and Varner (1980) contained no cysteine. Alternatively, Fry (1979) proposed that peroxidase stiffens the wall by catalysis of the oxidation of cell wall phenolic compounds to form the more hydrophobic biphenyls, polymers or quinones, any of which could protect wall polysaccharides against attack by wall glycanases (or transglycosylases). According to Fry (1980) under low wall peroxidase levels, phenols are maintained in their reduced state thus making the environment of the polymers less hydrophobic so that (i) covalently bound polysaccharides are rendered more susceptible to enzyme excision by existing hydro lases or transferases, (ii) hydrogen-bonded wall polysaccharides are freed owing to increased competition from water and (iii) growth is facilitated as a result of this loss of polysaccharides. Further, low wall peroxidase content could hinder the covalent cross linking of polysaccharides via diferuloyl bridges. Acknowledgements We are thankful to Professor S. C. Pandeya for providing the necessary facilities; Mr. C. S. R. Murti, Physical Research Laboratory, Ahmedabad for his generous help in statistical analysis; and Dr. M. S. Murthy for going through the manuscript. This work was supported by Univers· ity Grants Commission, New Delhi.
References BEASLEY,
C. A.: Hormonal regulation of growth in unfertilized cotton ovules. Science 179,
1003-1005 (1973).
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BEASLEY, C. A. and I. P. TING: Effects of plant growth substances on in vitro fibre development from unfertilized cotton ovules. Amer. J. Bot. 61, 188-194 (1974). BEASLEY, C. A., I. P. TING, A. E. LINKIN, E. H. BIRNBAUM, and D. P. DELMER: Cotton ovule culture: A review of progress and a review of potential. In: STREET, H. E. (Ed.): Tissue Culture and Plant Science, 169-192. Academic Press, London, New York, San Francisco, 1974. BRYNT, S. D. and F. LANE: Indole-3-acetic acid oxidase from peas. I. Occurrence and distribution of peroxidative and nonperoxidative forms. Plant Physiol. 63, 696-699 (1979). CHOY, Y., Y. WONG, K. LAU, and K. YUNG: Thermal activation of peroxidase from tobacco leaf mesophyll cell walls. Int. J. Pept. Protein Res. 14, 1-4 (1979). FIELDING, J. L. and J. L. HALL: A biochemical and cytochemical study of peroxidase activity in roots of Pisum sativum. II. Distribution of enzyme in relation to root development. J. Exp. Bot. 29, 983-991 (1978). FLINT, E. A.: The structure and development of the cotton fibre. Bot. Rev. 25, 414-434 (1950). FRY, S. c.: Phenolic components of the primary cell wall and their possible role in the hormonal regulation of growth. Planta 146, 343-351 (1979). - Gibberellin-controlled pectinic acid and protein secretion in growth cells. Phytochem. 19, 735-740 (1980). GALSTON, A. W. and P. J. DAVIES: Hormonal regulation in higher plants. Science 163, 1288-1297 (1969). GARDINER, M. G. and R. CLELAND: Peroxidase changes during the cessation of elongation in Pisum sativum stems. Phytochem. 11,1095-1098 (1974). GIPSON, J. R. and L. L. RAy: Fibre elongation rates in five varieties of cotton (Gossypium hir· sutum L.) as influenced by night temperatures. Crop Sci. 9, 339-341 (1969). GORDON, S. A. and R. P. WEBER: Colorimetric estimation of indoleacetic acid. Plant Physiol. 26,192-195 (1951). HAWKINS, R. S. and G. H. SERVISS: Development of cotton fibres in the Pima and Acala varieties. J. Agric. Res. 40,1017-1029 (1930). HELPER, P. K., R. M. RICE, and W. A. TERRANOVA: Cytochemical localization of peroxidase activity in wound vessel members of coleus. Can. J. Bot. 50, 977-983 (1972). HOYLE, M. c.: High resolution of peroxidase indoleacetic acid oxidase isoenzyme from horseradish by isoelectric focusing. Plant Physiol. 60, 787-793 (1977). JASDANWALA, R. T., Y. D. SINGH, and J. J. CHINOY: Auxin metabolism in developing cotton hairs. J. Exp. Bot. 28, 1111-1116 (1977). - - - Changes in components related to auxin turnover during cotton fibre development. Beitr. BioI. Pflanzen. 55, 23-36 (1980). JOSHI, P. c., A. M. WADHWANI, and B. M. ]OHRI: Morphological and embryological studies of Gossypium. Proc. Nat. Inst. Sci. India 388,37-93 (1967). KING, E. E.: Extraction of cotton leaf enzyme with borate. Phytochem. 10,2337-2341 (1971). LAMPORT, D. T. A.: The protein component of primary cell walls. In: PRESTON, R. D. (Ed.): Advances in Botanical Research 2, 151-218. Academic Press, New York, 1965. LANG, A. G.: The origin of lint and fuzz hairs of cotton. J. Agric. Res. 56, 507-521 (1938). LAUREMA, S.: Indoleacetic acid oxidases in resting cerial grains. Physiol. Plant. 30, 301-306 (1974). LOWRY, O. H., N. J. ROSE BROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193,265-275 (1951). MADER, M., J. UNGEMACH, and P. SCHLOB: The role of peroxidase isoenzyme groups of Nico· tiana tabacum in hydrogen peroxide formation. Planta 147, 467-470 (1980). ORNSTEIN, L. and B. J. DAVIS: Disc electrophoresis. Distillation Products Industries, Rochester, New Yark, 1962. OSBORNE, D. J., I. RIDGE, and J. A. SARGENT: Ethylene and the growth of plant cells. Role of per-
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oxidase and hydroxyproline-rich proteins. In: CARR, D. J. (Ed.): Plant Growth Substances 1970,534-542. Springer-Verlag, Berlin, Heidelberg, New York, 1972. RAA, J.: Indole-3-acetic acid levels and the role of indole-3-acetic acid oxidase in the normal root and club root of cabbage. Physiol. Plant. 25,130-134 (1971). RIDGE, I. and D. J. OSBORNE: Hydroxyproline and peroxidases in cell walls of Pisum sativum Regulation by ethylene. J. Exp. Bot. 21, 843-856 (1970). - - Role of peroxidase when hydroxyproline-rich protein in plant cell walls is increased by ethylene. Nature New BioI. 229, 205-209 (1971). RUBERY, P. H.: Studies in indole acetic acid oxidation by liquid medium from crown gall tissue culture cells; the role of malic acid and related compounds. Biochim. Biophys. Acta 261, 21-34 (1972). SADAVA, D. E. and M. J. CHiRSPEELS: Hydroxyproline-rich cell wall protein {ext ens in): Role in the cessation of elongation in excised pea epicotyls. Dev. BioI. 30,49-55 (1973). SADAVA, D. E., F. WALKER, and M. J. CHIRSPEELS: Hydroxyproline-rich cell wall protein (extensin): Biosynthesis and accumulation in growing pea epicotyls. Dev. BioI. 30, 42-48 (1973). SAHULKA, J.: Electrophoretic study on peroxidase, indoleacetic acid oxidase and o-diphenol oxidase fractions in extracts from different growth zones of Vicia faba L. roots. Biologia Plant. 12, 191-198 (1970). SAUNDERS, B. c., A. G. HOLMES-SIEDLE, and B. P. STARK: Peroxidase: the properties and uses of a versatile enzyme and of some related catalysts. Butterworths, London, 1964. SCHUBERT, A. M., C. R. BENEDICT, J. D. BERLIN, and R. J. KOHEL: Cotton fibre development. Kinetics of cell elongation and secondary wall thickening. Crop. Sci. 13, 704-709 (1973). SEQUEIRA, L. and L. MINEO: Partial purification and kinetics of indole acetic acid oxidase from tobacco roots. Plant Physiol. 41,1200-1208 (1966). SIEGEL, S. M.: The biochemistry of lignin formation. Physiol. Plant. 8, 30-32 (1955). SIEGEL, B. Z. and A. W. GALSTON: Indole-acetic acid oxidase activity of apoperoxidase. Science 157,1557-1559 (1967). SRIVASTAVA, O. P. and R. B. VAN HUYSTEE: IAA Oxidase and polyphenol oxidase activities of peanut peroxidase isoenzymes. Phytochem.16, 1527-1530 (1977). STAFFORD, H. A. and S. BRAVINDER-BREE: Peroxidase isoenzymes of first internodes of sorghum. Tissue and intracellular localization and multiple peaks of activity isolated by gel filtration chromatography. Plant Physiol. 49, 950-956 (1972). STONIER, T. and H. M. YANG: Studies on auxin protectors. XI. Inhibition of peroxidase - catalyzed oxidation of glutathione by auxin protectors and o-dihydroxyphenols. Plant Physiol. 51, 391-395 (1973). STUART, D. A. and J. E. VARNER: Purification and characterization of a salt-extractable hydroxyproline-rich glycoprotein from aerated carrot discs. Plant Physiol. 66,787-792 (1980). THIMANN, K. V.: The natural plant hormones. In: STEWARD, F. C. (Ed.): Plant Physiology A Treatise 6B, 3-365. Academic Press, New York, 1972.
Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
Summary Growth analysis, IAA oxidase and both cytoplasmic and ionic ally bound wall peroxidase activities were investigated during the entire period of cotton fibre development. IAA oxidase and peroxidase activities recorded low levels during the elongation phase while during the secondary thickening phase these activities increased significantly. It is suggested that IAA oxidase in vivo may regulate the levels of IAA. The close correlation between cessation of elongation growth and increase in wall peroxidase points to an important role of this enzyme in the termination of the elongation phase. Its role as a wall rigidifying factor is discussed. Electrophoresis data supported the view that both IAA oxidase and peroxidase activities are associated with a single protein molecule.
Key words: Gossypium barbadense, cotton fibre, peroxidase, fAA oxidase, wall rigidifying/actor.
Introduction Cotton lint fibres are initiated from epidermal cells of the seed coat from a day before to a day after anthesis (Lang, 1938; Joshi et aI., 1967). The growth of the fibre involves cell elongation and secondary wall thickening (Flint, 1950). It has been shown (Hawkins and Serviss, 1930) that the initial period of fibre growth involves elongation and that the secondary wall thickening does not start till the elongation phase is completed. However, the mechanisms regulating the development of cotton fibre are poorly understood. Importance of auxin in cotton fibre development has been demonstrated (Beasley, 1973; Beasley and Ting, 1974; Beasley et aI., 1974) and it has also been suggested Qasdanwala et aI., 1977, 1980) that auxin levels may play an important role in the termination of the elongation phase. Levels of IAA in plants are regulated via synthesis, binding, derivatisation and enzymic degradation, which is catalysed by a complex of enzymes termed the IAA oxidase system. The major constituent of IAA oxidising system is peroxidase, an enzyme which is capable of utilizing hydrogen peroxide to oxidise a wide variety of hydrogen donors including phenolic compounds and IAA (Saunders et aI., 1964). Further, this enzyme is often found in significant association with cell wall and particulate fractions of plant tissues (Fielding and Hall, 1978; Choy et aI., 1979; Mader et aI., 1980). Despite the fact that many researches have been devoted to the characterization of peroxidase activity, its function still remains obscure. Its activity has been Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
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implicated in hydroxylation of proline (Ridge and Osborne, 1971), lignification (Helper et aI., 1972; Siegel, 1955) and IAA oxidation (Galston and Davies, 1969). Therefore, in the present investigation, we have assayed IAA oxidase and peroxidase activities during the entire period of cotton fibre development, with a view to examine their role in the fibre development.
Material and Methods Cotton (Gossypium barbadense L. Cv. ERB 4492) plants were grown in the field. The cultural practices including irrigation, fertilizer, insecticides etc., were conducted to maximize the lint yield. On the day of anthesis, each individual flower was tagged and the bolls were harvested for analysis after the required periods.
Fibre length and dry weight measurements Fibre length was determined by the method of Gipson and Ray (1969). A locule from a boll was placed in boiling water to allow the seeds to separate from each other and each such separated seed was placed on the convex side of a watch glass and the fibre was streamed out with a jet of water. The length of the fibre was measured from the rounded side of the seed adjacent to the chalazal end. All the seeds from the three locules were measured from three bolls and an average was calculated for the fibre length. The fibre was removed from the seed with a scalpel without removing the seed coat and the dry weight was determined after drying in an oven at 80°C for 2 days. Each result represents an average of three bolls harvested randomly at a given age. The data (plotted versus boll age) were fitted to an appropriate curve by computer curvilinear regression analysis and the rate curves were obtained by differentiating the best fit equation (Schubert et aI., 1973).
Preparation 0/enzyme extract Freshly harvested bolls were opened with a sharp knife and hairs were immediately separated from the seed and frozen. The frozen material was crushed in a cooled mortar, with borate buffer (0.2 M pH 7.6) as recommended by King (1971) to yield maximum proteins and centrifuged. The crude extract was mixed with chilled acetone (1: 2 v/v) at O°C to precipitate soluble proteins. The precipitate was separated by centrifuging in the cold at 15,000 g and stored at -5°C. Till 5 days of postanthesis, it was difficult to separate fibres from the seed. Hence, young ovules were taken for analysis, while at 8 day and in subsequent periods only fibres were used. The acetone-precipitated proteins were dissolved in 0.02M phosphate buffer (pH 6.4) and the purified extract was used for measurement of IAA oxidase and cytoplasmic peroxidase activities.
fAA oxidase assay IAA oxidase activity was determined by the modified method of Gordon and Weber (1951). The reaction mixture (5 ml) consisting of 1 ml extract, 198 J.1g MnCh, 162 J.1g 2-4 dichlorophenol, 200 J.1g IAA and 1 ml 0.02 M phosphate buffer (pH 6.4), was incubated for 30 min at 28 ± 2 °C in dark. After incubation, 2 ml of the mixture was added to 4 ml of Salkowski reagent and the colour was allowed to develop for 20 min. The absorbance of the {'ink solution was measured at 530 nm. The enzyme activity is expressed as mg IAA oxidised h - mg -I protein.
Cytoplasmic peroxidase activity The activity of cytoplasmic peroxidase was measured by recording the change in absorbance at 470nm (~~70) to the oxidation of guaiacol in the presence of H202. The activity is expressed as ~~70 min- I mg- I protein.
Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
IAA oxidase and peroxidase activities in cotton fibre
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Wall peroxidase activity (ionically bound) The wall fraction was separated from the initial borate buffer crushing by centrifuging the homogenate at 1,500 g and washed thrice or till the washing was free of peroxidase reaction with guaiacol. The wall pellet was then resuspended with 5 ml of NaCI (1 M) solution (Ridge and Osborne, 1970) for 1 h and centrifuged at 12,000 g for 10 min. The supernatant served as the source of wall peroxidase. The activity of wall peroxidase was measured as described in cytoplasmic peroxidase. The activity is expressed as ~~70 min-I mg- I protein. The protein content of the extract was estimated using Folin reagent (Lowry et al., 1951). Three replicates were taken for biochemical analysis and the mean values with standard deviation are presented.
Gel electrophoresis Electrophoretic separations of purified cytoplasmic peroxidase and IAA oxidase were performed using the system of Ornstein and Davis (1962), using 7.5 % polyacrylamide gel (in cold). The applied current was 2 m Amp per tube for 10 min, followed by 4 Amp per tube until the dye marker (0.01 % bromophenol blue) had reached the ends of the gels. Ten samples from the entire period of cotton fibre development were run simultaneously. Peroxidase isoenzymes were detected with benzidine (Sahulka, 1970) while IAA oxidase isoenzymes were visualized by the method of Hoyle (1977). The results of electrophoretic separation have been presented schematically with mobilities calculated as the relative distance traversed by the band with respect to the distance travelled by bromophenol dye.
Results
The data for fibre length and dry weight versus boll age X (days) were fitted to a best-fit polynomial with the equation: Y
=
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Fig. 1 A: Best-fit curves for fibre length and dry ~ weight per seed against boll age. The regression u: 1.50 coefficients are 0.998, 0.979, and the multiple ~ correlation coefficients 0.998 and 0.989 for fibre ~ 0.75 length and dry weight respectively. - B: The 0: rate curves for fibre elongation and dry weight. Fibre length, continuous lines: fibre dry weight, broken lines.
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ANTHESIS
Z. Pjlanzenphysiol. Bd. 106. S. 157-165. 1982.
160
C.
N. RAMA RAo, S.
NAITHANI,
R. T. ]ASDANWALA and Y. D. SINGH
The best-fit curve for fibre length (Fig. 1 A) showed that the elongation starts 3 days after anthesis and continues up to 30 days. No significant increase was discernible in the subsequent period. The dry weight on the other hand showed a lag phase up to 20 days after which it increased exponentially till 43 days. The differential curve (Fig. 1 B) for fibre length indicated that fibre elongation stopped at 33 days after anthesis. The rate of fibre elongation was highest at 10 days. Dry weight increase was initiated around 15 days and maximum rate occurred at 30 to 40 days after anthesis or about the time elongation ceased. Low IAA oxidase activity was recorded on the day of anthesis while at day 3 it increased appreciably. Up to 10 days postanthesis a fall in its activity was discernible and it is interesting to note that the low level of IAA oxidase activity corresponded with maximum rate of fibre elongation (Fig. 1 Band 2). After day 10, IAA oxidase activity recorded an increasing trend, the increase being enormous after 25 days postanthesis. High level of activity was maintained till maturity (Fig. 2).
~~
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10 DAYS
20
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2000
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3000
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AFTER ANTHESIS
Fig. 2: IAA oxidase activity against boll age.
On the day of anthesis a very low level of cytoplasmic peroxidase activity was recorded. By day 5 it had increased significantly (Fig. 3 A). It was again low at 8 days postanthesis after which it showed a gradual rise up to 20 days, when a second peak was observed. The activity fell again at 25 days and recorded a sharp rise at maturity. Maximum cytoplasmic peroxidase was recorded in fully mature fibre. Substantial wall bound (ionically) peroxidase activity was observed which rose slightly at 3 days. Up to 15 days postanthesis it decreased, and the low level of this activity corresponded with maximum rate of fibre elongation (Fig. 1 Band 3 B). After 15 days wall peroxidase increased significantly and maximum activity was recorded on 40 days postanthesis. Again, it is interesting to note here that the increase in wall peroxidase corresponded with the rate of dry weight accumulation (Fig. 1 Band 3 B). In all, eleven isoenzymes of peroxidase and IAA oxidase were detected during the entire period of fibre development. A number of stage specific isoenzymes were detected e.g., band E4 which showed both IAA oxidase and peroxidase activity was present during elongation phase. Band E9 and EIO showed only peroxidase activity Z. Pjlanzenphysiol. Bd. 106. 5.157-165.1982.
IAA oxidase and peroxidase activities in cotton fibre
161 A
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o
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20
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DAYS AFTER ANTHESIS
I·Or----------------. E I • 9 K:l K:l K:l K:l K:l K:l K:l K:l K:l K:l E2 c:::::J K:l c:::::J K:l K:l K:l K:l K:l K:l K:l
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DAYS AFTER
ANTHESIS
and were present during early differentiation stages. Isoenzyme E7 with exclusive peroxidase activity was present during differentiation and elongation phases. While, band E6 , a peroxidative, and band E 8 , with both peroxidative and IAA oxidative activity, were detected only during dry matter accumulation stages. Isoenzyme E3 , an exclusive IAA oxidase band, was also detected during dry matter accumulation stages (Fig. 4). Isoenzymes E 1, E2 and E5 were present throughout and showed both IAA oxidase and peroxidase activity. Z. Pjlanzenphysiol. Ed. 106. S. 157-165. 1982.
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Discussion From the growth data presented here, cotton fibre development can be divided into four distinct phases; (I) initiation (0-3 days), (II) elongation (5-20 days), (III) secondary thickening (25-40 days), and (IV) maturation (40-45 days). The differential curve (Fig. 1 B) for fibre elongation showed that a maximum rate of fibre elongation was achieved on 10 days after anthesis and it ceased completely on 33 days. Before the elongation was completed the secondary thickening phase started around 15 days after anthesis and the maximum rate was observed from 30 to 40 days postanthesis indicating clearly that these rather distinct phases are continuous processes which can not be delimited from one another. IAA oxidase activity recorded low levels during the elongation phase (Fig. 2) while during the secondary thickening and maturation phases it increased sharply, confirming thereby the earlier suggestion (Thimann, 1972) that this enzyme may regulate plant growth by limiting the concentration of IAA. In contrast to IAA oxidase, cytoplasmic peroxidase activity recorded significant level during elongation phase while, during secondary thickening and maturation phases it almost paralleled the trends of IAA oxidase (Fig. 2 and 3 A). Peroxidases have been implicated in IAA oxidation. However, whether oxidation of IAA by peroxidase is a general phenomenon, is not well understood. There are three hypotheses regarding the relationship between IAA oxidase and peroxidase, (i) peroxidase and IAA oxidase are different enzymes (Sequeira and Mineo, 1966; Rubery, 1972), (ii) peroxidase and IAA oxidase activities are associated with the same protein molecule but have different active sites (Siegel and Galston, 1967, Laurema, 1974; Srivastava and Van Huystee, 1977) and (iii) only a few of several existing peroxidase isoenzymes possess IAA oxidase activity (Stafford and Bravinder-Bree, 1972). Our electrophoretic studies showed that isoenzymes of peroxidase and IAA oxidase can be classified into three categories, (I) with exclusively IAA oxidase or peroxidase activity, (II) with both IAA oxidase and peroxidase activity, and (III) initially showed peroxidase activity and later IAA oxidase activity. Recent studies on etiolated pea have also revealed the existence of exclusive peroxidase and IAA oxidase isoenzymes and isoenzymes with both peroxidative and IAA oxidative activities (Brynt and Lane, 1979). It is therefore, logical to believe that both IAA oxidase and peroxidase activities are associated with the same protein and that at a given time, depending upon the allosteric modifier, either one or both active sites may function. Reports in this direction indicated the allosteric (homotropic) nature of IAA oxidase and peroxidase (Srivastava and Van Huystee, 1977; Raa, 1971). In our studies with different hydrogen donors (Unpublished data) it was observed that the patterns of changes in peroxidase level and isoenzymes in developing cotton fibre differed with different hydrogen donors. It is therefore possible that exclusive IAA oxidase isoenzymes reported in the present study may turn out to be a peroxidase with another substrate. Studies in this direction are in progress.
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IAA oxidase and peroxidase activities in cotton fibre
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Changes in wall bound peroxidase, in the present investigation, showed that during the elongation phase its levels were low while, during the secondary thickening phase it increased significantly (Fig. 3 B). Similar correlation between wall peroxidase and elongation growth has been reported by Ridge and Osborne (1971), Osborne et ai. (1972). It has been suggested that wall peroxidase may playa causative role in cessation of elongation growth (Gardiner and Cleland, 1974). Further, it has been pointed out by these authors that cell elongation may not be under the control of a single factor, but may be influenced by a series of factors including peroxidase induced lignification, auxin destruction and «extensin» induced wall stiffening. Since a similar correlation between the cell wall bound hydroxyproline containing protein «extensin» and growth has also been reported (Sadava et aI., 1973). Evidences have been presented to link ethylene induced growth inhibition to «extensin» synthesis (Sadava and Chrispeels, 1973). Lamport (1965) proposed that the cross-liking of «extensin» by means of disulphide bridges may restrict cell expansion. That peroxidase can oxidise -SH groups has also been demonstrated (Stonier and Yang, 1973). Thus, it is possible that the increased levels of wall peroxidase during the secondary thickening phase may oxidise -SH groups of «extensin» which in turn may restrict extension growth. However, little evidence supporting the -SH oxidation theory of «extensin» growth control has appeared in the last fifteen years since Lamport's suggestion, and a putative «extensin» precursor purified by Stuart and Varner (1980) contained no cysteine. Alternatively, Fry (1979) proposed that peroxidase stiffens the wall by catalysis of the oxidation of cell wall phenolic compounds to form the more hydrophobic biphenyls, polymers or quinones, any of which could protect wall polysaccharides against attack by wall glycanases (or transglycosylases). According to Fry (1980) under low wall peroxidase levels, phenols are maintained in their reduced state thus making the environment of the polymers less hydrophobic so that (i) covalently bound polysaccharides are rendered more susceptible to enzyme excision by existing hydro lases or transferases, (ii) hydrogen-bonded wall polysaccharides are freed owing to increased competition from water and (iii) growth is facilitated as a result of this loss of polysaccharides. Further, low wall peroxidase content could hinder the covalent cross linking of polysaccharides via diferuloyl bridges. Acknowledgements We are thankful to Professor S. C. Pandeya for providing the necessary facilities; Mr. C. S. R. Murti, Physical Research Laboratory, Ahmedabad for his generous help in statistical analysis; and Dr. M. S. Murthy for going through the manuscript. This work was supported by Univers· ity Grants Commission, New Delhi.
References BEASLEY,
C. A.: Hormonal regulation of growth in unfertilized cotton ovules. Science 179,
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