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Carbohydrate Reserve and Dark Carbon Fixation in the Brown Macroalga, Laminaria hyperborea
Carbohydrate Reserve and Dark Carbon Fixation in the Brown Macroalga, Laminaria hyperborea BRUNO
P. KREMER
Universitat zu K61n, Institut fUr Naturwissenschaften und ihre Didaktik, Abt. f. Biologie, Gronewaldstr. 2, D-5000 K6ln 41 Received May 24, 1984 . Accepted August 25, 1984
Abstract Tissue discs originating from young, growing blade areas and from adult, mature frond regions of the brown macroalga Laminaria hyperborea (Fosl.) Gunn. (Phaeophyceae, Laminariales) were investigated with particular regard to photosynthesis, dark respiration, dark carbon fixation, and carbohydrate reserves. It was found that the mannitoi/laminaran reserve of the young, developing blade meets the requirements of dark respiratory metabolism for only 7 -10 d at 10±2 °C under continuous darkness. A concomitant decrease in the potential for {3carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) occurred along with the depletion of the stored carbohydrate. Restoring the intracellular pool of reserve carbohydrates by photosynthesis and by feeding of exogenously supplied mannitol resulted in a short term recovery of the rates of dark fixation. These findings support the view that (i) in the dark the substrate of {3-carboxylation is mainly derived from mannitol (along with glycolytic degradation of laminaran) and (ii) the young blade is not able to maintain its own carbon balance under the environmental conditions during midwinter and early spring, but relies on a carbon flow from the old blade.
Key words: Laminaria, Brown algae, Carbon fixation, {3-Carboxylation, Storage carbohydrates, Carbon budget, Ecophysiology.
Introduction
As in green plants primary CO 2 fixation in brown marine macroalgae occurs via ribulose-1,5-bisphosphate carboxylase. However, particularly in the species belonging to the brown algal orders Laminariales and Fucales an additional route of inorganic carbon assimilation is operational. It consists of /3-carboxylation of a C 3 unit to give the C4 compound oxaloacetate (OAA). The enzyme performing this carboxylation step has been recognized as a phosphoenolpyruvate carboxykinase (PEPCK) (Akagawa et al. 1972; Kiippers and Weidner 1980; Kremer 1981 a). A temporally and spatially different distribution of enzyme activity attributed to /3-carboxylation in vitro as well as in vivo was encountered with ontogenetically different frond tissue areas, topographically different frond regions, as well as with various stages in the life cycle of brown macroalgae (for literature review see Kremer 1981 band 1981 c). The process of /3-carboxylation of phosphoenolpyruvate (PEP) had hitherto mostly been referred to as dark or non-photosynthetic carbon fixation. Kinetic tracer studies
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indicated that 14C-Iabelling of ~ acids such as aspartate and malate immediately derived from OAA (d. Weidner and Kiippers 1982) is not restricted to dark periods, but also occurs during photosynthesis. Indeed, rates of carbon fixation into specific C4 compounds are approximately equal in the light and in the dark. The distribution of 14C between C 1 and C4 atoms of aspartate suggests a carbon flow from early occurring intermediates of the reductive pentose phosphate cycle such as 3-phosphoglycerate to ~ dicarboxylic acids. In brown macroalgae «dark,. carbon fIxation via is-carboxylation of PEP was assumed to be quantitatively and qualitatively integrated into photosynthetic C02 assimilation thus yielding appreciable 14C-Iabelling of aspartate and malate. Of course the underlying reactions and conversions are basically different from the ~ syndrome of vascular plants and were therefore preferably termed as C4 metabolism (Kremer 1981 a, 1981 d). It was suggested earlier that the metabolic basis for is-carboxylation of PEP in the dark is provided by sequential degradation of mannitol, the main soluble carbohydrate of brown algae. One line of evidence for this assumption were results from inhibition of the Krebs cycle with monofluoracetate and a concomitant increase in dark carbon fixation (Kremer 1981 a). This contribution also deals with the problem of substrate availability by comparing internal pools of stored carbohydrate (mannitol and laminaran) with the potentials for is-carboxylation within the same frond areas of the brown macroalga, lA·
minaria hyperborea.
Materials and Methods Plant Material Adult specimens of Laminaria hyperborea (Gunn.) Fos!. (Phaeophyceae, Laminariales) were collected by SCUBA diving from about 4 m below mean low tide at their natural habitat on the rocky shores near Helgoland (North Sea, Germany) in February 1984. The plants measuring between 1.20 and 1.60 m in length (including stipe and basal holdfast) and apparently free of epiphytes were maintained in 500 I tanks supplied with running seawater at 10 ± 2°C.
Experimental Young, growing blades (see Fig. 1) were cut off from the stipe and the old (second year) blade and stored for up to 7 d in black plastic bags in running seawater under the same temperature regime. Entire plants (without stipes and holdfasts) were kept individually under the same conditions. Tissue discs (2 cm diameter) were punched out from the areas as indicated in Fig. 1 and monitored for (i) photosynthetic and dark respiratory oxygen exchange and (ii) rates of light independent (dark) carbon fixation. Analytical
In addition to metabolic processes such as photosynthesis, dark respiration, and dark fixation the levels of PEPCK activity ( - phosphoenolpyruvate carboxykinase; EC 4.1.1.32) in vitro as well as contents of reserve carbohydrates (mannitol, laminaran) were determined from the same samples. All methods used for oxymetry, 14C-incubation, enzyme assays, and carbohydrate quantification have already been detailed earlier (Kremer 1981 a, 1981 d).
J. Plant Physiol. Vol. 117. pp. 233-242 (1984)
Carbohydrates and dark fixation in Laminaria
~
235
A-II-I- - - B - - - - - 1
~
nl .1 • •
10 em
L-----I
•
I
@ ~) I •
Fig. 1: Laminaria hyperborea with developing new blade (A) and mature, second year, senescent leafy frond region (B) - growth stage about February/March. Samples were taken from entire specimens (1 a - 4 a) as well as from isolated, detached blade regions (1 b - 4 b), each maintained under continuous darkness upon collection.
o Photoayntheela • o.k
I
IJ
respiration
Derk tlatkwt
3D
10
10
3D
60
II
JO
60 Time [mIn]
Fig.2: Metabolic performance (photosynthesis, dark respiration, dark fixation) of different frond regions of Laminaria hyperborea immediately after collection. Oxymetric measurements converted to carbon data assuming O 2/ CO 2 ::::: 1. Vertical bars indicate standard deviation
(n = 5).
J. Plant Physiol.
Vol. 117. pp. 233-242 (1984)
236
BRUNO
P. KRatER
Results
Metabolic performance of ontogenetically different frond tissue It may be seen from Fig. 2 that young, growing, and old, non-growing frond areas of Laminaria hyperborea are distinctly different with respect to photosynthesis, dark respiration, and light independent carbon fixation. Rates of photosynthesis in the leafy meristematic intercalary area of the Laminaria frond under light saturating conditions (photon flue nee density: 150 JLE m- 2 S- I) usually accounted for about 50 % of the respective rates recorded for mature frond areas. A closer inspection of dark respiratory and dark carbon fixation rates measured in vivo yields a different picture: Both rates recorded for the growing zone exceeded those obtained from old blade areas. These findings suggest that the net carbon gain achieved in the intercalary growing zone generating the new blade is presumably far less than the amounts actually needed for growth and development. This emphasizes the demand for further carbon sources supplementing or supporting the anabolic processes occurring in the growing tissue.
'C.-------,
~
•
Derk respiretlon
E$1 lli:;l Derk fixation
5
JFMAMJJA
~
2 -----'
' - - - 7 -----'
'---12 - - J Temperature ['e)
Fig. 3: Temperature profile of dark respiration and dark fixation rates of young (1 a) and old (4a) blade regions of Laminaria hyperborea. Insert: Long-term in situ seawater temperature in the environment of Laminaria hyperborea near Helgoland Qanuary-August) (Data kindly supplied by P. Mangelsdorf/BAH).
Fig. 3 presents information on the temperature profiles of dark respiration and dark fixation for the temperature range between 2 and 12°C. No marked differences in the QIO values between young and old fronds were obtained.
J Plant Physiol. Vol. 117. pp. 233-242 (1984)
Carbohydrates and dark fixation in Laminaria
237
Dark respiratory consumption 0/ reserve carbohydrates The consumption of mannitol and laminaran as substrates of dark respiration by young, excised blades was dependent on time. Fig. 4 describes the respiratory decay of both carbohydrates in continuous darkness as a percentage of the respective amounts determined in controls incubated in the light. The overall decrease of mannitol was more pronounced in tissue discs originating from the growing zone than from the adult blade. Since the latter tissue showed lower rates of dark respiration (d. Fig. 2), a comparatively faster respiratory depletion of the mannitol reserve occurred in the young blade. This also holds good for the laminaran reserves. As a consequence, the growing areas of Laminaria hyperborea were almost depleted within 6-7 d in continuous darkness at 10±2 °C thus contrasting with the old blade tissues
~
i
o.rI< incubation
1
[dl
Fig. 4: Dark respiratory consumption of stored mannitol and laminaran in young and old/intact and isolated blade tissues of Laminaria hyperborea. Insert Fig. 4 a: Decay of the mannitol reserve in a growing frond still connected with its old blade. Insert Fig. 4 b: Levels of PEPCKactivity in young and old fronds maintained in the light (controls) and cultured for 7 d in continuous darkness.
which still had a certain proportion of their original carbohydrate reserve. It is interesting to note that the soluble compound mannitol was obviously more readily metabolized than the polymeric reserve carbohydrate laminaran. Young blade tissue discs originating from entire, non-excised plants maintained in continuous darkness showed a significantly lower cumulative respiratory loss of soluble and insoluble carbohydrate reserves as compared to isolated growing blades (Fig.4). This again demonstrates that the intercalary growing blade is probably not able to maintain its own carbon balance upon the loss of the mature, old blade, at least not during midwinter and early spring. It therefore must rely on a continuous carbon flow which can be fed into its intermediary metabolism. j. Plant Physiol. Vol. 117. pp. 233-242 (1984)
238
BRUNO
I~
P. KREMER
10
100
D@ D®
"
N
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u
8
"0
E
~
6 50 4
2
\....--ld ----'
' - - 4d---.J
Control
I
L-Jd----'
Dark culture
Fig. 5: Dark carbon fixation (in vivo /3-carboxylation) in fronds maintained in continuous darkness, values expressed as a percentage of the initial rates of control specimens maintained in the light. For sample topography d. Fig. 1. Means of 5 replicates.
Dark carbon fixation in plants cultured in the dark Dark fixation of inorganic carbon appears to depend on the experimental pretreatment of the frond tissue samples. The data contained in Fig. 5 indicate that rates of dark fixation in tissue discs originating from the growing region of intact specimens showed only a slight decline in their in vivo potentials for i3-carboxylation. A much more pronounced drop was observed in discs from growing blades which had previously been cut off from stipe and older frond blade and were then kept in continuous darkness. In these samples, /3-carboxylation in vivo was reduced by about 80 % as compared to the controls (Fig. 5). No concomitant decline in the enzymatic potentials for /3-carboxylation (activity of PEPCK) was observed (data not shown). Linear regression analysis of rates of dark carbon fixation against total carbohydrate reserve showed a highly significant correlation (p < 0.01). Hence, the availability of the internal substrate for i3-carboxylation (= PEP) largely affected the rates of dark carbon incorporation via PEPCK.
Feeding of mannitol and dark carbon fixation In order to test the suggested relationship between carbohydrate content and rates of light independent carbon fixation further, it was attempted to partially restore the dark respiratory loss of reserve carbohydrates of the meristematic blades a) by exogenous feeding of mannitol and b) by photosynthetic incubation allowing for de
J Plant Physiol. Vol.
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Carbohydrates and dark fixation in Laminaria
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Table1: Uptake of exogenously supplied mannitol (100mM) and recovery of dark fixation potentials in meristematic frond tissue of Laminaria byperborea. Mean values ± standard deviation (n = 3). ----~-----------------------------------------------
Sample l )
Feeding of mannitol (h)
Mannitol content (I'mol dm -2)
Dark fixation (I'mol C dm -2 h -1)
1b
6 12 24
92±24 215±52 373±69
2.2±O.2 3.8±O.8 5.3±1.4
2b
6 12 24
78±18 229±54 315±61
204±O.2 3.5±O.6 6.2±1.5
I) d. Fig. 1.
novo synthesis of intracellular mannitol. Discs from young, growing blades depleted by continuous darkness were subsequently incubated in the dark in sterile filtered seawater medium containing 100 mM mannitol. Since it was earlier observed that brown macroalgae such as Laminaria spp. scarcely take up dissolved organic molecules from the incubation medium (d. Weidner and Kiippers 1982), tissue discs were divided lengthwise along their medullary layer and incubated separately. In a second set of experiments mannitol-depleted young blade tissue was allowed to photosynthesize under saturating light conditions. The results obtained are shown in Tables 1 and 2. It may be seen that the sliced tissue from the growing blade took up certain amounts of exogenously supplied mannitol dependent on time and thus restored the intracellular polyol pool. Similarly, photosynthesis gradually replenished the mannitol reserve. A concomitant increase in the rates of light independent carbon incorporation occurred together with restored mannitol contents of the same samples (Tables 1 and 2). Thus the overall increase in available mannitol within the tissue significantly enhanced the in vivo potential for a-carboxylation. Table 2: Photosynthetic de novo synthesis of mannitol and recovery of dark fixation potentials in meristematic and adult frond tissues of Laminaria byperborea. Mean values ± standard deviation (n = 3). ----~-----------------------------------------
Sample 1)
Photosynthesis (h)
Mannitol content (I'mol dm - 2)
Dark fixation (I'mol C dm- 2 h- 1)
1b
6 12 24
104±26 165±32 324±84
204±O.3 2.9±O.7 6.6± 104
4b
6 12 24
220±49 378±72 682±42
1.9±O.2 204±Oo4 3.2±O.5
I) d. Fig. 1.
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P. KREMER
Discussion In species of the genus Laminaria the transitional zone between the persistent stipe and the adult blade region exhibit peculiar features with respect to growth, development, and metabolism. This is particularly true for Laminaria hyperborea (Luning 1979; Kuppers and Weidner 1980), but also holds good for further Laminaria species (Chapman and Craigie 1978; Chapman et al. 1980; Kremer 1981 a, 1981 d). In Laminaria the annual growth is mediated by an intercalary meristematic zone and commences during a time of the year, when the temperature of the seawater (cf. Fig. 3) and light supply in the sublittoral zone are far below saturation (Luning 1979; Luning and Dring 1979). One of the most remarkable features of this meristematic zone (during midwinter and early spring still identical with the new blade in Laminaria hyperborea; cf. Fig. 1) is the comparatively high potential for {3-carboxylation of PEP yielding appreciable amounts of C dicarboxylic acids. Evidence for the relative efficiency of this pathway of inorganic carbon fixation was earlier derived from a comparison of photosynthetic and non-photosynthetic incorporation of H I4 C03" in vivo as well as by assays of the respective carboxylating enzyme mediating the entrance of CO 2 into organic compounds (Kremer 1981 a, 1981 d; Kerby and Evans 1983). In the light the substrate of PEPCK is derived from an early photosynthate, 3-phosphoglycerate. Upon {3-carboxylation of PEP, C4 dicarboxylic acids such as aspartate and malate were encountered with the rapidly 14C-Iabelled photoassimilatory products. Nevertheless, the respective metabolic events and conversions are different from those of C 4 photosynthesis in vascular plants, since the primarily fixed carbon is not transferred from the C4 compounds onto the initial photosynthetic carboxylation product, but in brown algae takes just the opposite direction (Kremer 1981 d). Since brown macroalgae usually have an appreciable mannitol reserve which is immediately available for respiratory metabolism, it was postulated that this polyhydroxy alcohol also serves as a source of C 3 units upon glycolytic conversion to PEP. Pyruvate kinase and PEPCK most probably compete for their common substrate PEP, since an inhibition of the Krebs cycle simultaneously results in a distinct enhancement of light independent carbon incorporation (Kremer 1981 a). This line of indirect evidence for the role of mannitol as a source of PEP in dark carbon metabolism is supported by the results of the present study. It was demonstrated that (i) frond tissues of Laminaria hyperborea depleted of mannitol (and laminaran) no longer perform light independent carbon fixation via {3-carboxylation (Figs. 4 and 5) and (ii) that restoring the intracellular mannitol pool(s) result in a concomitant rise of the potential for {3-carboxylation (Tables 1 and 2). Isolated growing blades disconnected from their subtending stipe and old senescent blade are obviously not capable of maintaining a positive carbon balance in continuous darkness or below compensation photon fluence density, since they consume the bulk of their carbohydrate reserve within a few days. It must be emphasized,
J Plant Pbysiol. Vol. 117. pp. 233-242 (1984)
Carbohydrates and dark fixation in Laminaria
241
however, that these findings were obtained with temperatures which occur in the environment of Laminaria hyperborea not before May/June (d. Fig.3). The biological half-life time of the convertible carbohydrate reserve (mannitol and laminaran; cell wall constituents such as alginate and further respiratory substrates not considered) increases with lower temperatures, since dark respiration is reduced to about 30 % at 2 - 3 °C seawater temperature (Fig. 3; see also for average field data near Helgoland in February/March). Nevertheless, a growing blade appears not to be able to maintain its own carbon balance under natural conditions during the season of beginning growth, and almost no growth occurs under conditions of continuous darkness in isolated blades (LUning 1969). However, in entire plants the growing blade regions receive a continuous flow of reduced carbon (as mannitol and amino acids) by long-distance translocation out of the old blade area (for literature review see Schmitz 1981). The pool at least of soluble carbohydrates of the growing blade is therefore permanently renewed. As a consequence, in growing blades not detached from their source of organic carbon, sufficient amounts of substrate needed for /3-carboxylation are available. The overall carbon strategy of brown macroalgae such as Laminaria hyperborea and its relatives appears mainly to consist of a metabolic polyfunctionality of mannitol (and even of laminaran, with which it is interconvertible). This compound is used as source of energy and reduction equivalent by sequential degradation via glycolysis and end oxidation. It also serves as a source of new organic compounds by ramification of the catabolic pathway(s} at the level of PEP - a subsequent carboxylation step yields C4 compounds occupuying a central position in intermediary metabolism. The latter reaction sequences providing Krebs cycle intermediates and amino acids are not exclusively operated in the dark, but also during photosynthesis (Kremer 1981 a) and thus appear to imply additional metabolic advantages. In brown macro algae, dark carbon fixation is an imporant and, evidently, indispensible metabolic element also in an ecophysiological context, but not necessarily an exclusive one to account for the various peculiar autecological features of Laminaria species. Acknowledgements The author wishes to express his sincere thanks to the staff members of the Biologische Anstalt Helgoland (BAH-C) for providing many facilities.
References AKAGAWA, H., T. lKAWA, and K. NISIZAWA: The enzyme system for the entrance of 14C02 in the dark C02-fixation of brown algae. Plant Cell Physiol. 13,999-1016 (1972). CHAPMAN, A. R. O. and J. S. CKAIGIE: Seasonal growth in Laminaria longicruris: relations with reserve carbohydrate storage and production. Mar. BioI. 46, 209-213 (1978). CHAPMAN, A. R. O. and J. E. LINDLEY: Seasonal growth of Laminaria solidungula in the Canadian High Arctic in relation to irradiance and dissolved nutrient concentrations. Mar. BioI. 57, 1-5 (1980). KERBY, N. W. and L. V. EVANS: Phosphoenolpyruvate carboxykinase activity in Ascophyllum nodosum (Phaeophyceae). J. Phycol. 19, 1-3 (1983).
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BRUNO P. KREMER
KREMER, B. P.: Metabolic implications of non-photosynthetic carbon fixation in brown macroalgae. Phycologia 20,242-250 (1981 a). - Aspects of carbon metabolism in marine macroalgae. Oceanogr. Mar. BioI. Ann. Rev. 19, 41-94 (1981 b). - Carbon metabolism. In: The Biology of Seaweeds (c. S. LOBBAN, M. J. WYNNE, eds.), pp. 493-533. Blackwell, Oxford, 1981c. - C4 metabolism in marine brown macrophytic algae. Z. Naturforsch. 36c, 840-847 (1981 d). KUPPERS, U. and M. WEIDNER: Seasonal variation of enzyme activities in lAminaria hyperborea. Planta 148, 222-230 (1980). LUNING, K.: Growth of amputated and dark-exposed individuals of the brown alga lAminaria hyperborea. Mar. BioI. 2, 218-223 (1969). - Growth strategies of three lAminaria species (phaeophyceae) inhabiting different depth zones in the sublittoral region of Helgoland (North Sea). Mar. EcoI. Prog. Ser. 1, 195-207 (1979). LUNING, K. and M. J. DRING: Continuous underwater light measurements near Helgoland (North Sea) and its significance for characteristic light limits in the sublittoral region. HelgoHinder Meeresunters. 32,403-424 (1979). SCHMITZ, K.: Translocation. In: The Biology of Seaweeds (c. S. LOBBAN and M. J. WYNNE, eds.), pp. 534-558. Blackwell, Oxford, 1981. WEIDNER, M. and U. KOPPERS: Metabolic conversion of 14C-aspanate, 14C-malate, and 14C_man_ nitol by tissue discs of lAminaria hyperborea: role of phosphoenolpyruvate carboxykinase. Z. PflanzenphysioI. 108, 353-364 (1982).
J. Plant Physiol. Vol.
117. pp. 233-242 {1984}
P. KREMER
Universitat zu K61n, Institut fUr Naturwissenschaften und ihre Didaktik, Abt. f. Biologie, Gronewaldstr. 2, D-5000 K6ln 41 Received May 24, 1984 . Accepted August 25, 1984
Abstract Tissue discs originating from young, growing blade areas and from adult, mature frond regions of the brown macroalga Laminaria hyperborea (Fosl.) Gunn. (Phaeophyceae, Laminariales) were investigated with particular regard to photosynthesis, dark respiration, dark carbon fixation, and carbohydrate reserves. It was found that the mannitoi/laminaran reserve of the young, developing blade meets the requirements of dark respiratory metabolism for only 7 -10 d at 10±2 °C under continuous darkness. A concomitant decrease in the potential for {3carboxylation of phosphoenolpyruvate by phosphoenolpyruvate carboxykinase (EC 4.1.1.32) occurred along with the depletion of the stored carbohydrate. Restoring the intracellular pool of reserve carbohydrates by photosynthesis and by feeding of exogenously supplied mannitol resulted in a short term recovery of the rates of dark fixation. These findings support the view that (i) in the dark the substrate of {3-carboxylation is mainly derived from mannitol (along with glycolytic degradation of laminaran) and (ii) the young blade is not able to maintain its own carbon balance under the environmental conditions during midwinter and early spring, but relies on a carbon flow from the old blade.
Key words: Laminaria, Brown algae, Carbon fixation, {3-Carboxylation, Storage carbohydrates, Carbon budget, Ecophysiology.
Introduction
As in green plants primary CO 2 fixation in brown marine macroalgae occurs via ribulose-1,5-bisphosphate carboxylase. However, particularly in the species belonging to the brown algal orders Laminariales and Fucales an additional route of inorganic carbon assimilation is operational. It consists of /3-carboxylation of a C 3 unit to give the C4 compound oxaloacetate (OAA). The enzyme performing this carboxylation step has been recognized as a phosphoenolpyruvate carboxykinase (PEPCK) (Akagawa et al. 1972; Kiippers and Weidner 1980; Kremer 1981 a). A temporally and spatially different distribution of enzyme activity attributed to /3-carboxylation in vitro as well as in vivo was encountered with ontogenetically different frond tissue areas, topographically different frond regions, as well as with various stages in the life cycle of brown macroalgae (for literature review see Kremer 1981 band 1981 c). The process of /3-carboxylation of phosphoenolpyruvate (PEP) had hitherto mostly been referred to as dark or non-photosynthetic carbon fixation. Kinetic tracer studies
J. Plant Physiol. Vol.
117. pp. 233-242 (1984)
234
BRUNO
P. K1lEMEll
indicated that 14C-Iabelling of ~ acids such as aspartate and malate immediately derived from OAA (d. Weidner and Kiippers 1982) is not restricted to dark periods, but also occurs during photosynthesis. Indeed, rates of carbon fixation into specific C4 compounds are approximately equal in the light and in the dark. The distribution of 14C between C 1 and C4 atoms of aspartate suggests a carbon flow from early occurring intermediates of the reductive pentose phosphate cycle such as 3-phosphoglycerate to ~ dicarboxylic acids. In brown macroalgae «dark,. carbon fIxation via is-carboxylation of PEP was assumed to be quantitatively and qualitatively integrated into photosynthetic C02 assimilation thus yielding appreciable 14C-Iabelling of aspartate and malate. Of course the underlying reactions and conversions are basically different from the ~ syndrome of vascular plants and were therefore preferably termed as C4 metabolism (Kremer 1981 a, 1981 d). It was suggested earlier that the metabolic basis for is-carboxylation of PEP in the dark is provided by sequential degradation of mannitol, the main soluble carbohydrate of brown algae. One line of evidence for this assumption were results from inhibition of the Krebs cycle with monofluoracetate and a concomitant increase in dark carbon fixation (Kremer 1981 a). This contribution also deals with the problem of substrate availability by comparing internal pools of stored carbohydrate (mannitol and laminaran) with the potentials for is-carboxylation within the same frond areas of the brown macroalga, lA·
minaria hyperborea.
Materials and Methods Plant Material Adult specimens of Laminaria hyperborea (Gunn.) Fos!. (Phaeophyceae, Laminariales) were collected by SCUBA diving from about 4 m below mean low tide at their natural habitat on the rocky shores near Helgoland (North Sea, Germany) in February 1984. The plants measuring between 1.20 and 1.60 m in length (including stipe and basal holdfast) and apparently free of epiphytes were maintained in 500 I tanks supplied with running seawater at 10 ± 2°C.
Experimental Young, growing blades (see Fig. 1) were cut off from the stipe and the old (second year) blade and stored for up to 7 d in black plastic bags in running seawater under the same temperature regime. Entire plants (without stipes and holdfasts) were kept individually under the same conditions. Tissue discs (2 cm diameter) were punched out from the areas as indicated in Fig. 1 and monitored for (i) photosynthetic and dark respiratory oxygen exchange and (ii) rates of light independent (dark) carbon fixation. Analytical
In addition to metabolic processes such as photosynthesis, dark respiration, and dark fixation the levels of PEPCK activity ( - phosphoenolpyruvate carboxykinase; EC 4.1.1.32) in vitro as well as contents of reserve carbohydrates (mannitol, laminaran) were determined from the same samples. All methods used for oxymetry, 14C-incubation, enzyme assays, and carbohydrate quantification have already been detailed earlier (Kremer 1981 a, 1981 d).
J. Plant Physiol. Vol. 117. pp. 233-242 (1984)
Carbohydrates and dark fixation in Laminaria
~
235
A-II-I- - - B - - - - - 1
~
nl .1 • •
10 em
L-----I
•
I
@ ~) I •
Fig. 1: Laminaria hyperborea with developing new blade (A) and mature, second year, senescent leafy frond region (B) - growth stage about February/March. Samples were taken from entire specimens (1 a - 4 a) as well as from isolated, detached blade regions (1 b - 4 b), each maintained under continuous darkness upon collection.
o Photoayntheela • o.k
I
IJ
respiration
Derk tlatkwt
3D
10
10
3D
60
II
JO
60 Time [mIn]
Fig.2: Metabolic performance (photosynthesis, dark respiration, dark fixation) of different frond regions of Laminaria hyperborea immediately after collection. Oxymetric measurements converted to carbon data assuming O 2/ CO 2 ::::: 1. Vertical bars indicate standard deviation
(n = 5).
J. Plant Physiol.
Vol. 117. pp. 233-242 (1984)
236
BRUNO
P. KRatER
Results
Metabolic performance of ontogenetically different frond tissue It may be seen from Fig. 2 that young, growing, and old, non-growing frond areas of Laminaria hyperborea are distinctly different with respect to photosynthesis, dark respiration, and light independent carbon fixation. Rates of photosynthesis in the leafy meristematic intercalary area of the Laminaria frond under light saturating conditions (photon flue nee density: 150 JLE m- 2 S- I) usually accounted for about 50 % of the respective rates recorded for mature frond areas. A closer inspection of dark respiratory and dark carbon fixation rates measured in vivo yields a different picture: Both rates recorded for the growing zone exceeded those obtained from old blade areas. These findings suggest that the net carbon gain achieved in the intercalary growing zone generating the new blade is presumably far less than the amounts actually needed for growth and development. This emphasizes the demand for further carbon sources supplementing or supporting the anabolic processes occurring in the growing tissue.
'C.-------,
~
•
Derk respiretlon
E$1 lli:;l Derk fixation
5
JFMAMJJA
~
2 -----'
' - - - 7 -----'
'---12 - - J Temperature ['e)
Fig. 3: Temperature profile of dark respiration and dark fixation rates of young (1 a) and old (4a) blade regions of Laminaria hyperborea. Insert: Long-term in situ seawater temperature in the environment of Laminaria hyperborea near Helgoland Qanuary-August) (Data kindly supplied by P. Mangelsdorf/BAH).
Fig. 3 presents information on the temperature profiles of dark respiration and dark fixation for the temperature range between 2 and 12°C. No marked differences in the QIO values between young and old fronds were obtained.
J Plant Physiol. Vol. 117. pp. 233-242 (1984)
Carbohydrates and dark fixation in Laminaria
237
Dark respiratory consumption 0/ reserve carbohydrates The consumption of mannitol and laminaran as substrates of dark respiration by young, excised blades was dependent on time. Fig. 4 describes the respiratory decay of both carbohydrates in continuous darkness as a percentage of the respective amounts determined in controls incubated in the light. The overall decrease of mannitol was more pronounced in tissue discs originating from the growing zone than from the adult blade. Since the latter tissue showed lower rates of dark respiration (d. Fig. 2), a comparatively faster respiratory depletion of the mannitol reserve occurred in the young blade. This also holds good for the laminaran reserves. As a consequence, the growing areas of Laminaria hyperborea were almost depleted within 6-7 d in continuous darkness at 10±2 °C thus contrasting with the old blade tissues
~
i
o.rI< incubation
1
[dl
Fig. 4: Dark respiratory consumption of stored mannitol and laminaran in young and old/intact and isolated blade tissues of Laminaria hyperborea. Insert Fig. 4 a: Decay of the mannitol reserve in a growing frond still connected with its old blade. Insert Fig. 4 b: Levels of PEPCKactivity in young and old fronds maintained in the light (controls) and cultured for 7 d in continuous darkness.
which still had a certain proportion of their original carbohydrate reserve. It is interesting to note that the soluble compound mannitol was obviously more readily metabolized than the polymeric reserve carbohydrate laminaran. Young blade tissue discs originating from entire, non-excised plants maintained in continuous darkness showed a significantly lower cumulative respiratory loss of soluble and insoluble carbohydrate reserves as compared to isolated growing blades (Fig.4). This again demonstrates that the intercalary growing blade is probably not able to maintain its own carbon balance upon the loss of the mature, old blade, at least not during midwinter and early spring. It therefore must rely on a continuous carbon flow which can be fed into its intermediary metabolism. j. Plant Physiol. Vol. 117. pp. 233-242 (1984)
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I~
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10
100
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"
N
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u
8
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~
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Control
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Dark culture
Fig. 5: Dark carbon fixation (in vivo /3-carboxylation) in fronds maintained in continuous darkness, values expressed as a percentage of the initial rates of control specimens maintained in the light. For sample topography d. Fig. 1. Means of 5 replicates.
Dark carbon fixation in plants cultured in the dark Dark fixation of inorganic carbon appears to depend on the experimental pretreatment of the frond tissue samples. The data contained in Fig. 5 indicate that rates of dark fixation in tissue discs originating from the growing region of intact specimens showed only a slight decline in their in vivo potentials for i3-carboxylation. A much more pronounced drop was observed in discs from growing blades which had previously been cut off from stipe and older frond blade and were then kept in continuous darkness. In these samples, /3-carboxylation in vivo was reduced by about 80 % as compared to the controls (Fig. 5). No concomitant decline in the enzymatic potentials for /3-carboxylation (activity of PEPCK) was observed (data not shown). Linear regression analysis of rates of dark carbon fixation against total carbohydrate reserve showed a highly significant correlation (p < 0.01). Hence, the availability of the internal substrate for i3-carboxylation (= PEP) largely affected the rates of dark carbon incorporation via PEPCK.
Feeding of mannitol and dark carbon fixation In order to test the suggested relationship between carbohydrate content and rates of light independent carbon fixation further, it was attempted to partially restore the dark respiratory loss of reserve carbohydrates of the meristematic blades a) by exogenous feeding of mannitol and b) by photosynthetic incubation allowing for de
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Table1: Uptake of exogenously supplied mannitol (100mM) and recovery of dark fixation potentials in meristematic frond tissue of Laminaria byperborea. Mean values ± standard deviation (n = 3). ----~-----------------------------------------------
Sample l )
Feeding of mannitol (h)
Mannitol content (I'mol dm -2)
Dark fixation (I'mol C dm -2 h -1)
1b
6 12 24
92±24 215±52 373±69
2.2±O.2 3.8±O.8 5.3±1.4
2b
6 12 24
78±18 229±54 315±61
204±O.2 3.5±O.6 6.2±1.5
I) d. Fig. 1.
novo synthesis of intracellular mannitol. Discs from young, growing blades depleted by continuous darkness were subsequently incubated in the dark in sterile filtered seawater medium containing 100 mM mannitol. Since it was earlier observed that brown macroalgae such as Laminaria spp. scarcely take up dissolved organic molecules from the incubation medium (d. Weidner and Kiippers 1982), tissue discs were divided lengthwise along their medullary layer and incubated separately. In a second set of experiments mannitol-depleted young blade tissue was allowed to photosynthesize under saturating light conditions. The results obtained are shown in Tables 1 and 2. It may be seen that the sliced tissue from the growing blade took up certain amounts of exogenously supplied mannitol dependent on time and thus restored the intracellular polyol pool. Similarly, photosynthesis gradually replenished the mannitol reserve. A concomitant increase in the rates of light independent carbon incorporation occurred together with restored mannitol contents of the same samples (Tables 1 and 2). Thus the overall increase in available mannitol within the tissue significantly enhanced the in vivo potential for a-carboxylation. Table 2: Photosynthetic de novo synthesis of mannitol and recovery of dark fixation potentials in meristematic and adult frond tissues of Laminaria byperborea. Mean values ± standard deviation (n = 3). ----~-----------------------------------------
Sample 1)
Photosynthesis (h)
Mannitol content (I'mol dm - 2)
Dark fixation (I'mol C dm- 2 h- 1)
1b
6 12 24
104±26 165±32 324±84
204±O.3 2.9±O.7 6.6± 104
4b
6 12 24
220±49 378±72 682±42
1.9±O.2 204±Oo4 3.2±O.5
I) d. Fig. 1.
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Discussion In species of the genus Laminaria the transitional zone between the persistent stipe and the adult blade region exhibit peculiar features with respect to growth, development, and metabolism. This is particularly true for Laminaria hyperborea (Luning 1979; Kuppers and Weidner 1980), but also holds good for further Laminaria species (Chapman and Craigie 1978; Chapman et al. 1980; Kremer 1981 a, 1981 d). In Laminaria the annual growth is mediated by an intercalary meristematic zone and commences during a time of the year, when the temperature of the seawater (cf. Fig. 3) and light supply in the sublittoral zone are far below saturation (Luning 1979; Luning and Dring 1979). One of the most remarkable features of this meristematic zone (during midwinter and early spring still identical with the new blade in Laminaria hyperborea; cf. Fig. 1) is the comparatively high potential for {3-carboxylation of PEP yielding appreciable amounts of C dicarboxylic acids. Evidence for the relative efficiency of this pathway of inorganic carbon fixation was earlier derived from a comparison of photosynthetic and non-photosynthetic incorporation of H I4 C03" in vivo as well as by assays of the respective carboxylating enzyme mediating the entrance of CO 2 into organic compounds (Kremer 1981 a, 1981 d; Kerby and Evans 1983). In the light the substrate of PEPCK is derived from an early photosynthate, 3-phosphoglycerate. Upon {3-carboxylation of PEP, C4 dicarboxylic acids such as aspartate and malate were encountered with the rapidly 14C-Iabelled photoassimilatory products. Nevertheless, the respective metabolic events and conversions are different from those of C 4 photosynthesis in vascular plants, since the primarily fixed carbon is not transferred from the C4 compounds onto the initial photosynthetic carboxylation product, but in brown algae takes just the opposite direction (Kremer 1981 d). Since brown macroalgae usually have an appreciable mannitol reserve which is immediately available for respiratory metabolism, it was postulated that this polyhydroxy alcohol also serves as a source of C 3 units upon glycolytic conversion to PEP. Pyruvate kinase and PEPCK most probably compete for their common substrate PEP, since an inhibition of the Krebs cycle simultaneously results in a distinct enhancement of light independent carbon incorporation (Kremer 1981 a). This line of indirect evidence for the role of mannitol as a source of PEP in dark carbon metabolism is supported by the results of the present study. It was demonstrated that (i) frond tissues of Laminaria hyperborea depleted of mannitol (and laminaran) no longer perform light independent carbon fixation via {3-carboxylation (Figs. 4 and 5) and (ii) that restoring the intracellular mannitol pool(s) result in a concomitant rise of the potential for {3-carboxylation (Tables 1 and 2). Isolated growing blades disconnected from their subtending stipe and old senescent blade are obviously not capable of maintaining a positive carbon balance in continuous darkness or below compensation photon fluence density, since they consume the bulk of their carbohydrate reserve within a few days. It must be emphasized,
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Carbohydrates and dark fixation in Laminaria
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however, that these findings were obtained with temperatures which occur in the environment of Laminaria hyperborea not before May/June (d. Fig.3). The biological half-life time of the convertible carbohydrate reserve (mannitol and laminaran; cell wall constituents such as alginate and further respiratory substrates not considered) increases with lower temperatures, since dark respiration is reduced to about 30 % at 2 - 3 °C seawater temperature (Fig. 3; see also for average field data near Helgoland in February/March). Nevertheless, a growing blade appears not to be able to maintain its own carbon balance under natural conditions during the season of beginning growth, and almost no growth occurs under conditions of continuous darkness in isolated blades (LUning 1969). However, in entire plants the growing blade regions receive a continuous flow of reduced carbon (as mannitol and amino acids) by long-distance translocation out of the old blade area (for literature review see Schmitz 1981). The pool at least of soluble carbohydrates of the growing blade is therefore permanently renewed. As a consequence, in growing blades not detached from their source of organic carbon, sufficient amounts of substrate needed for /3-carboxylation are available. The overall carbon strategy of brown macroalgae such as Laminaria hyperborea and its relatives appears mainly to consist of a metabolic polyfunctionality of mannitol (and even of laminaran, with which it is interconvertible). This compound is used as source of energy and reduction equivalent by sequential degradation via glycolysis and end oxidation. It also serves as a source of new organic compounds by ramification of the catabolic pathway(s} at the level of PEP - a subsequent carboxylation step yields C4 compounds occupuying a central position in intermediary metabolism. The latter reaction sequences providing Krebs cycle intermediates and amino acids are not exclusively operated in the dark, but also during photosynthesis (Kremer 1981 a) and thus appear to imply additional metabolic advantages. In brown macro algae, dark carbon fixation is an imporant and, evidently, indispensible metabolic element also in an ecophysiological context, but not necessarily an exclusive one to account for the various peculiar autecological features of Laminaria species. Acknowledgements The author wishes to express his sincere thanks to the staff members of the Biologische Anstalt Helgoland (BAH-C) for providing many facilities.
References AKAGAWA, H., T. lKAWA, and K. NISIZAWA: The enzyme system for the entrance of 14C02 in the dark C02-fixation of brown algae. Plant Cell Physiol. 13,999-1016 (1972). CHAPMAN, A. R. O. and J. S. CKAIGIE: Seasonal growth in Laminaria longicruris: relations with reserve carbohydrate storage and production. Mar. BioI. 46, 209-213 (1978). CHAPMAN, A. R. O. and J. E. LINDLEY: Seasonal growth of Laminaria solidungula in the Canadian High Arctic in relation to irradiance and dissolved nutrient concentrations. Mar. BioI. 57, 1-5 (1980). KERBY, N. W. and L. V. EVANS: Phosphoenolpyruvate carboxykinase activity in Ascophyllum nodosum (Phaeophyceae). J. Phycol. 19, 1-3 (1983).
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KREMER, B. P.: Metabolic implications of non-photosynthetic carbon fixation in brown macroalgae. Phycologia 20,242-250 (1981 a). - Aspects of carbon metabolism in marine macroalgae. Oceanogr. Mar. BioI. Ann. Rev. 19, 41-94 (1981 b). - Carbon metabolism. In: The Biology of Seaweeds (c. S. LOBBAN, M. J. WYNNE, eds.), pp. 493-533. Blackwell, Oxford, 1981c. - C4 metabolism in marine brown macrophytic algae. Z. Naturforsch. 36c, 840-847 (1981 d). KUPPERS, U. and M. WEIDNER: Seasonal variation of enzyme activities in lAminaria hyperborea. Planta 148, 222-230 (1980). LUNING, K.: Growth of amputated and dark-exposed individuals of the brown alga lAminaria hyperborea. Mar. BioI. 2, 218-223 (1969). - Growth strategies of three lAminaria species (phaeophyceae) inhabiting different depth zones in the sublittoral region of Helgoland (North Sea). Mar. EcoI. Prog. Ser. 1, 195-207 (1979). LUNING, K. and M. J. DRING: Continuous underwater light measurements near Helgoland (North Sea) and its significance for characteristic light limits in the sublittoral region. HelgoHinder Meeresunters. 32,403-424 (1979). SCHMITZ, K.: Translocation. In: The Biology of Seaweeds (c. S. LOBBAN and M. J. WYNNE, eds.), pp. 534-558. Blackwell, Oxford, 1981. WEIDNER, M. and U. KOPPERS: Metabolic conversion of 14C-aspanate, 14C-malate, and 14C_man_ nitol by tissue discs of lAminaria hyperborea: role of phosphoenolpyruvate carboxykinase. Z. PflanzenphysioI. 108, 353-364 (1982).
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