RESPIRATORY METABOLISM IN ISOLATED ORCHID PETAL CELLS

Transcription

1 New Phytol. (1987) 105, RESPIRATORY METABOLISM IN ISOLATED ORCHID PETAL CELLS BY C. S. HEW AND K. C. YIP Department of Botany, National University of Singapore, Republic of Singapore, Singapore 0511 {Accepted 25 November 1986) SUMMARY A respiratory drift has been demonstrated in cells isolated from petals of Aranda flowers at different stages of development. Tight buds had a respiratory quotient of 0-5; this increased to 0-7 in the first flower and reached 10 in the mature flower. There appeared to be a non-glycolytic pathway contribution in addition to the EMP pathway in tight bud and newly opened flower stages. Carbohydrate metabolism in the mature flower proceeds predominantly via the EMP pathway. Cyanide-insensitive respiration was demonstrated in the tight bud. There was a shift towards cyanide-sensitive respiration as the flower developed. Key words: Respiration, orchid, petal cells, Aranda. INTRODUCTION In general, respiratory metabolism in the flower is less well understood than that of the leaf, fruit and germinating seed (Coorts, 1973; Halevy & Mayak, 1979; Rhodes, 1980). A respiratory drift similar to that of ripening fruits has been reported in cut carnation flowers (Maxie et al., 1973). The same, however, has not been demonstrated conclusively in orchid flowers (Hew, 1986). Sheehan (1954) observed a decline in respiratory rate with age in Cattleya flowers, with the tight bud having the highest respiratory rate. The same was observed for Aranda flowers (Hew, 1980). It was also noted that floral respiratory rates in orchids varied with species. In all cases, the young flower had a higher rate of respiration (Hew, 1980). Hew (1980) showed that carbohydrate metabolism in the mature orchid flower proceeded via the EMP pathway. A respiratory quotient of 1 has been observed for the column and perianth of Coelogyne mooreana, Cymbidium lowianum (Hsiang, 1951) and labellum of Oncidium goldiana (Hew, 1980). Whether or not changes occur in the respiratory pathway of orchid flowers during development remains unclear. This paper examines the respiratory metabolism in cells isolated from Aranda flowers at different stages of development. It appears that there is a shift in metabolic substrate and pathway accompanying floral development. MATERIALS AND METHODS Petal cells were isolated from the floral bud, newly opened flower and fully opened flowers of Aranda Christine 130 [Arachnis hookerana x Vanda Hilo Blue) by enzymic digestion and mechanical agitation as described by Jensen, Erancki & Zaitlin (1971). Orchid petal tissue was flrst cut into strips of about 0-1 x 0-15 cm in size and 1 g of the tissue was then vacuum-infiltrated with 15 ml of maceration medium (Jensen et al., 1971). The tissue was agitated on a reciprocating shaker X/87/ $03.00/0 1^87 The New Phytoiogist

2 6o6 C. S. HEW AND K. C. YIP maintained at 29 C. The cell suspension was filtered through a nylon sieve (Hydro-Bios, Apparatebau, GmbH, FRG) with a pore size 2 /im smaller than the cell size. Cells retained on the sieve were washed gently with a washing medium and the cells centrifuged at 80 x ^ for 3 min. The pellet was resuspended in 5 ml Table L Respiratory quotient (R.Q.) of petal cells isolated from Arandsi fiowers at different stages of development Stage of development Flower bud Mean Just opened flower Mean R.Q Fully opened flower 1 ' Mean 1011 See text for an explanation of the stages of development. of incubation medium (Jensen etal., 1971). Assessment of cell membrane integrity was based on the Evans blue-dye exclusion method (Kanai & Edwards, 1973). Respiration was measured as oxygen uptake using a Warburg respirometer maintained at 28 C. Protein content was determined according to the method of Peterson (1977). All determinations were done in triplicate. RESULTS Isolation of petal cells Examination of a suspension of isolated petal cells showed mainly cells with an intact cell wall and very few broken cells. The membranes of most cells were intact, as indicated by the Evans blue-dye exclusion method. Intact idioblasts containing neatly stacked raphides were commonly observed. A yield of 60 % was obtained for cells isolated from orchid petals. Respiratory rate of isolated petal cells When compared with cells isolated from other developmental stages, cells from the bud stage were noticeably green. The respiratory rate of the bud was /^mol Og mg protein"^ h~\ while that of the first flower (just opened), fifth flower (fully opened) and seventh flower along a single inflorescence were 650-8, and 358-0/tmol O2 mg protein"^ h~\ respectively. Table 1 shows that there was a progressive increase in R.Q. from 0-5 in the bud to 0-7 in the just opened flower and 1-0 in the fully opened flower.

3 Respiration in orchid petals CM o 20 - Minutes Fig. 1. Effect of different concentrations of KCN on rate of oxygen uptake of petal cells isolated from the fully opened Aranda flower. # # Control; O O 1 mm; n d 5 mm; 10 mm. Effect of potassium cyanide {KCN) and salicylhydroxamic acid {SHAM) applied singly, or in combination, on the respiration of isolated cells The effect of various concentrations of KCN and SHAM on the respiration of cells isolated for mature orchid flowers was studied (Figs 1 and 2). At 1-0 mm, KCN inhibited respiration only slightly and the rate was linear. At 5 and 10 mm, complete inhibition occurred after 30 min (not inclusive of the 20 min equilibration period). In the first 30 min, the inhibition of respiration for 5 mm and 10 mm KCN was 12-5% and 30-9%, respectively (Fig. 1). Likewise, SHAM applied at 0-25 mm did not inhibit respiration significantly. At 2-5 mm, the respiratory rate was still linear but an inhibition of 52-5 % was observed 40 min after the start of the treatment. When the concentration of SHAM was increased to 25 mm, inhibition was apparent after 40 min (Fig. 2). In another series of experiments, the effects of KCN and SHAM on respiration of cells isolated from flowers at different stages of development were investigated. KCN and SHAM were applied either singly or in combination and their concentrations were 5 mm and 0-5 mm, respectively. For floral bud cells, there was 60-6 % and 73 % inhibition of respiration, respectively, in 40 min when KCN or SHAM was added alone. In both cases, inhibition was not complete and the rate of respiration was linear. When KCN and SHAM were added together, almost complete inhibition occurred after 10 min. The inhibition in the first 10 min was 45 % that of the control (Fig. 3). For the just opened flower cells, KCN when added alone blocked respiration by 55-6 %, while the addition of SHAM alone inhibited respiration by 19 % (Fig. 4). When KCN and SHAM were added in combination, there was 78-8% inhibition in 20 min after treatment, followed by a complete inhibition thereafter. For the fully opened flower cells, KCN when added alone led to complete inhibition after 30 min (Fig. 5). When added alone, SHAM inhibited respiration

4 6o8 C. S. HEW AND K. C. YIP CO O Minutes lilted f^^ th Ir 7j^^' "V^ ^"^^ " '^' '''' ^ isolated from the fully opened Aranda flower. #-# Control; D 25-0 mm; Q O 2-5 mm. D 0-25 mm Fig. 3. Efltect of difl^erent inhibitors on the rate of oxygen uptake of petal cells isolated from the floral bud oi Aranda. # # Control; O O SHAM; +- - KCN- D D NaF- A- -A SHAM + KCN;! malonate. ' ' only by 5-6 %. Adding KCN and SHAM together led to a complete inhibition after 30 min. The inhibition in the first 30 min was 38 % compared to that of the control. Effect of sodium fluoride (NaE) and malonate on respiration of isolated petal cells The concentration of NaF and malonate used was 100 mm. For floral bud cells, malonate completely blocked respiration after 20 min. NaF blocked respiration by 56-7% but it did not completely inhibit respiration (Fig. 3). For just opened flower petal cells, malonate and NaF inhibited respiration almost completely after 20 min. Respiration in malonate- and NaF-treated cells in the first

5 Respiration in orchid petals CM o Fig. 4. EflFect of different inhibitors on the rate of oxygen uptake of petal cells isolated from the just opened y4raw<fa flower. # # Control; O O SHAM; I- KCN; D D NaF; malonate; A SHAM -I- KCN Fig. 5. Effect of different inhibitors on the rate of oxygen uptake of petal cells isolated from the fully opened ^raw</a flower. Control; O O SHAM; malonate; -I KCN; A A SHAM + KCN; D D NaF. 20 min was 67-3 and 86-1 % of that of the control (Fig. 4). For the fully opened flower petal cells, malonate inhibited respiration completely only after 40 min. Before this, respiration of malonate-treated cells was 14-1 % that of the control (Fig. 5). Sodium fluoride completely blocked respiration after 10 min. DISCUSSION The results show that the respiratory rate of Aranda petal cells changes with the stage of development as has been observed previously using whole orchid petals (Sheehan, 1954; Hew, 1980). The highest rate of respiration, however, was in the

6 6io C. S. HEW AND K. C. YIP newly opened flower and not in the tight bud as reported in Cattleya and Aranda flowers. It is possible that the difference in developmental stage of the tight bud used could account for the discrepancy observed. A drift of R.Q. was observed during floral development. An R.Q. of 053 in bud cells may be an indication of the partial oxidation of fatty acids. Characteristically, complete oxidation of fat will yield an R.Q. of around 07 (James, 1953), but if this fat is partially converted to sugar, which uses oxygen but without carbon dioxide evolution, and if this sugar is formed faster than it is being respired, the R.Q. will be in the region of 0 57 instead (Stiles & Leach, 1952). That the orchid bud may have a high lipid content is interesting because the cells of the orchid embryo have many lipid bodies (Arditti, 1979). If the fatty acid was partly oxidized and partly converted to sugar, a concomitant rise in R.Q. would follow. This may explain the increase of R.Q. to 0*66 in the flrst flower and eventually to 1-0 in the flfth flower when carbohydrate becomes the sole respiratory substrate. However, one cannot rule out the possibility that other substrates, such as amino acids, organic acids or other substances were being utilized in respiration. The possibility of incomplete oxidation, utilization of multi-substrates in different proportions, the involvement of more than one chain of reaction in the breakdown of substrates and the diversity of flnal products prevent an accurate interpretation of the observed R.Q. (Stiles & Cocking, 1969). The complete inhibition of oxygen uptake by malonate and sodium fluoride after 20 min in isolated cells from the flrst flower (Fig. 4) suggests that the predominant metabolic pathway for the oxidation of respiratory substrate in the orchid flower is the glycolytic pathway. Sodium fluoride inhibits the conversion of phosphoglyceric acid to phosphoenolpyruvate, and malonate competitively inhibits succinate dehydrogenase in the TCA cycle (Beevers, 1960). It may be inferred from the 57 % inhibition of oxygen uptake of floral bud cells by sodium fluoride (Fig. 3) that half of the total respiratory metabolism may proceed via the glycolytic pathway, while the other half is via some other, non-glycolytic, pathway. It is possible that the alternative pathway may be the pentose phosphate pathway, as non-inhibition by sodium fluoride on respiratory metabolism has often been taken as an indication of the operation of this pathway (Stiles & Cocking, 1969). The operation of the pentose phosphate pathway might serve to generate NADPH which could be utilized in the biosynthesis of fatty acids (Beevers, 1960). This would support earlier suggestions that fat is metabolized in the floral bud. It is not surprising that the pentose phosphate pathway operates in the floral bud stage, as this stage of development involves predominantly biosynthesis. As the flower matures, more energy is channelled to maintenance rather than biosynthesis. Inhibition of respiration by malonate was greater in the bud and the just opened flower and less in the fully opened flower. Being a competitive inhibitor, the action of malonate on respiration would be strongly influenced by the concentration and turnover of succinate in cells, both of which might account for the different degree of inhibition by malonate observed in orchid flowers at different stages of development. A similar pattern of inhibition of respiration by malonate and fluoride has been reported in developing rose petals (Siegelman, Chow & Biale, 1958). Complete inhibition of oxygen uptake in the fully opened flowers by KCN indicated that the cyanide-sensitive pathway of the electron transport chain was operative predominantly in this flower. Apparently, this was not so in the bud and

7 Respiration in orchid petals 6ii in the just opened flower. It appears that the cyanide-resistant pathway was also operative concurrently in cells isolated from the bud and the just opened flower. Complete inhibition of respiration in the presence of KCN and SHAM, when added together, would further support this. As reported earlier (Passam, 1976), inhibition of respiration by KCN and SHAM when applied in combination was greater than the sum of inhibition when they were applied singly, suggesting that the addition of either KCN or SHAM separately caused some diversion of electron flow from one oxidase pathway to the other. Our present flndings suggest that there is a gradual shift towards the cyanidesensitive pathway as the flower develops. This was reflected in the concomitant decrease in the degree of inhibition when SHAM was applied alone. Siegelman et al. (1958) also observed that the respiration of fully expanded rose petals was strongly inhibited by cyanide, but this was not so in the slowly expanding petal. It is interesting to note the similarity between petal cells and some germinating seeds in their response to cyanide. Yentur & Leopold (1976) reported that, in the early germination stage of soybean seeds, respiration was predominantly cyanideresistant. This was followed by a gradual transition to cyanide-sensitive respiration at a later stage of seed germination. The same was observed in lettuce and Cicer seeds (Poljakoff-Mayber & Evenari, 1958; Burguillo & Nicolas, 1977). It is now important to study the enzymatic and biochemical changes accompanying the floral development of orchid in order to understand fully the respiratory drift observed in the present study and to conflrm our speculations. Another interesting area which awaits further investigation is whether or not cyanide-resistant respiration occurs when the flower begins to senesce. Cyanideresistant respiration precedes the climacteric rise in respiration of cut carnation flowers (Wulster, Sacalis & Janes, 1984). REFERENCES ARDITTI, J. (1979). Aspects of the physiology of orchids. Advances in Botanical Research, 7, BEEVERS, H. (1960). Respiratory Metabolism in Plants. Row-Patterson, USA. BURGUILLO, P. F. & NICOLAS, G. (1977). Appearance of an alternative pathway cyanide-resistant during germination of seeds of Cicer arietinum. Plant Physiology, 60, COORTS, G. D. (1973). Internal metabolic changes in cut flowers. Horticultural Science, 8, HALEVY, A. H. & MAYAK, S. (1979). Senescence and post harvest physiology of cut flowers. Part I. Horticultural Review, 1, HEW, C. S. (1980). Respiration of tropical orchid flowers. Proceedings of the Ninth World Orchid Conference, Bangkok (1978), HEW, C. S. (1986). Respiration in orchids. In: Orchid Biology: Reviews and Perspectives, vol. 4 (Ed. by J. Arditti), pp Cornell University Press. HSIANG, T. H. T. (1951). Physiological and biochemical changes accompanying pollination in orchid flowers. II. Respiration, catalase activity and chemical constituents. Plant Physiology, 26, JAMES, W. O. (1953). Plant Respiration. Clarendon Press, Oxford. JENSEN, R. G., FRANCKI, R. 1. B. & ZAITLIN, M. (1971). Preparation of photosynthetically active cells from tobacco. Plant Physiology, 48, KANAI, R. & EDWARDS, G. E. (1973). Purification of enzymatically isolated mesophyll protoplasts from C3, C4 and CAM plants using an aqueous dextran polyethylene glycerol two phase system. Plant Physiology, 52, MAXIE, E. C, FARNHAM, D. S., MITCHELL, F. G., SOMMER, N. F., PARSONS, R. A., SNYDER, R. G. & RAE, H. L. (1973). Temperature and ethylene eflfects on cut flower of carnation {Dianthus carophyllus L.). Journal of the American Society for Horticultural Science, 98, PASSAM, H. C. (1976). Cyanide insensitive respiration in root tubers of cassava. Plant Science Letters, 7, PETERSON, G. L. (1977). A simplification of the protein assay method of Lowrey et al., which is more generally applicable. Analytical Biochemistry, 83,

8 6i2 C. S. HEW AND K. C. YIP POLJAKOFF-MAYBER, A. & EVENARI, M. (1958). Some further investigations of oxidative systems of germinating lettuce seed. Physiologia Plantarum, 11, RHODES, J. C. (1980). Respiration and senescence of plant organs. In: Biochemistry of Plants, vol. 2, Metabolism and Respiration (Ed. by D. D. Davies), pp Academic Press, New York. SHEEHAN, T. J. (1954). Respiration of cut flowers of Cattleya mossiae. American Orchid Society Bulletin, 23, SiEGELMAN, H. W., CHOW, C. T. & BiALE, J. B. (1958). Respiration of developing rose petal. Plant Physiology, 33, STILES, W. & COCKING, E. C. (1969). An Introduction to the Principles of Plant Physiology, 3rd edn. Methuen, London. STILES, W. & LEACH, W. (1952). Respiration in plants, 3rd edn. Metbuen, London. WuLSTER, G. J., SACALIS, J. N. & JANES, H. W. (1984). Development of cyanide-resistant respiration in carnation flower petals. Journal of the American Society for Horticultural Science, 109, YENTUR, S. & LEOPOLD, A. C. (1976). Respiratory transition during seed germination. Plant Physiology, 57,

9