PHOTOSYNTHETIC AND RESPIRATORY CHARACTERISTICS OF MALAYAN SUN AND SHADE FERNS

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1 New Phytol. (1987) 105, PHOTOSYNTHETIC AND RESPIRATORY CHARACTERISTICS OF MALAYAN SUN AND SHADE FERNS BY A. NASRULHAQ-BOYCE AND M. A. HAJI MOHAMED Department of Botany, Universiti Malaya, Kuala Lumpur 59100, Malaysia (Accepted 9 September 1986) SUMMARY A comparative study of four Malayan ferns, (Bl.) Maxon, Tectaria singaporeana (Wall.) Ching, Abacopteris multilineata (Wall.) Ching and Hymenophyllutn polyanthos Sw. from shady habitats and another four, Dicranopteris linearis (Burm.) Und., Lygodium scandens (L.) Sw., Blechnum orientate Linn, and (Burm.) Bedd. from sunlit habitats showed that the total chlorophyll content expressed on a gram fresh weight basis was greater in the shade ferns. There was little difference in the chlorophyll content between the sun and shade ferns when it was expressed on a per unit leaf area basis. The protein and protohaem content was greater in the sun ferns. Measurements of the in vitro photochemical activities of the photosystems I and II in isolated chloroplasts by means of an oxygen electrode showed higher rates in the sun ferns. As determined by spectrophotometric analysis, the photosynthetic cytochrome content from isolated chloroplasts was greater in the sun ferns. The results indicate that the sun ferns have physiological characteristics favouring greater capacity for photosynthesis. Mitochondria isolated from the sun ferns showed faster rates of electron transport using exogenous NADH as substrate. Key words: Malayan ferns, photosynthesis, respiration, sun ferns, shade ferns. INTRODUCTION Plants may be classified into sun and shade plants depending on their preference for living in habitats with a particular irradiance. Extreme shade plants not only survive but thrive at much lower irradiance than sun species. Those plants which grow in shady habitats have been shown to possess characteristic differences m terms of morphology, anatomical structure and physiological activities when compared to those growing in full sunlight (Boardman, 1977). It is well documented that plants grown in the shade photosynthesize more efficiently at low irradiance than do the sun plants, although the rate of photosynthesis at light saturation is less (Boardman, 1977). Eurthermore, plants grown in the shade have been reported to have large, dark green chloroplasts with well-developed grana, in comparison with those from sun plants, apparently adapted for maximum efficiency in trapping light (Anderson et al, 1973). Two recent studies on temperate sun and shade ferns have shown that the chlorophyll content was greater in the shade ferns when compared to the sun ferns. (Hew & Wong, 1974; Ludlow & Wolf, 1975). These reported that the sun ferns had a higher stomatal density and showed greater capacity for in vivo photosynthesis and respiration than their shade counterparts. However, very little is known about the photosynthetic characteristics of tropical sun and shade ferns. The present investigation was carried out to remedy this dearth of information. The present paper reports an in vitro investigation of the photochemical rates of chloroplasts and respiratory X/87/ S03.00/ The New Phytologist

2 82 A. NASRULHAQ-BOYCE AND M. A. HAJI MOHAMED activities of mitochodria of Malayan ferns selected from two habitats with a different light irradiance. The protohaem and photosynthetic cytochrome contents of the plants were also studied. MATERIALS AND METHODS Plant material The ferns chosen in this study were classified as sun and shade plants depending on their preference for living in a particular irradiance. The irradiance measurements of the shady habitats were in the region of 7 to 11 W m~^, while those of the sunny habitats varied from 600 to 710 W m~^. Irradiance measurements were carried out daily at midday for a week using a lux meter. To minimize the effect of other factors which might affect the results, all the plants studied were collected from a single locality at an elevation of 300 m on the outskirts of Kuala Lumpur. The ferns chosen from shady habitats were (Bl.) Maxon, (Wall.) Ching, Abacopteris multilineata (Wall.) Ching and Hymenophyllum polyanthos Sw. The ferns from sunlit habitats studied were Dicranopteris linearis (Burm.) Und., Lygodium scandens (L.) Sw., Blechnum orientate Linn, and (Burm.) Bedd. Isolation of organelles and measurements made Chlorophyll was extracted and determined using the method of Arnon (1949). Protein was measured using the method of Lowry et al. (1951). Amounts of protohaem in the leaves were measured as described by Castelfranco & Jones (1975), with some modifications. The acid acetone extract was further washed with an equal volume of ether and distilled water. This was necessary when difficulty was encountered in obtaining good spectra of protohaem from acid acetone extracts. The spectrum of protohaem was plotted from a Shimadzu UV-visible double beam spectrophotometer. Chloroplasts were isolated from the leaves using the method of Griffiths (1975) and mitochondria by that of Bonner (1967). Cytochromes /, b 559jjp and b 559LP plus b 563, were determined in isolated chloroplasts by difference spectroscopy, as described by Bendall et al. (1971). Cytochromes and b 563 were estimated together, as their spectra were not separable. Assay of photochemical and respiratory activities Activities of photosystems I and II in isolated chloroplasts were assayed at 25 C at saturating irradiance using a Hansatech oxygen electrode according to the method described by Plesnicar & Bendall (1973). Photosystem II activity was assayed as the rate of light-dependent oxygen evolution observed in the presence of potassium ferricyanide as the electron acceptor in a reaction mixture rendered anaerobic by pre-fiushing with nitrogen. Photosystem I activity was assayed as the rate of light-dependent oxygen uptake in the presence of 3, 4 dichlorophenyl- 1, 1-dimethyl urea and ascorbate plus tetramethyl-p-phenylenediamine as the electron donor and methyl viologen as the electron acceptor. In the latter assay, the reaction mixture was rendered aerobic by pre-fiushing with air. Respiratory activity was assayed with the Hansatech oxygen electrode in a buffer medium containing 0 2 M sucrose, 10 mm KH2PO4, ph 7 2, 10 mm KCl, 5 mm MgCla, aerated prior to use. To 2 0 ml of the reaction buffer contained in the electrode vessel, 100 to 500 /i\ of the mitochondrial extract was added. The rate of oxygen

3 Photosynthesis and respiration in ferns uptake was monitored after each addition of 25 /il 20 mm NADH, 25 /^l cytochrome c (lomgml"^), 50/^1 100 mm ascorbate, 50 jltl 100 mm tetramethyl phenylene diamine (TMPD) and finally 20 jul 50 mm KCN. The activity was determined as the rate of oxygen uptake in the presence of NADH and cytochrome c and was expressed as /tmol Og consumed mg~^ protein h~^ RESULTS Total chlorophyll and chlorophyll a and b ratios in the shade and sun ferns are shown in Table 1. There was a slightly greater amount of chlorophyll expressed on a fresh weight in the shade ferns. However, the chlorophyll content of the shade ferns per unit leaf area was about the same as that in the sun species. The variations in fresh weight per unit leaf area are probably related to diflferences in leaf morphology and structure. Irrespective of the level of chlorophyll in the leaves, the soluble protein content (Table 1) was considerably higher in the sun species as reported earlier by Boardman (1977). Table 1. Chlorophyll and soluble protein content in sun and shade ferns Fresh weight per unit leaf area (g dm~^) Chlorophyll content (mg g"' f. wt) Chlorophyll content per leaf area (mg dm~^) Chlorophyll a/b ratio Ratio soluble protein/ chlorophyll (mg mg"*) Abacopteris multilineata Tectaria vasta ± ± ± ±O ± ± Blechnum orientate Dicranopteris linearis Lygodium scandens 2-10± l-60± ± ±0-08 l-49±0-21 l-55±o-16 l-79± Means±SD (w - 5). Since the amount of chlorophyll calculated on a fresh weight basis was greater in the shade ferns than the sun ferns (although not necessarily so on a per unit leaf area basis), we decided to compare the content of another tetrapyrrole, protohaem, of the ferns from the two types of habitats. This is particularly relevant, since both chlorophyll and protohaem are believed to share a common pathway (Castelfranco & Beale, 1983). Furthermore, protohaem makes up the prosthetic group of cytochromes which participate in electron transfer activities of the chloroplasts and mitochondria. The amount of protohaem in the sun ferns was greater than that of the shade ferns (Table 2). The protohaem value m the sun and shade ferns was comparable to that reported in higher plants (Nasrulhaq- Boyce & Jones, 1981). Plants grown under high irradiance exhibit a greater photosynthetic rate at light saturation than shade plants, but lower rates at low irradiance (Bohnmg & Burnside, 1956; Boardman, 1977). Ludlow & Wolf (1975) reported similar findings for temperate sun and shade ferns. However, few data are available

4 84 A. NASRULHAQ-BOYCE AND M. A. HAJI MOHAMED Table 2. Protohaem content of some Malayan sun and shade ferns Abacopteris multilineata Blechnum orientale Dicranopteris linear is Lygodium scandens Protohaem (nmol g~' f. wt) 1-60 ±0-36 l-70± ± Means+ SD (w = 3). 50 "401- _L I _L I I I Irradiance (W m~^) Fig. 1. The effect of varying irradiance on the in vitro photosystem I activity of chloroplasts isolated fronn sun and shade ferns: (A); Dicranopteris linearis (#); Stenochlaena palustris (O); (A). concerning the rate of electron transport driven by both photosystems I and II in isolated chloroplasts from sun and shade ferns. Furthermore, most of the rates reported in the literature have been observed by measuring the rate of reduction of 2, 6 dichlorophenolindophenol (DCPIP). We report here our findings on the photochemical activities of isolated chloroplasts from sun and shade ferns using a Clarke-type oxygen electrode, a more sensitive and accurate method. Chloroplasts from the sun ferns showed greater photochemical rates than did chloroplasts isolated from shade ferns at saturating irradiance (Fig. 1 and Table 3). However, at low irradiance, the rate of electron transport was greater in the chloroplasts from the shade ferns. Saturation was reached at about 4 W m~^ for the two shade ferns (C. aesculifolia and T. singaporeana), whereas it was reached at around 25 W m~^ for the two sun ferns {D. linearis and S. palustris; Fig. 1). The lower rates of photochemical activity observed in the shade ferns might be assumed to be a result

5 Photosynthesis and respiration in ferns of expressing the rate of photochemical activity on the basis of per unit of chlorophyll, since the shade plants had more chlorophyll when compared to the sun ferns. However, this assumption does not seem to be true, because the difference in the rates of photochemical activities between the sun and shade ferns does not correlate with the differences in the amounts of chlorophyll. Following reports that sun ferns show greater photochemical as well as photosynthetic rates at light saturation than shade ferns, we investigated the possible relationship between this phenomenon and an increase in electron transport components. Both sun ferns had considerably more cytochromes /, b 559HP and b 559LP plus b 563 when compared to the two shade ferns (Table 4). This indicates that the sun ferns possess greater capacity for electron transport and thus probably explains the greater rate of photochemical activity observed at light saturation. In isolated mitochondria, the rate of electron transport was greater in the sun ferns when the rate was determined by the oxidation of exogenous NADH as well as with ascorbate and tetramethyl phenylene diamine (TMPD) as electron donors (Table 5). Although the oxidation of exogenous NADH does not flow through the entire respiratory chain, it nevertheless reflects the respiratory capabilities of the mitochondria. Table 3. Total photochemical activities of chloroplasts isolated from some shade and sun species Dicranopteris linearis Photochemical activity (jj. equivalent e~ transferred ^ chlorophyll) min"' O ± Means ±SD {n-a). Table 4. Photosynthetic cytochrome content in chloroplasts isolated from and shade ferns sun Photosynthetic cytochrome content (nmol mg""' chlorophyll) / 6 559HP. b 559LP + b ± ± ± ± ± ±0-86 Dicranopteris linearis 5-46 ± ± ± ± ±4-21. Means + SD {n 3).

6 86 A. NASRULHAQ-BOYCE AND M. A. HAJI MOHAMED Table 5. Respiratory activity of mitochondria isolated from shade and sun ferns Respiratory activity (nmol O2 uptake mg~' protein min~*) Dicranopteris linearis Means+ SD (n = 3). With NADH and exogenous cytochrome c as electron donors 3-67 ± ± ±0-90 With ASC/TMPD as electron donors ±l O DISCUSSION The chlorophyll content expressed on a fresh weight basis was higher in shade ferns than sun ferns (Table 1). Similar findings have been reported by Ludlow & Wolf (1975) and Boardman (1977). However, the chlorophyll content per unit leaf area was about the same in both the shade and sun ferns except in the case of C. aesculifolia which had a higher chlorophyll content. This exception is to be expected, since the leaves of C. aesculifolia are much thicker than the other ferns. However, leaves of sun plants are generally thicker than leaves of shade plants (Boardman, 1977; McMillen & McClendon, 1983). A higher ratio of soluble protein to chlorophyll was exhibited by sun ferns compared to the shade ferns, irrespective of the differences in the chlorophyll level between the two types of ferns. The higher soluble protein content in the sun ferns probably reflects the higher concentration of ribulose bisphosphate carboxylase (RUBISCO). Although the concentration of RUBISCO was not determined here, it has been reported that high RUBISCO activity is associated with high photosynthetic rates observed in sun plant leaves at light saturation (Boardman, 1977). This result is consistent with our findings in that the sun ferns have greater protohaem and photosynthetic cytochrome content than their share counterparts (Tables 2 and 4). The haemoproteins include cytochromes and enzymes such as catalase, peroxidase and nitrate reductase. The greater protohaem content in the sun ferns is probably due to the greater photosynthetic cytochrome content found in these plants (Tables 2 and 4). This in turn possibly explains the higher photochemical activities observed in the chloroplasts isolated from the sun plant leaves (Table 3). Although it is believed that both chlorophyll and protohaem share a common pathway in plants (Castelfranco & Beale, 1983), the results obtained here show that there is a big difference in the protohaem levels in the two types of ferns. It is highly probable that more protoporphyrin IX (an intermediate in chlorophyll and protohaem synthesis) is channelled into protohaem synthesis in the sun ferns, thus reducing chlorophyll levels. A study of the photochemical activities of the chloroplasts isolated from the ferns show that the sun ferns have greater in vitro photosystems I and II activities in saturating irradiance than chloroplasts from shade ferns. However, at low

7 Photosynthesis and respiration in ferns 87 irradiance, shade chloroplasts showed greater rates of oxygen uptake. Similar results were obtained by Ludlow & Wolf (1975), who studied photosystem II activity with DCPIP. They reported considerably higher Hill reaction activity in the sun ferns. These results confirm that the shade chloroplasts are more efficient at low irradiance, while the sun chloroplasts have a greater capacity for electron transport at higher irradiance. The greater capacity for electron transport may be attributed to the higher level of electron transport carriers present. Cytochromes /, b 559HP, b 559LP and b 563 were present in greater amounts in chloroplasts isolated from the sun ferns (Table 4). When the total cytochrome b content is compared with the total cytochrome / content, the shade ferns were found to have a b 559jjp + ft S59^^ + b 563 :/ ratio of 5, while the ratio for the sun ferns is 4. Cramer & Whitmarsh (1977) reported that the sun plant Atriplex, when grown at moderate irradiance, had a b :f ratio of 4; when grown at high irradiance, the ratio of total b./was only 3. This confirms that the shade plants possess greater amounts of the b cytochromes relative to cytochrome/than the sun ferns. Why this is so is not clear and is also complicated by the fact that the functions of b cytochromes in photosynthetic electron transport have yet to be elucidated. The sun ferns have a higher mitochondrial respiratory rate compared to the share ferns (Table 5). The greater rate of electron transport observed in the sun plants is probably a result of the greater quantity of redox components present in their mitochondria. ACKNOWLEDGEMENTS We thank A. J. Omar, Shamshunnisa Yakob and Rabiah Bidin for the various forms of assistance rendered during this investigation. REFERENCES ANDERSON, J. M., GOODCHILD, D. J. & BOARDMAN, N. K. (1973). Composition of the photosystems and chloroplast structure in extreme shade plants. Biochimica et Biophysica Acta, 325, ARNON, D. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase Beta vulgaris. Plant Physiology, Lancaster, 24, 1-5.» c D- \ BENDALL, D. S., DAVENPORT, H. E. & HILL, R. (1971). In: Methods in Enzymology (Ed. by A. ban Pietro), 23, Academic Press, New York. BOARDMAN, N. K. (1977). Comparative photosynthesis of sun and shade plants. Annual Review of Plant Physiology, 28, BoHNiNG, R. H. & BuRNSiDE, C. A. (1956). The effect of light intensity on the rate ot apparent photosynthesis in leaves of sun and shade plants. American Journal of Botany, 43, L BoNNER, JR, W. D. (1967). A general method for the preparation of plant mitochondria. In: Methods in Enzymology (Ed. by R. E. Estabrook & M. E. Pullman), 10, Academic Press, New York. CASTELFRANCO, P. A. & BEALE, S. I. (1983). Chlorophyll biosynthesis: recent advances and areas ot current interest. Annual Review of Plant Physiology, 34, CASTELFRANCO, P. A. & JONES, O. T. G. (1975). Protohaem turnover and chlorophyll synthesis m greenmg barley tissue. Plant Physiology, Lancaster, 55, 485^90.. CRAMER, W. A. & WHITMARSH, J. (1977). Photosynthetic cytochromes. Annual Review of Plant I^hysiology, 28, GRIFFITHS, W. T. (1975). Characterisation of the terminal stages of chlorophyll (lde) synthesis m etiopiast membrane preparations. Biochemical Journal, 152, u K K' HEW, C. S. & WONG, Y. S. (1974). Photosynthesis and respiration of ferns in relation to their habitat. American Fern Journal, 64, ,, LOWRY, D. H., ROSEBROUGH, N. J., F.^RR, N. L. & RANDALL, R. J. (1951). Protein measurement with the FoVm phenol reagent. Journal of Biological Chemistry, 193, LUDLOW, C. J. & WOLF, F. T. (1975). Photosynthesis and respiration rates of ferns. American tern Journal, 65,

8 A. NASRULHAQ-BOYCE AND M. A. HAJI MOHAMED McMiLLEN, G. G.& MCCLENDON, J. H. (1983). Dependenceof photosynthetic rates of leaf density thickness in deciduous woody plant grown in sun and shade. Plant Physiology, Bethesda, 72, NASRULHAQ-BOYCE, A. & JONES, O. T. G. (1981). Tetrapyrrole biosynthesis in greening etiolated barley seedlings. Phytochemistry, 20, PLESNICAR, M. & BENDALL, D. S. (1973). The photochemical activities and electron carriers of developing barley leaves. Biochemical Journal, 136,

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