PHOTOSYNTHESIS OF Y O U N G ORCHID SEEDLINGS

Transcription

1 New Phytol. (1980) 86, PHOTOSYNTHESIS OF Y O U N G ORCHID SEEDLINGS BY C. S. H E W * AND S. L KHOO Department of Biology, Nanyang University, Singapore 22 {Accepted 1 December 1979) SUMMARY Adult leaves, protocorms and seedlings of succulent tropical orchids exhibit diurnal fluctuations in acidity. The titratable acidity varied at different stages of growth. The fluctuation in acidity was barely detectable in protocorms but as they grew older, their capacity for acid accumulation increased. The O2 exchange in orchid protocorms and seedlings was similar to that of adult leaves in their responses to temperature, bicarbonate and light intensity. It is concluded that protocorms and seedlings resemble adult organs in having the characteristics of crassulacean acid metabolism (CAM). INTRODUCTION The crassulacean acid metabolism (CAM) pathway of photosynthesis has attracted considerable interest recently (Ting, Johnson and Szarek, 1972; Kluge, 1976; Osmond, 1978). This is because of the biochemical similarity between the CAM and C4 pathways of photosynthesis and because CAM is now increasingly understood as an adaptive mechanism that enables succulent plants to maintain a positive carbon balance under arid conditions. Its biochemistry, physiology and ecological significance have been extensively reviewed (Ranson and Thomas, 1960; Ting et al., 1972; Kluge, 1976; Osmond, 1978)..,... x In the past, measurement of CAM in plants has been done with whole plants (Joshi, Boyer and Kramer, 1965; Osmond and Bjorkman, 1975; Kaplan, Gale and PoljakofF-Mayber, 1976), intact leaves (Osmond and Bjorkman, 1975; Hew, 1976), detached shoots (Jones and Mansfield, 1972) or leaf slices (Deniusand Homann, 1972). The use of whole plants or leaves has limitations because gas exchange may be restricted by the stomata and by the diffusion of gas through bulky tissue. Sectioning the leaves should lessen or alleviate such problems and facilitate the entry of gas or of added inhibitors. However, Bruinsma (1958) observed that CAM decreased when Bryophyllum leaves were sliced. This decrease has been attributed to the action of tannin or other phenolic compounds (Denius and Homann, 1972). Tropical orchids exhibit two types of COg fixation (Nuernbergk, 1963; Wong and Hew, 1973; Neales and Hew, 1975; Hew, 1976). In thin-leaved orchids, photosynthetic CO2fixationtakes place through Cg photosynthesis whereas thick-leaved orchids exhibit typical features of CAM. In this report, we have examined CAM activities of seedlings at various stages of development and have also explored the possible use of orchid protocorms or seedlings. To whom reprint requests should be sent X/80/ $02.00/ The New Phytologist. ANP 86

2 35O C. S. H E W A N D S. I. KHOO MATERIALS AND METHODS Plants Dendrobium taurinum was mainly used. For comparative purposes, a number of otber orchid species and hybrids were also used. Dendrobium taurinum sets pods readily after artificial self-pollination of its flowers, thus seeds required for experiments can easily be obtained. The pods become ripe in a period of about 4 months. Various growth stages of D. taurinum were selected for present studies, i.e. protocorms and seedlings of various lengths, 0-5 to 1 mm, 1 to 1-5 mm, 2 to 3 mm, 5 to 7 mm, 15 to 20 mm (Plate 1). Other orchid species and hybrids used were Arundina graminifolia (Don) Hochr, Dendrobium crumenatum Sw, Dendrobium Schulleri, Dendrobium Mei Lin {Dendrobium Guandalcanbai x Dendrobium Sunset), Vanda dearei, Vanda 'Tan Chay Yan' {Vanda Josephine Van Brero x Vanda dearei), Vanda Ruby Prince {Vanda Ruby x Vanda cooperi var. Cho Yam Neo) and Spathoglottis plicata BI. Adult plants of Dendrobium crumenatum and Dendrobium taurinum were grown under partial shade conditions; all other adult orchids were grown under full sunlight. Orchid seeds were sown immediately after harvesting. They were cultured asymbotically on an agar medium (Vacin and Went, 1949) to which mashed banana (5% w/v) had been added. After sowing, the seeds were kept in darkness for 1 week; later they were grown under a light intensity of 30/tE m"^ sec~i at 28 C with a cycle of 12 h light, 12 h darkness. Determination ofo^ evolution O2 evolution was measured polarigraphically as described previously (Wong and Hew, 1973). The chamber was made of plexiglass with a central compartment surrounded by a water jacket at 25 C. A Clark type oxygen electrode was inserted from the side of the chamber into a central compartment which had a volume of either 1 ml or 5 ml. Leaf discs (3 mm), protocorms or seedlings were washed, placed in distilled water and pre-illuminated at a light intensity of 800 fie m-^ sec~i for 1-5 h. They were then placed in the central compartment for measurements. The light source was a Kodak Carousel S Projector and the intensity during measurements was Oxygen measurements on acidified leaves or seedlings were made at 9.00 h and those on deacidified plants at h. Determination of titratable acidity and chlorophyll content Titratable acidity was measured by the method of Szarek and Ting (1974) and chlorophyll by the method of Arnon (1949). Isolation ofprotocorm chloroplasts Chloroplasts were isolated following the method of Izawa and Good (1968). The reaction mixture for the Hill reaction contained 100 mm sucrose, 100 mm tricine, ph 7-8, 5 mm MgClg, 20 mm NaCI and 10 mm K3Fe(CN)6. Oxygen evolved was measured polarigraphically. I.}'-.-.;:.-,?,?.--.-^.. ^- ; :.. ^ - i ^ ' R E S U L T S ' "" " '" /^" ^' '" ' ' ' ' ' ' ' ' [ " " Titratable acidity : Diurnal changes of acidity in orchid protocorms/seedlings of different ages are

3 CAM in orchids 351 shown in Figure 1. In very young protocorms, the fluctuations were low and barely detectable. They increased with an increase of age of the seedlings. The overall change in acidity, as calculated from the difference between maximum and minimum acidities, increased with the age of the plants. The protocorms of D. taurinum (0-5 to 1 mm) had a net acid change of 2-5 /^equiv/g FW, while the seedlings (15 to 20 mm) had a net change of 19-5 /tequiv/g FW. The seedlings (40 to 60 mm) of D. Mei Lin had a net acid change of 23'0/^equiv/g FW. Seedlings of Z). taurinum and D. Schulleri, of the same length (2 to 3 mm), had acid fluctuations of about the same s i z e. - ' ' - ' - ': ' - " - - ' - - ' Time (h) Fig. 1. Diurnal fluctuation of titratable acidity in young protocorms and seedlings of three Dendrobium orchids, (a) Dendrobium taurinum, ^, 0-5 to 1 n\m; #, 1 to 1-5 mm; A> 2 to 3 mm; D, 15 to 20 mm. (b) M, Dendrobium Schulleri (2 to 3 mm);, Dendrobium Mei Lin (4 to 6 mm). Table 1 shows acid fluctuations in seedlings of a number of succulent and non-succulent tropical orchids. The acid fluctuations in seedlings of succulent orchids at various stages of growth were similar to those of adult plants but the young seedlings accumulated less acid. Generally, mature leaves showed a net acid fluctuation of about 100 to 150/*equiv/g FW. In contrast, thin-leaved orchids did not show acid fluctuations and generally had low acidities (less than 20/*equiv/g FW). Oxygen evolution and uptake The protocorms and the fourth leaf of Dendrobium taurinum were studied. Leaves were harvested just before use. Prior to this work, the presence of polyphenolic compounds in these leaves was investigated (Hawker et al., 1972) but no significant amounts of these compounds were found. Effect of age Figure 2 compares the gross O2 evolution and O2 uptake by Dendrobium taurinum at various stages of growth (2 to 4 mm protocorms, 5 to 7 mm seedlings and fourth leaves of adult plants) and at 9.00 and h. Values for gross O^ evolution have been corrected for dark respiration. Fully acidified plant materials had the highest capacity for Og evolution in light; the rate declined towards the end of the day and no net O2 evolution was observed at h. Gross O2 evolution was lowest in the protocorm

4 352 C. S. HEW AND S. 1. KHOO stage. The acidified protocorm had a Og evolution of 20 /imol Og/mg chl/g FW; when deacidified it was zero. No net Og evolution was observed at either 9.00 h and h.. i Table 1. Acidity fluctuation in tropical orchids,.»rtw.,:..;...* ' -i.^.;..^..r..v;:,., /tequiv/g FW M... '.* ^t 9.30 h '^ ''' X: h ' ''.<^"^*t.*l ' *' Orchids: Succulent, thick leaved orchids Leaves: : Dendrobium taurinum. Dendrobium crumenatum, Vanda dearei Vanda 'Ruby Prince' Protocorms (0-5 to 1 mm): Dendrobium taurinum '; Dendrobium crumenatum Vanda dearei Non-succulent, thin leaved orchids Leaves: Spathoglottis plicata Arundina graminifolia Protocorms (1 to 3 mm): Spathoglottis plicata Arundina graminifolia Oxygen uptake was highest in the protocorms (30 to 55 fimox Og/mg chl/h). There was a slight decrease in Og uptake at h. In contrast, the mature leaves exhibited an increase in Og uptake at h. Since 2 to 4 mm protocorms of Dendrobium taurinum were found to have a negative net O^ evolution throughout the day, a study of the photochemical activity of the chloroplasts was carried out. The chloroplast preparation showed rapid Og evolution in light; in darkness there was Og uptake. Similar results were obtained with chloroplasts isolated from protocorms of D. crumenatum. The rate of net Og evolution in D. taurinum and D. crumenatum was 1 /tmol Og mg chl~i h~^ and 0-5 /imol mg chl~i h~^ respectively. Ejfect of addition of bicarbonate.= Figure 3 shows the effect of bicarbonate on gross Og evolution of D. taurinum. Both the deacidified mature leaves and 2 to 4 mm protocorms responded to the addition of bicarbonate. Deacidified protocorms showed no gross Og exchange [Figure 3(a)] until 0-02 mmol bicarbonate were added. The rate then steadily increased with increase of bicarbonated concentration until it reached a plateau when 0-06 mmol of bicarbonate were added. The deacidified leaf showed significant gross Og evolution and this rate increased with addition of bicarbonate up to 5 mmol. Further addition of bicarbonate resulted in a slight inhibition of Og evolution. The acidified plant materials saturated at a much lower concentration of bicarbonate, i.e. approximately 100 times less than the amount needed for deacidified materials ffigure 3(a), (b)]. The Og evolution of the protocorms levelled off at 4 x 10~^ mmol

5 CAM in orchids I... *. ;. L. - :....,., - I of added bicarbonate. Og evolution of the mature leaves was inhibited when more than 0-04 mmol of bicarbonate were added. ; 6' 0 Fig. 2. Gross O2 evolution and O2 uptake of Dendrobium taurinum at various stages of growth. A, adult; B, seedling; C, protocorm; D, gross O2 evolution;, Og uptake, (a) 9 h; (b) 17 h Bicarbonate added (mmol) Fig. 3. EfTect of various bicarbonate concentrations on O^ evolution by young seedlings and leaves of Dendrobium taurinum. (a) deacidified; (b) acidified. Effect of temperature and light intensity Gross Og evolution of both mature leaves and seedlings (5 to 7 mm) oi Dendrobium taurinum increased with an increase in temperature (Figures 4 and 5). Figure 6 shows the effect of different light intensities on 5 to 7 mm seedlings and leaves of Dendrobium taurinum. The effect of light was studied at h when the plants were partially deacidified. Increase of light intensity increased the gross O^ evolution. Gross Og evolution of seedlings appeared to saturate at a lower light intensity than did the mature leaf discs. With seedlings, light intensities higher than 400/*E m~2 sec~^ produced slight inhibition. No inhibition was observed in the mature leaf discs at this light intensity..,.^,

6 354 C. S. HEW AND S. I. KHOO (a) 30 Gross Og evolution - Gross Og evolui-ion " 20 E <B 10 a> I(J X I 02 uptake Temperature CC) Fig. 4. Effect of temperature on O2 exchange of young seedlings of Dendrobium taurinum. (a) deacidified; (b) acidified. 30 (a) Gross Og evolution (b) Gross O2 evolution s: 1 20.A' u o> E (II O ^mol 10 0 exct"lange ( " 15 Temperature ( C) Fig. 5. EfTect of temperature on O2 exchange of leaves of Dendrobium taurinum. (a) deacidified; (b) acidified. DISCUSSION This investigation shows that not only the adult leaves, but also the protocorms and seedlings of succulent CAM orchids exhibit diurnal fluctuations in acidity. The total titratable acidity of orchids at various stages of growth also differs with the youngest protocorms having the least capacity for acid accumulation. As the protocorms grow older, they accumulate more acid and have a more pronounced fluctuation of acidity. As with acidity, the rate of gross Og evolution in protocorm/seedlings increased with age. A decline in titratable acidity correlated closely with a decrease in gross Og evolution. A similar observation has been reported by Denius and Homann (1972) using Aloe leaf slices. There was a substantial difference in the stimulatory effect of bicarbonate on the rate of Og evolution in the acidified and deacidified orchid leaves and protocorms. A difference in the enhancement by addition of bicarbonate has also

7 CAM in orchids 355 been noted in acidified and deacidified Aloe leaf tissue. This has been explained on the basis of stored acid providing an additional source for COg assimilation in light (Denius and Homann, 1972). Also similar to the behaviour of Aloe leaf tissue (Denius and Homann, 1972), is that an increase of temperature accelerated Og evolution and uptake by orchid seedlings in a more or less linear manner. Acceleration of Og evolution by higher temperatures has been attributed to a faster mobilization and decarboxylation of the malate pool (Denius and Homann, 1972)., 0" 200 Light intensity (microeinsteins 400 ' sec"') Fig. 6. EfTect of light intensity on Og evolution of young seedlings and leaves of Dendrobium taurinum. (a) adult; (b) seedling. The finding that in succulent orchids acidity fluctuations appear only at a certain stage of ontogeny is worth noting. It may indicate that a switch in COg fixation pattern occurs during ontogeny. Such a change from Cg to C4 photosynthesis and vice versa during ontogeny has been reported (Downton, 1971; Kennedy and Laetsch, 1973; Khana and Sinha, 1973). In succulent plants showing CAM features, a COg assimilation pathway comparable to that of Cg photosynthesis exists (Osmond, 1978). The pathway of carbon in very young protocorms of succulent orchids could well be a Cg photosynthesis. Harrison and Arditti (1978) recently measured the RUDP carboxylase activity in germinating Cattleya seedlings. The level of activity correlated well with the ability of the young seedling to photosynthesize. The occurrence of a Cg photosynthesis in young Bryophyllum leaves has recently been reported (Nishida, 1978). It would therefore be of interest to investigate whether the pathways of photosynthesis change during the ontogeny of orchid seedlings. However this may be, young protocorms and seedlings of Dendrobium taurinum exhibit prominent titratable acid fluctuation and they closely resembled the adult leaves in their responses to various environmental factors, e.g. temperature, light

8 356 C. S, HEW AND ^. I. KHOO intensity and addition of bicarbonate. Thus it appeared that young CAM orchids^ could be good material for the study of the biochemistry of CAM. * I,,,,.._.^, ^.,,.j.,, R E F E R E N C E S ^,,,,;,. > ^., j.>,j I. (1949). Copper enzyme in isolated chloroplasts polythenoloxidase in Beta vulgaris.t Plant Physiology, 24,1-15. BRUINSMA, J. (1958). Studies on the Crassulacean acid metabolism. Botanica Neerlandica, 7, DENIUS, H. R., Jr. & HOMANN, P. H. (1972). The relation between photosynthesis, respiration, and Crassulacean acid metabolism in leaf slices of Aloe arborescens Mill. Plant Physiology, 49, DowNTON, W. J. S. (1971). Adaptive and evolutionary aspects of C4 photosynthesis. In: Photosynthesis and Photorespiration (Ed. by M. D. Hatch, C. B. Osmond & R. O. Slayter), pp WileyInterscience, Chichester. HAWKER, J. S., BUTTROSE, M. S., SIEFFKY, A. & POSSINGHAM, J. V. (1972). A simple method for demonstrating macroscopically the location of polyphenolic compounds in grape berries. Sonderdrick aus der Zeitschrift' VITIS', 11, HARRISON, C. R. & ARDITTI, J. (1978). Physiological changes during the germination of Cattleya auranftaca (Orchidaceae). Botanical Gazette, 139, HEW, C. S. (1976). Patterns of COj fixation in troical orchid species. In: Proceedings of Sth World Orchid Conference (Ed. by K. Senghas), pp German Orchid Society Ltd. IZAWA, S. & GOOD, N. E. (1968). The stoichiometric relation of phosphorylation to electron transport in isolated chloroplasts. Biochimica et Biophysica Acta, JONES, M. J. & MANSFIELD, T. A. (1972). A circadian rhythm in the level of carbon dioxide compensation in Bryophyllum fedtchenkoi with zero values during the transient. Planta, JosHi, M. C, BoYER, J. S. & KRAMER, P. J. (1965). Growth, carbon dioxide exchange, transpiration, and transpiration ratio of pineapple. Botanical Gazette, 126, KAPLAN, A., GALE, J. & POLJAKOFF-MAYBER, A. (1976). Resolution of net dark fixation of carbon dioxide into its respiration and gross fixation components in Bryophyllum daigremontianum. Journal of Experimental Botany, 27, KENNEDY, R. A. & LAETSCH, W. M. (1973). Relationship between leaf development and primary photosynthetic products in the C4 plant Portulaca oleracea. Planta, 115, KHANA, R. & SINHA, S. K. (1973). Change in predominance from C4 to C3 pathway following anthesis in Sorghum. Biochemical and Biophysical Research Communication, 52, KLUGE, M. (1976). Crassulacean acid metabolism (CAM): CO2 and water economy. In: Water and Plant Life (Ed. by O. L. Lange, L. Kapper & E. D. Schulze). pp Springer Verlag, Berlin. NEALES, T. F. & HEW, C. S. (1975). Two types of carbon fixation in tropical orchids. Planta, 123, NiSHiDA, K. (1978). Effect of leaf age on light and dark "CO2 fixation in a CAM plant, Bryophyllum Calycinum. Plant and Cell Physiology, 19, NuERNBERGK, E. L. (1963). On the carbon dioxide metabolism of orchids and its ecological aspects. Proceedings of 4th World Orchid Conference, Singapore, OSMOND, C. B. (1978). Crassulacean acid metabolism: a curiosity in context. Annual Review Plant Physiology, 29, 379^14. OSMOND, C. B. & BJORKMAN, O. (1975). Pathway of COg fixation in the CAM plant Kalanchoe daigremontiana. II. Effects of O2 and CO2 concentration on light and dark CO2 fixation. Australian ' Journal of Plant Physiology, 2, RANSON, S. L. & THOMAS, M. (1960). Crassulacean acid metabolism. Annual Review of Plant Physiology, 11, SZAREK, S. R. & TING, I. P. (1974). Seasonal patterns of acid metabolism and gas exchange in Opuntia basilaris. Plant Physiology, 54, T I N G, P. I., JOHNSON, H. B. & SZAREK, S. R. (1972). Net CO2 fixation in Crassulacean acid metabolism plants. In: Net Carbon Dioxide Assimilation in Higher Plants (Ed. by C. C. Black), pp Southern Section of the American Society of Plant Physiologists/Cotton. VACIN, E. & WENT, F. W. (1949). Some ph changes in nutrient solution. Botanical Gazette, 110, WONG, S. C. & HEW, C. S. (1973). Photosynthesis and photorespiration in some thin-leaved orchid species. Journal of Singapore National Academy of Science, 3, ARNON, D.

9 The New Phytologist, Vol. 86, No. 4 C. S. HEW AND S. r. KHOO Plate 1 {Facing p. 356)

10 CAM in orchids EXPLANATION OF PLATE PLATE 1 Dendrobium taurinum at various stages of growth. No mm. - ' No mm. No cm. No cm.

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