C4-Metabolism in Marine Brown Macrophytic

Bruno P. Kremer Seminar für Biologie und ihre Didaktik der Universität, Gronewaldstraße 2, D-5000 Köln 41

Z. Naturforsch. 36 c, 840-847 (1981); received June 19, 1981

Photosynthesis, Dark Fixation, /?-Carboxylation, C4-Metabolism, Marine brown macroalgae including , luetkeana, Lessoniop- sis littoralis, saccharina, serratus and some further representatives of the Laminariales and (Phaeophyta) have been investigated with respect to their remarkably high potential for /?-carboxylation of phosphoenolpyruvate supplementing photosynthetic C02 fixation. Kinetic tracer studies indicate that 14C-labelling of C4 acids such as aspartate and malate is not restricted to dark periods, but also occurs during . Rates of carbon fixation into C4 compounds are approximately equal in the light and in the dark. Distribution of 14C between C1 and C4 atoms of aspartate suggests carbon flow from early occurring photosynthates such as 3-phosphoglycerate to C4 compounds including aspartate and malate. In brown macro­ algae dark carbon fixation via /?-carboxylation of phosphoenolpyruvate is therefore assumed to be quantitatively and qualitatively integrated into photosynthetic C 02 assimilation thus yielding appreciable 14C-labelling of C4 dicarboxylic acids. The underlying reactions and conversions are basically different from C4 photosynthesis and should preferably be termed as C4 metabolism.

Introduction prokaryotic and eukaryotic algae [5 - 7] as well as in in general [8 ]. The basic questions surrounding the pathway of Among marine algae, particularly brown macro- inorganic carbon assimilation during photosynthesis phytes belonging to the orders Laminariales and have been resolved by experiments mostly using Fucales were recognized to exhibit notibly high unicellular green algae [1]. The essential features of potentials for /?-carboxylation of phosphoenolpyru­ the reactions and conversions proposed have later vate. This process has hitherto mostly been quoted been extended to all photoautotrophic organisms. as dark or non-photosynthetic carbon fixation [9— Only one decade after finally establishing the uni­ 12]. It is now accepted that the enzyme performing versally operating reductive pentose phosphate cy­ the respective carboxylation of a C3-unit to give the cle providing photosynthetic carbon fixation and re­ C4-compound oxaloacetate is a phosphoenolpyru­ duction, a further carboxylating mechanism in­ vate carboxykinase [13—15]. Temporally and spa­ volved in light dependent harvesting and assimila­ tially different distribution of enzyme activity at­ tion of inorganic carbon was found [2]. A complete tributed to /^-carboxylation in vitro as well as in biochemical reaction scheme to account for the par­ vivo was encountered with ontogenetically different ticular pattern of 14C-labelled C4 dicarboxylic acids frond areas, topographically different frond tissues, observed in ecologically specialized plant species and various developmental stages in the life cycle of immediately after exposure to 14C 0 2 has been pre­ macrophytic brown algae [16—18]. The peculiar in­ sented [3]. Although initially discussed as a possibly terface between /?-carboxylation and photorespira­ alternative pathway, the series of metabolic reac­ tion has been characterized for a variety of species tions constituting the so-called C 4 pathway of pho­ [19]. tosynthesis is now widely understood to be super­ The present investigation has been undertaken to imposed on the indispensible reactions of the reduc­ evaluate the problem as to what extent /?-carboxyla- tive pentose phosphate cycle. Since its original de­ tion via phosphoenolpyruvate carboxykinase might scription, C 4 photosynthesis basically involving the definitely contribute to total inorganic carbon assi­ /?-carboxylation of a C 3 acid to C4 compound (s) is milation. In particular, experiments have been con­ not restricted to terrestrial vascular plants, but was ducted to determine i) whether /?-carboxylation is also reported to occur in a seagrass [4], in unicellular fully independent from photosynthesis and there­ fore continues in the light without rate depression, Reprint requests to Dr. B. P. Kremer. ii) which are the minimal rates of ^-carboxylation 0341-0382/81/0900-0840 $01.00/0 during photosynthesis, and iii) from which source is B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae 841 the specific substrate of phosphoenolpyruvate carb- X-100 to the still frozen mixture. The resulting su­ oxykinase metabolically derived in the light. The pernatant obtained after centrifugation at 40000 x g findings presented suggest that marine brown algae and 4 °C was regarded as crude enzyme extract and possess C 4 metabolism rather than C4 photosynthe­ used for assays of ribulose-l,5-bisphosphate carb­ sis. oxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxykinase (EC 4.1.1.31). For details on the ex­ traction and assay procedures see Kremer and Küp­ Materials and Methods pers [1 1 ]. Organisms Extraction and chromatography o f14C-labelled Macrocystis integrifolia Bory, Nereocystis luet- compounds keana (Mert.) P. & R., Lessoniopsis littoralis (Tild.) Reinke, Cymathere triplicata (P. & R.) J. Ag., and Frond samples 14C-incubated in the light or in the Eisenia arborea Aresch. (Laminariales, Phaeophyta) dark were exhaustively extracted in aqueous ethanol were collected from stands in Barkley Sound, (50%) and a mixture o f methanol-chloroform-for­ Vancouver Island, B. C., Canada near Bamfield Ma­ mic acid ( 6 N) = 12:5:1. The combined extracts rine Station. (Huds.) Lamour., were partitioned into an aqueous/alcoholic and a Laminaria saccharina (L.) Lamour. (Laminariales) chloroform phase and counted for total 14C-activity as well as nodosum (L.) Le Jol. and Fu- incorporated. The aqueous phase was evaporated to cus serratus L. (Fucales, Phaeophyta) originated dryness in vacuo, redissolved in 30% ethanol, and from the rocky shores of Helgoland, North Sea, further analyzed by thin-layer chromatography on German Bight, where they were collected during cellulose (MN 300, 200-400 nm layer thickness), low tide. Experiments were performed with care­ either directly or following pre-fractionation into fully checked specimens within 6 — 1 2 h after collec­ neutral, anionic, and cationic compounds by passing tion and maintaining in running seawater at am­ through columns of Dowex-50 (H+) and Dowex-1 bient temperature. (formate) resins. Details on these procedures have already been described in an earlier technical paper C 0 2 Fixation [20]. Distribution o f radiocarbon between the Q Sections of about 2 cm diameter or 2 x 1 cm edge and C4 atoms of aspartate was determined by degra­ length were cut from the leafy parts of the fronds, dation of the chromatographically isolated com­ pound to ^-alanine using a ninhydrin treatment ac­ repeatedly rinsed in membrane-filtered seawater and equilibrated in the light (photon flux density cording to ref. [ 2 1 ]. 200 *iE m ~ 2 s_1; 15 min) or in the dark (30 min) at ambient temperature (10-15°C). Following the Results equilibration period, frond samples were submerged in a H 14CO 7 -containing seawater medium (50 \iC i/ Gross patterns of carbon fixation 100 ml; specific activity > 55 mCi/mM) and allowed Fig. 1 describes the time courses of carbon fixa­ to fix the radioactive bicarbonate for various lenghts tion in the light and in the dark for two macro­ of time. The assimilation was terminated by immer­ phytic brown algae including the giant kelp, Macro­ sion of the samples into liquid N2. Carbon fixation cystis integrifolia, for incubation periods ranging up rates were calculated by measurements of acid- to 90 min. Several features are noteworthy. All stable 14C incorporation and referred to the total in­ frond regions specifically investigated reveal linear­ organic carbon content (2.7 mM) of the incubation ity o f C 0 2 incorporation. Some depression of the al­ media used. most constant rates is observed after longer expo­ sure periods exceeding 3 - 5 h (data not included in Enzyme extraction and assays Fig. 1) thus suggesting presumable depletion of in­ Frond samples (1 -5 g fresh weight) were deep organic radiocarbon in the incubation medium. frozen in liquid N 2 and homogenized by grinding Photosynthetic rates in ontogenetically young (i.e. with quartz sand adding polyvinylpolypyrrolidone, growing) frond areas comprising the meristematic EDTA, D-araboascorbate, MgCl2, DTT and Triton- intercalary region of the leafy thallus part account 842 B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae

3 10 30 60 90 3 10 30 60 90 Incubation [min] Fig. 1. Macrocystis integrifolia (M), Lessoniopsis littoralis (L). Rates of photosynthetic (a) and light independent = dark (b) carbon assimilation in growing (= young) and differentiated (= old) frond areas. Inserts: Activity of RubP-C and PEP-CK, respectively, in (imol substrate carboxylated on a fresh weight basis.

for generally less than 60% of those recorded for Assimilates 14C-labelled in the dark fully differentiated tissue. It is also obvious from In contrast to photosynthesis, radiocarbon intro­ Fig. 1 that the frond samples investigated show in­ duced via ^-carboxylation in the dark is recovered organic carbon incorporation and fixation into acid- from only a few low-molecular weight assimilates as stable organic compounds even in the dark. On the is shown in the autoradiograph (Fig. 2). Among average, in adult frond areas about 5% of maximal them, aspartate along with malate and some further carbon assimilation in the light are due to non- compounds including alanine and glutamate as well photosynthetically performed C 0 2 uptake. In grow­ as citrate and fumarate form the bulk of 14C-la- ing zones, this proportion is considerably higher. belled material. Traces of other assimilates mostly Expressed in terms of relative as well as absolute representing intermediates of the tricarboxylic acid amounts of radiocarbon incorporated, /?-carboxyla- cycle are also encountered. Fig. 3 includes data dem­ tion as measured by in vivo dark carbon assimilation onstrating the time course of 14C-labelling in the is more significant in growing (young) than in adult, dark. Kinetics o f 14C-incorporation into individual non-growing frond areas. compounds suggest that the C 4 acids aspartate and Activities of the respective enzymes responsible malate occur among the first labelled metabolites, for photosynthetic and dark carbon fixation as de­ whereas other dark assimilatory products receive termined by in vitro carboxylation are inserted in radiocarbon distinctly later. This observation is con­ Fig. 1. Again, appreciably higher activity of phos- sistent with the assumption that the reaction prod­ phoenolpyruvate carboxykinase (= PEP-CK below) uct of /^-carboxylation via PEP-CK is oxaloacetate is confined to meristematic frond regions, whereas which then is immediately converted to aspartate by more activity of ribulose-1,5-biphosphate carboxy­ transamination and to malate upon a simple reduc­ lase (further quoted as RubPC) is encountered with tion step (cf. [1 0 , 1 2 ]). the fully differentiated parts. The gross pattern of in vivo and in vitro carbon fixation is thus basically the Photosynthetic uC-labelling of C4-compounds same, though the enzymatic carboxylation per­ formed with the isolated system cannot account for Under steady state conditions of photosynthesis, the equivalent rates obtained from intact frond sam­ certain amounts of 14C are consistently recovered ples. Very similar results have been found for all from aspartate and malate even after short-term ex­ further algal species investigated in this connection. posure of frond samples to H14COä in the light. Dis- B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae 843 tribution of photosynthetically fixed 14C among ear­ ly 14C-labelled metabolites is exemplified in Fig. 4 for growing and fully differentiated frond areas of Laminaria saccharina and Fucus serratus. It may be seen that percentage of 14C-labelling in organic ■Q phosphates including diverse sugar phosphates and 3 3-phosphoglycerate rapidly decreases from approxi­ mately 80% to about 25% of total 14C recovered O from soluble photosynthates. Kinetics of 14C-label- a* ling thus show a characteristic negative slope curve. Time dependent shifts in percentage 14C-labelling of C4-compounds such as aspartate and malate are basically different. Both photosynthates achieve in-

Incubation [mini Fig. 3. Laminaria digitata. Time dependency of 14C-label- mal ling in single assimilates during dark incubation. For ab­ ala breviations see Fig. 2. glu'

asp creasing percentage up to 10-30 s after exposure to H 14CC>3 . Hence, maximum relative 14C-labelling confined to these assimilates is distinctly delayed as compared to the organic phosphates. Again, it is in­ teresting to note that in young, growing frond areas the initial photosynthetic 14C-labelling of aspartate 0 and malate exceeds that recorded for samples not taken from the intercalary (Laminaria) or apical (Fucus) growing zones. fum Position of radiocarbon in 14C-aspartate m mal ala Provided that 14C-labelling of aspartate occurring glu in the assimilate pattern after photosynthesis and light independent carbon fixation is actually initiat­ ed by the carboxylation of phosphoenolpyruvate via asp PEP-CK, then it might be assumed that the distri­ bution of 14C between both carboxyl groups must be helpful for some insight into the metabolic origin of phosphoenolpyruvate required as a carboxylation substrate. The results of appropriate experiments and analyses are shown in Fig. 5. Most of the r e ­ activity of aspartate, photosynthetically 14C-labelled © during short-term photosynthesis, is released upon Fig. 2. Macrocyst is integrifolia. Autoradiographs showing ninhydrin-mediated decarboxylation to ^-alanine. the patterns of photosynthetically (a) and non-photosyn- thetically (b) 14C-labelled assimilates as demonstrated after This indicates preferential location of 14C in the Q - two-dimensional TLC: Dark assimilates are qualitatively atom (= a-carboxyl group) of aspartate. After longer integrated into pattern of photoassimilatory compounds; asp = aspartate, glu = glutamate, ala = alanine, mal = ma­ photosynthetic incubation exceeding 2 -3 min the late, fum = fumarate. For further details ( cf. ref. [20]). percent 14C-activity, which can be removed by nin,- 844 B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae

. LAMINARIA Q t FUCUS ©

X \ • O ORGANIC P-ESTER \ • ■ old

■ □ ASPARTATE + MALATE \\ O □ young Y \ 50 \ 'S J?

10 ------* ------10

5 10 30 60 5 10 30 60 Photosynthesis [s] Fig. 4. Laminaria saccharina, Fucus serratus. Time dependent 14C-labelling of C4-acids (aspartate amd malate) and organic phosphates in growing (= young) and differentiated (= old) frond areas after short-time photosynthesis.

hydrin, distinctly decreases to approximate an ap­ and malate via /?-carboxylation. The data compiled parent theoretical level of 50%. This suggests in­ in Fig. 6 show the appropriate rates of carbon flow creasing 14C-amounts within the /?-carboxyl group into C 4 acids as calculated for several brown macro­ of the aspartate molecule which must be due to ß- algae. When the values for photosynthetically de­ carboxylation. Symmetrical 14C-labelling of the Cx rived aspartate and malate are reduced by 50% and C4-atoms of aspartate therefore suggests /?-carb- (cf Fig. 5) to negate the contribution of 14C in the oxylation progressing in the light during photosyn­ thesis. From aspartate 14C-labelled in the dark rather low amounts of radiocarbon can be released by ninhydrin treatment. This result indicates pre­ pondering (if not exclusive) 14C-labelling in the ex­ position of aspartate.

Integration of ß-carboxylation into photosynthesis

From the results contained in Fig. 5 the question arises as to what extent /?-carboxylation of phospho- enolpyruvate contributes to photosynthetic carbon fixation. How much carbon is thus additionally provided via /?-carboxylation as compared to photo­ synthetic C 0 2 fixation via RubP-C? More precise information on this problem may be obtained from a calculation of the respective carboxylation rates as based on percentage 14C-labelling of aspartate and malate as well as carbon fixation achieved in the light and the dark. If it is assumed that both carb­ Incubation [s] oxyl groups of malate are 14C-labelled in a very similar way to those of aspartate, then photosyn­ Fig. 5. Macrocystis integrifolia, Nereocystis luetkeana, Eise- nia, arborea. Percentage 14C-labelling of the Q-atom (= a- thetic and dark C 02 assimilation may be expressed carboxyl group) of aspartate during short-term photosyn­ as amount of carbon fixed into the C 4 of aspartate thesis and in the dark. ^imol C O 2 g "1 tresh wt h 1 □ abxlto o hsheoprvt i particular­ is phosphoenolpyruvate of carboxylation C4-acids into fixed carbon inorganic of terms in scales. expressed as ordinate dark the different in ote and N light the photosynthesis. in to relation in /?-carboxylation of Rates 6. Fig. y hc seis f h Lmnrae ad Fucales and Laminariales the areas, of (blade) species frond which growing by developping, in high ly ifr rm h mjrt of ute age [ algae further f o majority the from differ 0 C the in fixation carbon non-photosynthetic and thesis s ae o te omto ofaprae n malate, and aspartate f o formation the on based As 18, 22] (Fig. 1). (Fig. 22] 18, tion tion eaoi faue atclry curn i brown in occurring particularly feature metabolic abxltn ezms eitn te nrne of entrance the respective mediating the f o enzymes measurements by carboxylating as well photosyn­ as f o dark comparison a from derived is process well. reasonably in compare dark groups the /?-carboxyl in the and into quan­ light the the fixed that then carbon of obvious is tities It /?-carbox- light. via the in introduced ylation directly carbon f o amounts n rw age ?croyain n h lgt t least at (Fig. dark light the in the in achieved that equals /?-carboxylation algae brown in phyceae. Evidence for the relative efficiency o f this f o efficiency relative the for negligible Evidence is phyceae. extent its whereas algae, macrophytic Discussion rpsto, t eoe psil t apoiae the approximate to possible becomes it position, Cr n ersnaie fte hoohca n Rhodo- and Chlorophyceae the of representatives in 20 Algae Macrophytic Brown Marine in C4-Metabolism • Kremer P.B. 10 siiain fiognc abn y /?-carboxyla- by carbon inorganic of Assimilation 2 into organic compounds. The potential for for potential The compounds. organic into via hsheoprvt croyiae s a is carboxykinase phosphoenolpyruvate o / / ((/ / , n 9 6 / ). .y r « 0 1 — 2 1 ß- ,

$ ae pae n h lgt n i tu ovosy not obviously thus is and light actually the yS-carboxylation in that place takes suggests finding this photosynthe­ fixed increasing tically However, 5). (Fig. s 60 than probably derived from from derived probably count the photosynthetic photosynthetic the count yuae i a ery curn pooytae most photosynthate occurring phosphoenol­ early an PEP-CK, is may f o pyruvate, substrate conclusion The Another drawn: be periods. dark to restricted relative kinetic in from resulting obtained [12,23,24] been had experiments light tracer the in tion ^croy gop ofaprae A frt approach, first a At aspartate. f o group) (^-carboxyl less periods incubation partic­ group), short-term a-carboxyl following (= ularly -atom Q the in located f o degradation Chemical ie aa ee ihro vial alwn fr a for the in allowing progressing available /?-carboxylation f o hitherto quantification were data cise strong strong also but initiated), dark is the growth in only blade not (when carbon period fixed contributes tion d ht n prcal pooto ofrdoabn is radiocarbon f o proportion appreciable an that ed light. pre­ no However, 4). and 2 (Fig. malate and partate in n h poal priiain /?-carboxyla- f o participation probable the on tion uig htsnhtcC0 C photosynthetic during h qeto aoe hte o nt /?-carboxyla- not or whether arose question The n hs oncin t s neetn t tk it ac­ into take to interesting is it connection this In 14 -aeln of yia C typical f o C-labelling 14 -ciiy s lo en n h C4-atom the in seen also is C-activity ^ f CROYAIN/ DARK CARBOXYLATION / fi | PHOTOSYNTHESIS | ß -CROYAIN/ LIGHT - / CARBOXYLATION 14 2 14 -aeld 3-phosphogly- C-labelled 14 euto. frt indica­ first A reduction. -aeln of aspartate. f o C-labelling -satt demonstrat­ C-aspartate j / 4 cd sc a as­ as such acids

845

846 B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae cerate. Photosynthetic C 0 2 fixation via RubP-C photosynthesis was reported to operate in blue- yields 3-phosphoglycerate 14C-labelled in the Q- green algae [5]. Proofs for such generalizing qualifi­ atom [1]. This compound is presumably not quanti­ cations of algal photosynthetic pathways have main­ tatively fed into the reductive pentose phosphate ly been derived from 14C-labelling patterns of short­ cycle, but undergoes further conversion to 14Cr term assimilates. In essence, however, there is no phosphoenolpyruvate. Upon carboxylation by PEP- immediate and clear cut experimental evidence for CK 14Ci,4 -oxaloacetate is readily obtained, which in any algal species strongly suggesting that radiocar­ turn gives rise to the formation of aspartate and bon in the C4-position of aspartate and/or malate is malate and thus accounts for the radiocarbon being actually transferred to the exposition of 3-phospho- symmetrically distributed between Q- and C4- glycerate through decarboxylation and subsequent atoms (Fig. 5). Therefore, photosynthesis preferen­ refixation - metabolic events invariably linked with tially provides the substrate for ^-carboxylation in C4 photosynthesis in ecologically specialized ter­ the light. This also explains the delay in 14C-label- restrial plants [26]. Moreover, findings presented in ling of the C4-atom as compared to the a-carboxyl this contribution indicate that, at least in brown group (Fig. 5). Earlier investigation on brown ma­ algae, photosynthetically fixed radiocarbon obvi­ croalgae provided evidence that in the dark phos- ously flows in the opposite direction from 3-phos­ phoenolpyruvate is preponderingly derived from phoglycerate to the C4-acids (Figs. 3 and 5). Ocur- mannitol catabolism via glycolysis [15]. rence of 14C-labelling in aspartate and malate is thus The position of radiocarbon within the aspartate a secondary event which, in the light, clearly de­ molecule is not only indicative for /^-carboxylation pends on primary carbon fixation via RubP-C in­ progressing in the light, but also provides a basis for stead of preceeding this essential step. the tentative quantification of this process. Since it There is no doubt that, particularly in brown ma­ is rather likely to assume that the distribution of 14C crophytic algae, C 4 acids are important intermedia­ between the Q and C4-atoms of malate is very simi­ tes of primary metabolism certainly supplementing lar to the pattern recorded for aspartate, the occur­ the universally occurring reactions of the reductive rence o f i 4C-labelling in the exposition may be pentose phosphate cycle. Biosynthesis and turnover taken as a measure of /^-carboxylation during photo­ of these compounds are more intense in brown than synthesis. A comparison of respective calculations in green or red pigmented algae [10—12, 14—18]. (Fig. 6 ) suggests that rates of /^-carboxylation in the However, the participation of /^-carboxylation ac­ light are not basically different from those observed counting for rapid formation of C 4 acids is basically in the dark (cf Fig. 1). Hence it may be concluded different from that encountered with vascular plants. that products arising from ^-carboxylation via PEP- Therefore the respective metabolic reactions and CK under dark conditions are qualitatively and conversions involved should be associated with the quantitatively comparable to those provided by certainly less ambiguous term C 4 metabolism in­ the same set of reactions in the light. “Dark” carbon stead of C4 photosynthesis [26]. Marine brown algae fixation is thus fully integrated into photosynthetic are thus C4 plants in a very modified sense (cf. ref. C 0 2 assimilation contributing fixed carbon primari­ [19]). It is now recognized that the high potential for ly in the form o f C 4-compounds. This additional /^-carboxylation is an indispensable component of pathway of C0 2 fixation particularly occurring in the overall carbon strategy of brown macrophytes brown seaweeds [11, 12,24] at least partly compen­ [16, 17,27,28] providing the metabolic basis for the sates for respiratory carbon loss [15]. It is therefore remarkable seasonality of their growth performance. justified to consider (dark) /^-carboxylation for cal­ culations of primary productivity [25]. The well documented occurrence of C 4 acids Acknowledgements among the early 14C-labelled photosynthates of ma­ rine algae [11,23,24] caused considerable confu­ This investigation was supported by the Deutsche sions and uncertainties with respect to the mecha­ Forschungsgemeinschaft. The author wishes to thank nisms primarily involved in C 0 2 fixation. Marine Prof. J. Willenbrink (Köln) for continuous interest algae in general have been claimed to exhibit C 4 in this work and Mrs. C. Zander for skillful techni­ photosynthesis [ 8 ]. Similarly, the C 4 pathway of cal assistance. B. P. Kremer • C4-Metabolism in Marine Brown Macrophytic Algae 847

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