Photosynthesis, dark respiration and light independent ... - Springer Link

Polar Biol (1991) 11:329-337

c SpringerNerlag 1991

Photosynthesis, dark respiration and light independent carbon fixation of endemic Antarctic macroaigae* D.N. Thomas and C. Wiencke Alfred-Wegener Institut ffir Polar-und Meeresforschung, Columbusstrasse, W-2850 Bremerhaven, Federal Republic of Germany Received 12 February 1991; accepted 19 May 1991

Summary. The light saturated photosynthesis, dark respiration and light independent carbon fixation of macroalgal species endemic to the Antarctic were measured. Five brown algae. Ascoseira mirabilis, Desmarestia anceps, D. antarctica, Phaeurus antarcticus, Himantothallus 9randifolius and the red alga Palmaria decipiens were included. Rates of these three parameters at 0~ were very similar to those measured in other studies on temperate algae at higher temperature. This indicates a high degree of physiological adaptation to the Antarctic environment within these species. A comparison was made of polarographic and chemical means of measuring oxygen flux during photosynthesis and dark respiration at low temperature. There was a good correlation between measurements of oxygen evolution and carbon fixation, although apparent photosynthetic quotient values were in most cases high. Introduction Apart from a very few studies (Drew 1977; Gutkowski and Maleszewski 1989; Ohno 1984), there has been little physiological investigation on the marine macroalgae endemic to the Antarctic region. Recently freezing tolerance of a freshwater Zygnema species from the region has been studied (Hawes 1990). This lack of research activity is surprising, since these algae belong to a biogeographically important flora, and are clearly capable of prolific productivity under harsh environmental conditions (Dieckmann et al. 1985). It is most likely that metabolic processes in these algae are adapted to facilitate growth and reproduction at very low temperatures, and possibly such adaptation results in the observed endemism. Physiological temperature adaptation within Antarctic fauna has * Contribution No. 4! 5 from the Alfred-Wegener-lnstitut f/Jr Polaru. Meeresforschung Offprint requests to.- D.N. Thomas Abbreviations: HEPES = N-(Hydroxyethyl) piperazine-N'-(2 ethanesulphonic acid); RuBP = D-ribulose 1,5-bisphosphate

been intensively studied (Clarke 1990), and it is necessary to extend such investigations to other constituents of the Antarctic ecosystem such as the macroalgae. In recent years growth characteristics of several Antarctic macroalgal species have been studied (Wiencke 1990a,b; Wiencke and Fischer 1990; Wiencke and tom Dieck 1989). They have shown that growth optima in these algae are very much restricted to low temperatures, although their actual distribution (endemism) is not limited by temperature tolerance. It has also been shown that endemic species are able to grow under conditions of very low light, saturation levels for growth being extremely low. Seasonal growth starts in September under ice cover, still during the period of very low light (Hastings 1977; Wiencke 1990a,b). The study reported here was designed to investigate some basic parameters of the carbon metabolism of some of these species. Photosynthetic performance above saturating light levels, dark respiration and rates of light independent carbon fixation were measured. Assimilation of carbon in the dark is thought to be important in the carbon metabolism of temperature macroalgae (Kremer 1981). In brown algae rates are on average 5% of light saturated carbon assimilation values, although rates up to 46% have been reported in the growing region of Laminaria hyperborea (Kfippers and Kremer 1978). Negligible rates of light independent carbon fixation are found in red and green algae (Kremer 1978; Kfippers and Kremer 1978). The pathways responsible for dark carbon fixation have been well studied, and much of this work is summarised by Kremer (1981) and Kerby and Raven (1985). Clearly an ability to assimilate carbon through pathways other than photosynthesis is of ecological importance in situations where environmental constraints reduce photosynthetic capability. This is especially true for periods when prevailing light conditions are poor. There are difficulties in measuring oxygen production/ consumption at low temperatures using polarographic electrodes, and often procedures based on determination of oxygen content using Winkler or micro-Winkler techniques are used in preference (Peck and Uglow 1990).

330 However, u n d e r stable c o n d i t i o n s electrodes have b e e n s h o w n to m e a s u r e o x y g e n c o n c e n t r a t i o n s a c c u r a t e l y over wide t e m p e r a t u r e r a n g e s (Wise a n d N a y l o r 1985). It was d e c i d e d to i n c l u d e a c o m p a r i s o n of p o l a r o g r a p h i c a n d c h e m i c a l m e t h o d s for m e a s u r i n g o x y g e n fluxes p r o d u c e d b y A n t a r c t i c m a c r o a l g a e at low t e m p e r a t u r e s . T o c o m p l e m e n t these m e a s u r e m e n t s c a r b o n a s s i m i l a t i o n was also e s t i m a t e d by m e a s u r i n g 1"C u p t a k e . T h i s p r o v i d e d supplem e n t a r y p h y s i o l o g i c a l i n f o r m a t i o n to t h a t given by the o x y g e n m e a s u r e m e n t s , a n d also e n a b l e d a m o r e c o m p l e t e c o m p a r i s o n of the m a j o r m e t h o d s used for e s t i m a t i n g photosynthetic production.

Material and methods Algal species included in this investigation are endemic to the Antarctic region, and were isolated as spores on King George Island, South Shetland Islands, Antarctica (Clayton and Wiencke 1986; Wiencke 1988). Plants were maintained in the laboratory at Bremerhaven at 0~ under fluctuating daylength conditions representative of the seasonal variation incident in the field. Details of culture conditions employed have been described by Wiencke (1990a, b) and Wiencke and tom Dieck 0989). All experiments were conducted between mid September and early December, the period during which highest growth rates occur within these species (Wiencke 1990a, b). For some species, plants of different stages of growth, and material grown at different irradiances were used. Five brown algae were studied: a) Ascoseira mirabilis Skottsberg- Young, 6 month old plants (2 cm), and two year old plants (25 cm) were compared. In the latter, measurements were made on both basal and apical regions of thalli. The region of growth in this species is at the base of the blade (unpublished data). All material was grown at a photon fluence rate of 1 0 # m o l m 2s 1. b) Desmarestia anceps. Montagne- Very young, partially corticated sporophytes (Fig. 1), as well as 12 month old plants (Fig. 2) were used. In the latter, the effect of preculture irradiance was studied using plants grown at 0.6, 3, 10, 25 and 50/~mol m - 2 s -~. c) Desmarestia antarctica Moe & Silva- One year old plants (Fig. 4) grown at 3 and I0 ~mol m-2 s 1, as well as very young filamentous, uncorticated plants (Fig. 3) grown at 1 0 # m o l m - 2 s 1 were included. d) HimantothaUus 9randifolius (A. et E. S. Gepp) Zinova- 18 month and 6 month old material were compared. The former was grown at 10 #mol m - 2 s- ~, and the latter at 3 #mol m 2 s 1. e) Phaeurus antarcticus Skottsberg.-Very young plants (Fig. 5), grown at 10/~molm-2s i were used. A red algal species, Palmaria decipiens (Reinseh) Ricker, grown at l0 #molm -2 s-1. was also included. Parts of plants from the previous season and very young actively growing tissue (Fig. 6) were compared. For all experiments, Provasoli enriched seawater (Mc Lachlan 1973) buffered to a pH of 8.0 with 5 molm -3 HEPES/NaOH was used. The total carbon dioxide content (all forms) of the seawater before enrichment was 2.18molm -a (measured by methods of Strickland and Parsons 1968). A further 3 molto -3 NaHCO3 were added to ensure that dissolved inorganic carbon was saturating for photosynthesis (Johnston and Raven 1986a). For several species it was necessary to cut pieces of thallus for experimentation. All plant material was selected and cut (if necessary) 24 h before use, and stored in fresh aerated media at 0~ in the dark. Similar parts of plants were used for replicates and to compare different methods. It is realised that there are conflicting views about using cut pieces of algal thalli for measurements of photosynthesis and respiration (Hatcher 1977). However, Drew (1983a) has shown that cutting of experimental plant material may not have such

adverse effects on subsequent measurements of photosynthesis and respiration as has sometimes been assumed. Bidwell and McLachlan (1985) also showed wound respiration to disappear when tissues were 'aged' in sea water for 12 h. Oxygen electrode measurements Measurements of photosynthesis and dark respiration were made in incubation chambers (volume -14 ml) fitted with oxygen electrodes (Eschweiler GmbH). The chambers and electrodes were maintained at 0~ by immersing the whole apparatus in a water bath maintained at 0+0.01~ The light source was a slide projector (Leica P2000 Pradovit, with 24 V 250 W halogen bulb) equipped with a range of neutral-grey glass filters (Schott GmbH). Light was reflected using a mirror onto the transparent top of the chamber. The photon fluence rate incident at the position of the thallus surface had been previously measured using a Quantummeter (Licor Inc., Li-185B equipped with a Li-190SB Quantum sensor). All experiments were performed using blue-green light (Schott GmbH, BG-38. Lfining 1980) at 200 #mol m - 2 s- 1. This irradiance has been found in preliminary investigations to be well above the saturation level for photosynthesis, but below photoinhibitory levels in all investigated species. The electrode was calibrated at 0~ before each measurement with media saturated nitrogen and air. Oxygen levels for calibration were taken from Truesdale et al. (1955). Experimental plant material was carefully blotted, weighed and then tied with fine nylon thread to a removable glass stage which allowed free movement of a magnetic stirrer at the bottom of the chamber. Media, which had been sparged briefly with nitrogen (oxygen content reduced to approximately 80%), was then introduced into the chamber. Dark respiration rates were measured, and continued until a constant rate of oxygen consumption was obtained (15 20 rain). The rate of photosynthesis was then measured, until a constant rate of oxygen production was recorded (aprox. 20 min). Eight replicates were used for each sample.

14C assimilation measurements After measurement with the oxygen electrode, samples were transferred still tied to the glass stage to an identical chamber without an electrode, and fresh media introduced. By moving the whole stage it was possible to ensure that the same thallus surface was incident to the light in both incubations. Rates of dark carbon fixation were then determined using samples pre-incubated in the dark for 30 rain, and rates of light assimilation measured in samples pre-incubated at a photon fluence rate of 200 #tool m -2 s-1 for 15 rain. Both dark and light assimilation rates were measured for 30 min after addition of 9.1 KBq 14Cm1-1 as NaH14CO3 (Amersham Buchler GmbH). Following incubation, plants were removed from the chambers, rinsed quickly in three washes of unlabelled media, and then placed into liquid nitrogen. After evaporation of the nitrogen, samples were solubilised by adding 200 #l of perchloric acid (70 %) followed by 500 #l of hydrogen peroxide (35%). Samples were heated at 70~ until the tissue was solubilised and a colourless solution remained (Johnston and Raven 1990; Lobban 1974). When cool, 5 ml of Hionic Fluor (Packard Inc.) scintillation cocktail was added, and the samples then left overnight in the dark. Activity was measured using a Packard Tri-Carb 460C liquid scintillation co: L: er. Uniformly labelled 14C-hexadecane (Amersham Buchler GmbH) was used for quench correction. From the eight replicate samlzL~ used for oxygen electrode measurements, four replicates were available for light and dark assimilation measurements. Winkler determinations Plant material was placed into glass stoppered bottles filled with experimental media that had previously been sparged with nitrogen.

331

Figs. 1-6. Different developmental stages of some of the endemic Antarctic macroalgae used for experimentation. 1 Partially corticated Desmarestia anceps sporophyte (Scale b a r = 0 . 5 mm). 2 One year old D. anceps sporophyte (Scale b a r = 5 cm). 3 Very young uncorticated D. antarctica sporophyte (Scale b a r = 0 . 5 mint. 4 One

year old D. antarctica sporophyte (Scale b a r = 2 cm). 5 Young uncorticated Phaeurus antarcticus (Scale b a r = 0.5 mm). 6 Palmaria decipiens- showing previous seasons and new growth (Scale bar = 3 cm)

Four replicate bottles were incubated at a photon fluence rate of 200/2mol m - 2 s - 1 under blue-green light as described above. Four replicate bottles were incubated in the dark, and four bottles that did not contain plants were used as controls. The latter were incubated in the light for the same length of time as the light-bottles. Incubations were carried out in a constant temperature room maintained at 0~ I~ However, due to the irradiance used and the subsequent proximity of the light source to the bottles, photosynthetic measurements were in fact made at 2~ Incubation periods varied between experimental runs depending on the amount of material available (light-2 to 4 h; dark-5 to 6 h), although within one experimental run plants were incubated for the

same period. During incubations bottles were shaken periodically. Plants were subsequently removed and the oxygen content of bottles determined using a modified Winkler method (Strickland and Parsons 1968). Oxygen contents of control bottles were subtracted from those containing plant material. It was thought that the introduction and removal of plant material may introduce oxygen into the incubation bottles therefore over-estimating photosynthetic rates, and under-estimating dark respiration rates. However, there was no significant difference in photosynthetic and respiration rates in an experiment were the oxygen contents of bottles were 'fixed' before or after removal of plant material. It was considered better to remove plant material in

332 order to awfid any abnormal oxygen fluxesor compound exudation on killing plants by addition of Winkler reagents. The latter problem has caused complications in studies based on Winkler methods (Drew 1977; 1983a).

Results and discussion The oxygen production/consumption measured by polarographic and Winkler methods, together with light and dark carbon assimilation rates are given in Table 1. The experimental design used during this study enables a reliable estimate of PQ (molar ratio of the rate of oxygen production to the rate of carbon dioxide utilization) to be made, since the same tissue was used to measure oxygen production (electrode) and light ~4C assimilation under identical incubation conditions. In order to calculate PQ, dark respiration rates have been added to net photosynthetic values thereby giving an estimate of gross photosynthetic oxygen production. Clearly it is uncertain how accurate a correction for respiration in the light this is (c.f. Bidwell and McLachlan 1985). Values of dark ~4C assimilation have also been subtracted from those of light 14C assimilation. Due to difficulties of measuring rates in absolute terms it is better to treat the values as "apparent PQ" (Platt et al. 1987).

Oxygen production measured in the light is an estimate of net photosynthetic production, however, it is a matter of debate as to what is measured using the 14C method. It has been reported that 14C assimilation in the light estimates gross photosynthesis, net photosynthesis or something in between (Raven 1990). In this study the rates of 14C assimilation consistently gave the lowest estimates of photosynthetic production, although the HCO~ concentration was identical in all assays. Differences between the 14C method and methods based on measuring oxygen production have been discussed by Williams et al. (1979, 1983) in regard to phytoplankton productivity. They have rightly emphasised that the methods are measuring two different parts of the photosynthetic process: "the 14C method ideally determines carbon flux, whereas the oxygen method gives results more associated with energy flux." Drew (1977), reported that the 14C method can give up to two times higher measurements of 'production' than simultaneous oxygen measurements using a Winkler method. However, physiologically it is difficult to understand how this can be so. There was only one instance in the present study, when ~4C values significantly exceeded oxygen estimations of photosynthesis (young, partially corticated Desmarestia anceps), although these measurements were not made simultaneously, and only Winkler determinations of oxygen production were made.

Methodological considerations

Photosynthesis and dark respiration

It is pertinent to consider first the variation in rates obtained using different methodologies. Due to the 2~ temperature difference in incubation temperature, measurements of net photosynthesis using the electrode and Winkler titrations are not strictly comparable. It is expected that rates measured at this higher temperature are greater than would be recorded at 0~ However, in Ascoseira mirabilis (6 month), Desmarestia anceps. D. antarctica and Himantothallus grandifolius (18 month), rates of net photosynthesis obtained using the chemical method are slightly lower or similar to those measured with the electrode. In the other plants rates obtained from the Winkler determinations are only slightly higher. Only in two cases were much greater rates recorded with the Winkler method (Palmaria decipiens and young H. grandifolius.) In general, therefore, the polarographic method gave higher estimates of photosynthesis than the chemical method. This is certainly true if the effect due to temperature difference is taken into consideration. However, dark respiration rates measured using the Winkler method were in all cases much lower than those measured with the electrode. Interestingly, Drew (1983b) obtained respiration rates with an oxygen electrode about half those measured with a Winkler sytem. One obvious source of error associated with the Winkler method used during the present study that will cause underestimation of gas exchange is that the bottles were only shaken periodically during the experiment, and there was no constant water movement around the plants (Drew 1977; Dromgoole 1978).

Rates of net photosynthesis measured during this study compare well with the range of saturated net photosynthetic rates measured in other studies on temperate macroalgal species at higher temperatures (Surif and Raven 1989, 1990; Raven et al. 1979, 1989). Drew (1977) and Healey (1972) also found photosynthetic rates of Antarctic and Arctic macroalgae respectively, to be of a similar magnitude to temperate species. Drew (1977) argued that no marked physiological adaptation to the Antarctic environment had taken place since temperature optima for these algae were the same as those of winter temperate material. However, it is felt that the ability of Antarctic algae at 0~ to photosynthesize at rates similar to temperate species at higher temperature certainly must reflect considerable adaptation to low temperature. Such adaptation is also suggested by the growth measurements of Wiencke (1990a, b), and Wiencke and Fischer (1990) in which rates at 0~ are of a similar magnitude to those of temperate species at higher temperatures (Bolton and L/ining 1982; Fortes and L/ining 1980). Descolas-Gros and de Billy (1987) have found evidence for molecular adaptation for the regulation and maintenance of RuBP carboxylase in Antarctic diatoms at low temperatures. They found that RuBP carboxylase has a minimum K m for substrate at 4.5~ in Antarctic species and at 20~ for temperate ones. Similar adaptation may have taken place in these endemic Antarctic macroalgae, and investigations at the biochemical/molecular level are clearly called for. Rates of dark respiration measured with the oxygen electrode were high, compared to some of the values given

4.4(0.8) 9.3(2.1)

7.2(1.5) 13.9(2.7) 23.9(6.3) -2.1 (1.3) --3.0(1.3)

4.6(0.4) -5.2(1.3) -7.9(2.6)

-2.4(1.1) -7.4(2.6)

-4.7(1.7}

-5.4(1.7)*** -6.9(2.5) N.D.

N.D -6.1(2.8) 9.9(2.8) --6.0(2.2)*** -7.0(1,7) N.D.

9.0(0.8) 16,4(1.3)

14.0(1.8) 17.8(1.8) 21.4(0.8)

17.2(1.7) 27.5(3.7)

N.D.

7.8(1.6) N.D. 43.5 (2.9)

19.6(3.3) 21.2(2.4) 20.4(1.9) 21.5(1.4) 23.2(0.7) 16.3(2.0)

-0.4(0.1) -0.3(0.1)**

-0.7(0.1) - 0.3 (0.2)** -0.7(0.3)

-1.3(0.1) -0.9(0.2)

N.D.

-1.0(0.7) N.D. 3.2 (1.8)

-1.4(0.6) -1.6(0.2) 1.0(0.3) -1.3(0.1) -l.2(0.0) -2.2(0.8)

Dark

4.7(0.6) l 1.6(1.5)

7.1 (1.3)* 11.4(3.6)* 19.0(2.8)**

11.0(2.7) 14.5(4.6)

5.9(2.6)

6.6(1.1) N.D. 43.9 (9.6)

24.8(2.9) 15.0(1.7) 18.9(3.3) 18.4(3.6)** 19.7(2.7) 29.7(7.2)

Light

~4C assimilation

~O2 values: ~tmol O2g l FW h 1; 14 C assimilation: F~mol C g ~ FW h hO 2 electrode n = 8; Winkler n = 4; 1"~Cassimilation n = 4 (unless otherwise indicated: *n = 2, **tl = 3, ***n= 7j r deviation given in parenthesis dN.D.: Not Determined

Old growth New growth

Palmaria decipiens

2 year plants-Top (non growing) -Base (meristem) 6 month plants

Ascoseira mirabilis

18 month plants 6 month plants

Himantothallus grandilblius

14.2(2.2) 18.7(4.2)

9.5(3.3)

Young uncorticated plants

Phaeurus antarcticus

7.5(1.7) 9.5(3.3) N.D.

N.D 27.7(2.5) 24.2(2.0) 26.0(2.4)*** 22.7(2.0) N.D

Light

Light

Dark

O2-Winkler

O2-Electrode

1 year from 3 #molm-2s i 10 ,umol m 2s-1 Very young uncorticated plants

Desmarestia antarctica

1 year from 0.6 p m o l m - 2s ~ 3/tmolm 2s i 10/zmol m 2s-~ 25 #molm 2s-l 50~molm 2s 1 Young plants (Partly corticated)

Desmarestia anceps

Species

0.2(0.1) 0.2(0.1)

0.5(0.0)* 0.7(0.1)* 2.5(1.0)

0.4(0.1) 0.4(0.1)**

0.7(0.3)

0.3(0.1) N.D. 1.2 (0.2)

0.7(0.1) 1.5(0.6) 0.5(0.1) 0.6(0.1) 0.4(0.4) 1.0(0.3)

Dark

1.4 1.1

1.8 1.8 1.9

1.6 1.9

2.7

2.1 N.D. N.D.

N.D. 2.5 1,9 1.8 1.5 N.D.

Apparent PQ

3.6 1.3

7.0 6.2 13.0

4.0 3.0

12.0

5.0 N.D. 2.8

2.7 9.7 2.6 3.4 2.1 3.5

Dark 14C assimilation% of light ~4C assimilation

8.0 4.9

10.8 13.4 31.1

18.6 5.8

15.1

6.1 N.D. N.D.

N.D. 24.1 4.9 10.6 6.0 N.D.

Dark a4C assimilation% of dark respiration

Table I. Net 0 2 exchange of endemic Antarctic macroalgae measured in the light and dark, using polarographic and Winkler titration methods. Rates of light and dark ~4C assimilation are given. Dark a4C assimilation values are also expressed as a percentage of light x4C assimilation, and dark respiration (only those measured with the electrode). Apparent photosynthetic quotient (PQ) values have been calculated using oxygen production measured with the electrode and ~4C assimilation in the light

L,,a

334 m the literature for temperate algae at higher temperatures. Drew (1977) measured respiration rates in Antarctic macroalgae in the same range as those from temperate waters, although Healey (1972) found dark respiration to be less than half that of temperate algae. In the current study the ratio of net photosynthesis to dark respiration lay between 2 and 6. These ratios are low compared to those for many marine algae (often between 5 and 10t, at higher temperature (Raven et al. 1979, 1989, 1990). However, values of approximately 3 are not uncommon (Kirst 1981; Raven and Samuelsson 1988). In mature Desmarestia antarctica and non growing apical regions of Ascoseira mirabilis ratios less than 2 were obtained. The dark respiration rates measured using the Winkler method are in all cases very low, and much lower than recorded in most studies with temperate algae. If these values are compared to net photosynthesis values obtained from the electrodes very high photosynthesis to dark respiration ratios were obtained, which are considered to be unrealistic. This is thought to be a further indication of the underestimation of dark respiration rates by the Winkler method employed. Dark respiration rates of Ascoseira mirabilis measured with both polargographic and Winkler methods were not particularly greater than those measured for the other species investigated. This is contrary to the much higher dark respiration rates measured by Drew (1977) for this species compared to other Antarctic plants. This may have resulted from the methodological problems he encountered using this species. There was little variation in the photosynthetic activity, or dark respiration of Desmarestia anceps grown at different photon flux densities. This was also true of D. antarctica grown at 3 and 10 #molm -2 s- 1 (Table 1). The similarity of the photosynthetic capacity in plants grown over such a range of irradiance, indicates that the plants have a very plastic response to light conditions, and it would appear that the photosynthetic metabolism of these plants is well adapted to low light conditions. However, in the absence of more detailed information such as photosynthesis-irradiance curves and information about chlorophyll content at the different irradiances it is not possible to comment more precisely about the light acclimation potential in these plants. Rates of carbon assimilation in the light were greater in very young Desmarestia plants than in older material. Young, partially corticated D. anceps plants had the highest rates of carbon assimilation measured for this species. Very young, uncorticated D. antarctica had four times greater rates of carbon assimilation and oxygen production (Winkler) compared to one year old plants (Table 1). The morphologies of young plants of both species are quite similar, although such large differences between young and mature D. anceps were probably not recorded, since cortication of the tissues had already started to take place. New season growth of Palmaria decipiens also had rates of oxygen production about two times higher than those found in older material. Carbon assimilation rates were also greater in younger tissue. There was no evidence of differences in dark respiration between the different aged plants of these three species.

Kiippers and Kremer (1978) showed differential patterns of photosynthetic and dark carbon fixation activity in the thalli of several members of the Fucales and Laminariales. In Fucus spp. highest photosynthetic rates were found in growing tips. Higher photosynthetic rates were recorded in the non growing apical regions of the thalli of Laminaria spp, compared to meristem regions. Much higher rates of net photosynthesis and carbon assimilation were found in the bases of Ascoseira mirabilis compared to apical tissues (Table 1). No significant variation in dark respiration was recorded for different parts of the thallus. Although A. mirabilis resembles Laminaria spp. in gross morphology, there are ultrastructural differences between the plants (Clayton and Ashburner 1990), that may account for this variation. There does appear to be a wide variation in spatial and temporal differentiation in carbon metabolism in the thalli of large brown macroalgae: Young tissue of Macrocystis inteyrifolia had higher photosynthetic rates than older tissue (Smith et al. 1983), although the opposite was found in Nereocystis luetkeana (Wheeler et al. 1984). Drew (1983a), in contrast to K/ippers and Kremer (1978) showed there to be very little variation in photosynthesis and dark respiration along the lamina of three Laminaria spp. Very few macroalgal studies have measured both carbon asimilation and oxygen production, and therefore estimates of PQ are rare. Hatcher et al. (1977) reported a seasonal variation in the PQ of Laminaria lonyicruris, ranging between 0.7 and 1.5. Many more estimates of PQ have been made for phytoplankton populations, and although the expected value is often taken to be 1.2, much higher values of up to 2.0 are often measured (Williams et al. 1979). The state of reduction of the nitrogen source has been shown to have a significant effect on PQ. Higher PQ values are to be expected when nitrate is the primary nitrogen source, and lower values when ammonium is mainly taken up (Williams et al. 1979; Williams and Robertson 1991). The high values obtained here for most species would suggest that nitrate is the principle nitrogen source under these conditions. This is certainly likely since the media used were enriched with nitrate. It is realised, however, that variation in protein, lipid and nucleic acid composition will also influence the value of PQ (Kirk 1983; Williams and Robertson 1991). Desmarestia anceps plants grown at lower photon flux density had higher apparent PQ values than those grown at higher irradiances. Palmaria decipiens had lower values of PQ compared to the brown algae, which was particularly so of the new growth. Light independent carbon fixation High rates of light independent carbon fixation were found in older Palmaria decipiens tissue compared to younger tissue (Table 1). Although the percentages of assimilation in the dark to that in the light are not very high, they are higher than is generally reported for marine red algae (Kirst 1981; Kremer 1978; Kremer and kfippers 1977). The metabolic basis of carbon fixation in the dark is stored reduced carbon (Kremer 1981), which will be present in the

335 older tissues in large quantities. In P. decipiens the substrate utilised will mostly be the low molecular weight heteroside floridoside (Karsten et al. 1991). There was no evidence of differences in light independent fixation between the meristem region and non growing tissue in two year old Ascoseira mirabilis plants (Table 1). Again this is in contrast to that found in Laminaria spp. (Kfippers and Kremer 1978). In these it is thought that stored mannitol is translocated from non growing regions of the frond, where it acts as s substrate for the high rates of light independent fixation found in the meristem region. This is an explanation for the possibility for initiating rapid growth during adverse light conditions at the beginning of the growth season (Kremer 1981). It is evident that the same temporal and spatial distribution of carbon metabolism found in Laminaria spp. may not be true for A. mirabilis. Much higher rates of photosynthesis and light independent carbon fixation were recorded in young Ascoseira mirabilis compared to any region of the mature plants. In fact these rates of light independent carbon fixation were the highest measured during this study (13% of light 14C assimilation). In contrast, Kremer and Markham (1979) reported that different developmental stages (gametophytes, zygotes and different ages of sporophyte) of Laminaria saccharina had very similar rates of photosynthesis and light independent carbon fixation. The different aged Himantothallus 9randifolius sporophytes likewise showed no difference in photosynthetic and light independent carbon fixation. However, higher rates of dark carbon fixation were also found in the very young Desmarestia anceps and D. antarctica compared to the older plants (Table 1). In the latter species the tremendously high light assimilation rates meant that light independent carbon fixation accounted for only a small percentage of that assimilated in the light. The reverse is true for the older plants, where dark fixation rates as well as light assimilation values were low. The uncorticated young Phaeurus antarticus (Table 1), had a relatively high light independent carbon fixation to light assimilation ratio, although the actual rate of fixation in the dark was not great. The highest rates of light independent carbon fixation in Desmarestia anceps were found in plants grown at a photon fluence rate of 3/~ mol m - 2 s- ~ (Table 1). However there was no apparent trend with respect to preculture irradiance. Johnston and Raven (1986b) have stated that the role of dark carbon fixation in the overall carbon budget of the Phaeophyceae may have been overstated. This certainly seems to be the case for Ascophyllum nodosum on which they based the statement. The measurements in this study were made at a time when the high rates of light independent fixation would be expected, because of the seasonal development within these species (Wiencke 1990 a, b). It could also be imagined that due to the harsh environmental conditions experienced by these algae and their seasonal growth patterns, that they would have had a higher capacity for light independent carbon fixation than their temperate counterparts. Despite this, the rates of light independent carbon fixation are very much in the order of what has been found in many other investigations

of temperate algae. There was therefore no evidence for greater light independent carbon fixation activity, resulting from adaptation to Antarctic low light conditions. Rates of light independent carbon fixation are most often compared to rates of carbon assimilation in the light. However, maybe a more relevant comparison is to dark respiration. Light independent carbon fixation accounts for between 5 and 31% of the dark respiration rates measured with the electrode. Certainly dark fixation reduces the overall losses of carbon during dark periods. Therein probably lies its major value, together with the fact that it provides important anabolic metabolites such as aspartate and malate. Rates of light independent carbon fixation are in all cases above 30% of respiration rates measured by the Winkler method. In young, and basal parts of older Ascoseira mirabilis plants, the relative rates suggest a considerable net uptake of carbon during the dark. This seems unlikely and serves to further highlight the probable underestimation of dark respiration by this method. Conclusions 1) Rates of light saturated photosynthesis in these selected endemic Antarctic species were of a similar range to those of comparable temperate species. Measured rates of dark respiration were a higher than recorded in some temperate species but of a similar magnitude. That these metabolic processes proceed at 0~ at similar rates to temperate species at higher temperatures, is evidence that these endemic algae are particularly adapted to the low temperatures of Antarctic waters. 2) Rates of light independent carbon fixation were also in the order of rates found in many other species of algae at higher temperature. However, these endemic algae evidently do not have any enhanced capacity for assimilating carbon through this pathway. 3) There is evidence for differences in the temporal and spatial carbon metabolism in Ascoseira mirabilis compared to that found in Laminaria spp. that at least superficially, have a similar gross morphology and growth pattern. 4) Despite experimental limitations rates of photosynthesis measured using the polarographic electrode and by the Winkler method were broadly comparable, the electrode giving the higher rates. There was a large variation in rates of dark respiration measured with the two methods. Differences between the methods can be partly accounted for by shortcomings in the incubation conditions used for Winkler determinations. 5) The ~4C method gave the lowest estimates of photosynthetic activity, although in conjunction with the other methods it provided information not available from the oxygen measurements. Estimates of the apparent PQ were high, although they indicated a good agreement between the oxygen and 14C method for measuring macroalgal photosynthesis at 0~ Acknowled.qements. We are very grateful to G.O. Kirst for his advice and cooperation in providing facilities at Bremen University for some of the analyses. His critical reading of the manuscript is much

336 appreciated. We also thank M. Gleitz for his assistance. The Deutsche Forschungsgemeinschaft gave financial support and the lnstituto Antartico Chileno and R. Westermeier, Valdivia provided the opportunity for C. W. to participate on two expeditions to Antarctica. C. Langreder gave excellent technical support during the study. This work was carried out while D.N.T. held a Guest Research Position at the A.W.I.

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