Photoassimilatory Products and Osmoregulation in Marine Rhodophyceae

Botanisches Institut der Universitat zu Koln, Bundesrepublik Deutschland

Photoassimilatory Products and Osmoregulation in Marine Rhodophyceae BRUNO P. KREMER With 2 figures Received November 30, 1978 . Accepted December 23, 1978

Summary A variety of intertidal and subtidal marine representatives of the Rhodophyceae including Porphyra perforata, Porphyra umbilicalis, Chondrus crispus, Iridaea flaccida, Dumontia incrassata, Rhodymenia palmata and some other species have been investigated with special regard to their synthesis of accumulated photoassimilatory products in relation to the osmotic value (salt content) of the incubation medium. Percentages of 14C-Iabelling of floridoside (2-0-D-glycerol-a-D-galactopyranose) as well as of digeneaside (1-0-D-glycerate-a-D-mannopyranose) show that biosynthesis of these heterosides during photosynthetic carbon assimilation is not affected by osmotic stress over a wide salinity range. Further evidence from pulse-chase experiments and quantitative determinations suggests that in marine Rhodophyceae osmotic regulation is not achieved on the level of soluble carbohydrates involving floridoside or related compounds. Key words: Rhodophyceae, red algae, photosynthesis, heterosides, floridoside, osmotic stress, osmoregulation.

Introduction

Algae show a Irather dear-cut differentiation into halophilic and non-halophilic species occupying biotopes of a wide range of water potentials. These may include very saline or rather dry environments and even habitats with fluctuating salinities (d. GESSNER and SCHRAMM, 1971). Several specific osmoregulatory or osmoadaptive mechanisms have been evolved to compensate for the externally induced water stress and to maintain the intracellular osmotic balance either by ion (water) tlransport or by biosynthesis of certain organic molecules. Physiological aspeates of osmoregulaltion particularly in fungi and algae have recently been reviewed by HELLEBUST (1976), whereas the biochemistry of osmotic regulation has been dealt with by KAUSS (1977) and structural aspects have been considered by WIENCKE and LXUCHLI (1977). KAUSS (1967 a, 1967 b, 1973) reported that in the chrysomonad flagellate Ochromonas malhamensis (as well as in Ochromonas danica) an increase or decrease in the osmotic value of the eXiternal medium is rather rapidly balanced by the

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synthesis or degradation, respectively, of isofloridoside (1-0-glycerol-a-D-galactopyranose). Since compounds of this type (heterosides) are widely distrubuted among the representatives of the Rhodophyceae as well (AUGIER and DuMERAc, 1954), it seemed reasonable to include species of this algal class for comparison. In Iridophycus flaccidum (Gigartinales) and Porphyra perforata (Bangiales) KAuss (1968, 1969) observed an increased incorporation of He into floridoside and isofloridoside, when the salt concentration was increased, and suggested that this might possibly indicate a role of these heterosides in species of the Rhodophyceae as well. Experiments on photosynthetic 14C02-assimilation using a variety of red algal species (MAJAK et aI., 1966; NAGASHIMA et aI., 1969; KREMER 1976 a, 1978 a, 1978 b, 1978 c) showed that floridoside ,is strongly HC-labelled in the light and thus represents the most important and striking solubLe carbohydrate among the accumulated photosyntha.tes. However, since this particular compound is encountered in freshwater red algae as well as in marine forms, a general funotion in osmoregulation (KAuss, 1968) seems to be questionable. Furthermore, it is evident that the representatives of the Ceramiales are lacking floridoside as a photosynthate, but synthesize digeneaside (2-0-D-glycerate-a-D-mannopyranose) instead (KREMER, 1978 b). This compound has not yet been considered for osmoregulatory balance. A critical reinvestig,ation of red algal heterosides was therefore needed with particula;r regard to their photosynthetic 14C-labelling, when the appropriate algae were previously kept under various osmotic conditions.

Material and Methods Algal Material

The red algal species listed in Tables 1 and 2 originated from Helgoland (North Sea, Germany) and/or from Roscoff (Brittany, France) and were usually collected from their intenidal or subtidal habitats during low tide. The Pacific species Iridaea flaccida (S. & G.) SILVA (= Iridophycus flaccidum) and Porphyra perforata J. Ag. were collected in the vicinity of the Barnfield Marine Station, Vancouver Island, B.C., Canada. All specimens were carefully checked for epibionts, cleaned, and maintained in flow tanks for not longer than about 12 h before being used in the experiments. 14e-Assimilation

Selected thallus pieces of about 1-3 g fresh weight were allowed to photosynthesize in H 14 COs--containing incubation media for various periods (cf. KREMER 1978 a, 1978 b). The incubation media used comprised a) normal membrane-filtered seawater (S = 33 0/00), b) hyposaline media (S = 2-27 %0) prepared by dilution of normal seawater with 20 mM carbonate buffer, and c) hypersaline media (S = 33-65 %0) which were obtained either by evaporation of normal seawater or by addition of equivalent amounts of NaC!. The CO 2content of all media was determined according to STRICKLAND and PARSONS (1972). During the photosynthesis experiments (20,000 Ix; 10-15 0c) the algal samples were continuously stirred with a magnetic stirrer to avoid concentration gradients. After the appropriate incubation periods the specimens were briefly rinsed in freshwater and immediately fixed either in boiling 80 Ufo ethanol or in liquid N2 and then lyophilized. All experiments were run with several replicates.

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Analytical

Extraction and chromatographic analyses of the constituents of the soluble fraction of assimilates were performed as has already been described earlier (KREMER, 1978 a, 1978 b). Heteroside contents of the samples investigated were quantitatively determined according to the method of DUBOIS et al. (1956) using calibrated galactose standards.

Results Rates of Photosynthesis Photosynthetic carbon assimilation in marine Rhodophyceae is distinctly influenced by the osmotic value of the incubation medium. Fig. 1 presents the

• •

PORPHYRA UMBILICALIS PORPHYRA PERFORATA

... 1S .s:

~

-b

...", 10 E

8

~

s.. 5

"0

•

CHONORUS CRISPUS

9172735

45

55

65

S Wool Fig. 1: Porphyra umbilicalis, Porphyra per/arata, Chondrus crispus. Dependency of photo-

synthetic carbon fixation from the osmotic value (salinity) of the incubation medium.

equivalent data for three euryhaline species, Porphyra perforata, Porphyra umbilicalis, and Chondrus crispus, showing that photosynthetic efficiency over a wide range of salinity (9-65 0 /0) obviously follows an optimum curve. Apart from species-dependent features of maximum photosynthetic rates, the light dependent carbon fixation in all species investigated is highest in the range of about 33 0/ 00 , but is markedly depressed in hyposaline as well as in hypersaline media representing exltremes that are rather unlikely to be encountered in the normal habitat of the species regarded. Osmotic stress in hyposaline media seems to be much better tolerated than a hypersaline environment. Salinity and 14C-Labelling of Floridoside Earlier investigations on a variety of red algae had shown that in species e. g. of the red algal orders Cryptonemiales and Gigartinales about 30-50 Ofo or even more

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of the photosymhetically assimilated 14C are recovered from 2-0-D-glycerol-a-D-galactopyranose (floridoside) after 30-60 min photosynthesis in normal seawater (cf. MAJAK et a!., 1966; CRAIGIE et a!., 1968; KREMER, 1978 a, 1978 b). Further experiments showed that a pretreatment of the species in a non-,radioactive incubation medium of a changed osmotic value did not affect the distribution of radiocarbon among the soluble low-molecular weight assimilaJtes. Therefore all experiments designed to evaluate the influence of salinity on the HC-labelling charact'eristics of floridoside included 30 min preillumination in a H 12 COa--incubation medium of defined salinity and further 30 min photosynthesis in the same, but radioactive (HHCOa--)medium. The amounts of HC in floridoside after 30 min photosynthesis of some selected marine algae of different osmotic values are presented in Table 1. It may be noted that in all red algae investigated the Table 1: Radiocarbon recovered from 2-0-D-glycerol-a-D-galactopyranose (floridoside) in different algae after 30 min photosynthesis in media of different salinity. Values expressed in Ofo of total HC in the soluble fraction of assimilates. Species Bangiales Porphyra perforata J. AG. Porphyra umbilicalis (L.) J. AG. Gigartinales Calliblepharis jubata KiiTZ. Chondrus crispus STACKH. Iridaea flaccida (S. & G.) SILVA Cryptonemiales Dilsea camosa (SCHMID) KUNTZE Dumontia incrassata (0. F. MiiL.) LAM OUR. Rhodymeniales Rhodymenia palmata (L.) GREV.

S (%0) 2 9

34.1

18.6 31.8

17

27

35

45

55

65

17.2 28.7

19.1 33.9

16.8 35.7

17.4 20.5

19.8

17.8

42.4 26.1

37.5 45.2 24.4

37.7

34.0

31.8

33.9

34.2

23.5 30.9

32.7 39.4

38.9

41.2

42.3

40.8

25.2

27.8

24.3

23.9

25.1

percentage of floridoside is rather invariable and does not show significant correlations with the salinity of the appropriate incubation medium. Even if the algal samples are allowed to photosynthesize HC from H 14COa- in a medium not exceeding 2 0/ 00 salinity, appreciable amounts of floridoside of proportionally the same order of magnitude as in normal (33 0/ 00 ) seawater are synthesized. Biosynthesis of Other Photosynthates Floridoside is a widespread constituent of freshwater and marine species of the Rhodophyceae, but is occasionally replaced by other specific compounds. While all

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members of the order Cerami ales such as Rhodomela confervoides have been found to photosynthesize digeneaside (1-0-D-glycerate-a-D-mannopyranose) (KREMER, 1978 b), speoies of the red algal genus Bostrychia accumulate two hexirtols, dulcitol (galactitol) and sorbitol (gluciwl) (KREMER, 1976 b). Since polyhydroxy alcohols such as hexitols have genel1ally been considered as osmoregulatory compounds of algae (cf. HELLEBUST, 1976), similar experiments on the influence of salinity on the biosynthesis of these photosynthates were conducted using Rhodomela confervoides and Bostrychia scorpioides. The resuIts are given in Fig. 2. In Bostrychia scorpioides the HC-la:belling percentage of both hexitols remains widely unaffected over a salinity range, but shows a slight depression
BOSTRYCHIA SCORPIOIOES

30

•

RHODOMELA CONFERVOIDES

29172735

1.5

55

65

S l%ol

Fig. 2: Bostrychia scorpioides, Rhodomela confervoides. Dependency of photosynthetic He-labelling of low-molecular weight carbohydrate photosynthates (Bostrachia: dulcitol + sorbitol; Rhodomela: 1-0-D-glycerate-a-D-mannopyranose = digeneaside) from osmotic value (salinity) of the incubation medium.

Pulse-Chase Experiments

Osmoregula.tion in the marine Rhodophyceae might possibly involve the interconversion of osmotically ineffective polymeric storage material (e. g. glucan-type polysaccharides) and of osmotically active floridoside(s) following a regulation scheme which has been proposed for the chrysomonad Ochromonas malhamensis (KAuss, 1974). Therefore specimens of Chondrus crispus were first allowed to photosynthesize HC for 12 h in normal seawMer (33 %0) and were then incubated in H 12 CO a--seawater for further 12 h, which provided strong HC-Iabelling even of the insoluble constituents including floridean starch. When the samples were Z. Pflanzenphysiol. Bd. 93. S. 139-147. 1979.

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subsequently transferred to hypersaline media and thus subjected to considerable osmotic st,ress for 30 min, no increase in the amounts of radiocarbon recovered from floridoside as compared to controls was observed. Quantitative Determinations Table 2 presents the results of quantitative determinations of the total floridoside content in Porphyra umbilicalis and Chondrus crispus after 60 min incubation in media of different osmotic pressure and includes average v,alues of the specific Table 2: Porphyra umbilicalis, Chondrus crispus. Total amounts of floridoside in algal samples after 60 min treatment in media of different salinity and specific radioactivity of the same compound after 30 min photosynthesis in H14CO a-. Average values of three replicates. Species

Porphyra umbilicalis (L.)

S(O /00) 17 9

J. AG.

Chondrus crispus STACKH.

,25 a) 52 b ) 16 a) 16b)

27

35

45

55

65

27 55

28 64

28 70

23 61

24 49

20 42

23 22

15 20

20 34

20 13

17 10

15 8

a) values expressed in ,umol floridoside/100 mg dry wt. values expressed in nCi 14e/ ,umol floridoside.

b)

radioactivity of floridoside achieved after 30 mm photosynthesis. Apart from the obviously species-dependent differences in total amounts of floridos,ide (> 20 ,umol/100 mg dry Wlt; equivalent to about 5.9 Ofo on a dry wt basis in Porphyra; < 20 ,umol/100 mg dry wt in Chondrus) no linear proportionality of the total content to ,the osmotic value of the incubation medium is observed, at least not after relatively short term incubations ranging up to 60 min. A distinot enhancement of the flor,idoside 'COIl!tent over the salinity range regarded should occur in the species investigated, if it is assumed that this heteroside actually serves as an osmoregulatory substrate.

Discussion

According to earlier findings w~th Iridophycus (Iridaea) and Porphyra (KAUSS, 1968, 1969) a distinct correlation of the rate of synthesis of floridoside-type carbohydrates and of ,the water potential (i. e. osmotic value) of the external incubation medium should generally he expected. However, in our experiments the rates and percentages of He-labelling of floridoside proved to be rather unaffected. This is, in our experiments, not only true for Iridaea flaccida and Porphyra

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perforata, the species originally investigated, hut also for a variety of further species including repr,esenta.tives of ~he intertidal zone as well ,as some subtidal species. It is of special interest nh 60 °/(0) (Table 1). Similar results are obtained when 14C-Iabelling of digeneaside (mannosidoglyoerate) in members of the Ceramiales (e. g. in Rhodomela confervoides) is regarded (Fig. 2). It remains an open question whether variability of the algal material or modified incubation techniques may account for the different findings obtained by KAUSS (1968).

Su and HASSID (1962) report that species of the Bangiales such as Porphyra contain both floridoside (2-0-D-glycerol-a-galactopyranose) as well as isofloridoside (1-0-D-glycerol-a-D-galactopyranose) which are 'both suggested to be involved in osmoregulation in Porphyra perforata according to KAUSS (1968, 1969). In a survey on the photosynthetic products of a variety of Rhodophyceae including more than 100 species KREMER, 1978 a; KREMER and VOGL, 1975), we were unable to trace 14C-Iabelled isofloridoside though a rel~able differentiation of both isomers by means of chromatographic analyses is readily achieved (d. KAuss, 1968). This is consistent with the findings 'by CRAIGIE et al. (1968) who report that very low l4C-Iabelling in isofloridoside is observed even after relatively long periods of photosynthetic carbon fixation. According ~o the results 'Obtained from the chrysomonad Ochromonas malhamensis KAuss, 1974), the pools of low-molecular weight carbohydrates such as isofloridoside and of storage glucan are interoonvertible depending on external osmotic pressure. Our experiments using the r.ed algal speci,es Chondrus crispus, which had been allowed to photosynthesize H 14CO S- for 12 h, yielded no significant increase in 14C~I
Provided that floridoside serves as an osmoregulatory effector in species of marine Rhodophyceae, proportionality between the osmotic value of the incubation medium (salinity) and the total content of floridoside in the algae should be .encountered. Determinations of the absolute quantiuies of floridoside present in different marine Z. Pflanzenphysiol. Bd. 93. S. 139-147. 1979.

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BRUNO P. KREMER

species after different pretreatment did not reveal such correlat[ons (Table 2). The amounts of floridoside determined proved to he r,ather independent from the osmotic stress to which the specimens investigated Ihad been subjected. Since earlier investigations showed that floridoside generaIIy occurs ·as a photosynthate in the Rhodophyceae and has hitheflto never been found 'to be synthesized under oonditions of light independent carbon assimilation (KREMER, 1978 b), the rate of synthesis of this particular compound may best be aIlied with the rates of net photosynthesis achieved by the lappropriate species under various osmotic conditions. Fig. 1 shows that photosynthesis in some selected species is markedly influenced by the salinity of the incubation medium. Particularly higher salinity exceeding 33 %0 causes considerably decreasing raltes of photosynthesis. If reciprocaIIy lower rates of photosynthetic carbon assimilation in hypersaline media are expressed in terms of floridoside biosynuhesis, it becomes evident that at higher salinity much less flor,idoside is synthesized de novo than in normal seawater (cf. Table 2). This, too, is contradictory to Kauss' hypothesis of osmoregulation via fIoridoside(s) in Rhodophyceae. On the whole, the experimental results presented in this contribution seem to provide sufficient evidence ,that osmoregulation in mar,ine Rhodophyceae is most probably not mediated by means of low-molecuLar weight carhohydrates including e. g. fIoridoside-type assimilates. Osmotic stress by incubation of algal samples in hypersaline or hypos aline media is obviously not balanced 'by internal regulation of organic constituents. In this special regard, marine representatives of the Rhodophycee are scarcely companable to the uniceIIular flageHate Ochromonas malhamensis (cf. KAuss 1974, 1977). Therefore, floridoside and digeneaside might be regarded as constitutive photo assimilatory products of the red algae, which occur independently from external physical conditions and which are hence even u,e[ul and reliable pammeters in taxonomic considerations (KREMER, 1978 b). Acknowledgements The author wishes to thank Prof. Dr. H. KAUSS (Kaiserslautern) and Prof. Dr. J. WILLENllRINK (Koln) for valuable criticism, The help of Dr. J. W. MARKHAM (Helgoland) with preparing the Engl.ish version of the manuscript is gratefully acknowledged.

References AUGIER, J. et M. 1. DU MhAC: Les sucres solubles des Rhodophycees. C. R. Acad. Sci. Paris 238, 387-389 (1954). CRAIGIE, J. S., J. McLACHLAN, and R. D. TOCHER: Some neutral constituents of the Rhodophyceae with special reference to the occurrence of the floridosides. Can. J. Bot. 46, 605-611 (1968). DUBOIS, M., K. A. GILLES, J. K. HAMILTON, P. A. REBERS, and F. SMITH: Colorimetric method for determination of sugars and related substances. Anal. Chern. 28, 350-356 (1956).

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GESSNER, F. and W. SCHRAMM: Plants. In: O. KINNE (Ed.): Marine Ecology, Vol. I12 (Environmental Factors), p. 705-820. Springer, Berlin-Heidelberg-New York, 1971. HELLEBUST, J.: Osmoregulation. Ann. Rev. Plant Physiol. 27, 485-505 (1976). KAUSS, H.: Isofloridosid und Osmoregulation bei Ochromonas malhamensis. Z. Pflanzenphysiol. 56, 453-465 (1967 a). - Metabolism of isofloridoside (O-a-D-galactopyranosyl-(1-+1)-glycerol) and osmotic balance in the freshwater alga Ochromonas. Nature 214,1129-1130 (1967 b). - a-Galaktosylglyzeride und Osmoregulation in Rotalgen. Z. Pflanzenphysiol. 58, 428-433 (1968). - Osmoregulation mit a-Galaktosylglyzeriden bei Ochromonas und Rotalgen. Ber. Dtsch. Bot. Ges. 82, 115-125 (1969). - Turnover of galactosylglycerol and osmotic balance in Ochromonas. Plant Physiol. 52, 613-615 (1973). - Osmoregulation in Ochromonas. In: U. ZIMMERMANN, J. DAINTY (Eds.): Membrane Transport in Plants, p. 90-94. Springer, Berlin-Heidelberg-New York, 1974. - Biochemistry of osmotic regulation. In: D. H. NORTHCOTE (Ed.): International Review of Biochemistry, Plant Biochemistry II, Vol. 13, p. 119-140. University Park Press, Baltimore, 1977. KREMER, B. P.: Mannitol in the Rhodophyceae - a reappraisal. Phytochemistry 15, 1135-1138 (1976 a). - 14C-Assimilate pattern and kinetics of photosynthetic 14C02-assimilation of the marine red alga Bostrychia scorpioides. Planta 129, 63-67 (1976 b). - Patterns of photoassimilatory products in Pacific Rhodophyceae. Can. J. Bot. 56, 1655-1659 (1978 a). Studies on 14C02-assimilation In marine Rhodophyceae. Marine Biology 48, 47-55 (1978 b). - Aspects of CO 2-fixation in some freshwater Rhodophyceae. Phycologia 17, 430-434 (1978 c). KREMER, B. P. and R. VOGL: Zur chemotaxonomischen Bedeutung des 14C-Markierungsmusters bei Rhodophyceen. Phytochemistry 14, 1309-1314 (1975). MAJAK, W., J. S. CRAIGIE, and J. McLACHLAN: Photosynthesis in algae. I. Accumulation products in the Rhodophyceae. Can. J. Bot. 44, 541-549 (1968). NAGASHIMA, H., S. NAKAMURA, and K. NISIZAWA: Isolation and identification of low-molecular weight carbohydrates from a red alga, Serraticardia maxima. Bot. Mag. (Tokyo) 82, 379-386 (1969). STRICKLAND, J. D. H. and T. R. PARSONS: A practical handbook of seawater analysis. Fish. Res. Bd. Can. Bull. 167, p. 1-310, Ottawa 1972. Su, J.-c. and W. Z. HASSID: Carbohydrates and nucleotides in the red alga, Porphyra perJorata. I. Isolation and identification of carbohydrates. Biochemistry 1, 468-474 (1962). WIENCKE, C. and A. LXUCHLI: Structural aspects of osmoregulation in Porphyra. In: 9th Int!. Congr. Electron Microscopy, Vol. II, p. 412-413, Toronto 1978. Dr. BRUNO P. KREMER, Botanisches Institut der Universitat, III. Lehrstuhl, Gyrhofstralle 15, D-5000 Koln 41, Bundesrepublik Deutschland.

Z. PJlanzenphysiol. Bd. 93. S. 139-147. 1979.