Planta

Planta 144, 497-501 (1979)

9 by Springer-Verlag 1979

Carbon Assimilation by Different Developmental Stages of Laminaria saccharina Bruno P. Kremer and James W. Markham Botanisches Institut der Universitfit, Gyrhofstrage 15, D-5000 K61n 41, and Biologische Anstalt Helgoland, Meeresstation, D-2192 Helgoland, Federal Republic of Germany

Abstract. Various stages of the life cycle of the marine brown alga Laminaria saccharina (L.) Lamour. (Lami-

nariales, Phaeophyta) including male and female gametophytes, female gametes, zygotes and young sporophytes of different age were investigated for their potentials of carbon dioxide (1~CO2) fixation. Rates of photosynthesis attain the same order of magnitude in all stages. Photosynthetic 14CO2-fixation is accompanied by a substantial light independent carbon assimilation. This is confirmed by rate determinations of the equivalent carboxylating enzymes present in the plants, ribulose-l,5-bisphosphate carboxylase (EC 4.1.1.39) and phosphoenolpyruvate carboxykinase (EC4.1.1.32) as well as by chromatographic analyses of the appropriate [14C]-assimilate patterns. Key words: Algae (brown) - Carboxylation - CO2 assimilation L a m i n a r i a - Photosynthesis (laminaria).

Introduction

The rates and products of photosynthetic and light independent carbon assimilation of several species of the Laminariales (Phaeophyta) have hitherto been investigated using the fully differentiated adult sporophytes (Kremer and Willenbrink, 1972; Kremer and Kfippers, 1977; Ktippers and Kremer, 1978). Comparative measurements for the gametophytes and very early developmental stages of Laminaria sporophytes have not been reported. In part this was due to the difficulty of obtaining sufficient quantities of the microscopic gametophytes for experimental purposes. Under optimal conditions the gametophytes become reproductive when they consist of one or a few cells

and then do not grow further vegetatively. The development of a technique whereby male and female gametophytes can be cultured to macroscopic dimensions in a vegetative state under red light (Lfining and Dring, 1975) has now made it possible to obtain sufficient quantities of vegetative and reproductive gametophytes as well as of early developmental stages of sporophytes. Hence the question can be evaluated whether e.g. t h e gametes and zygotes of the Laminariales mainly depen d on internal energy reserves or whether they are fully competent to maintain a positive carbon balance by carbon dioxide assimilation. At least the capacity for light dependent carbon fixation should be expected, since the zygotes of Laminaria normally contain well developped chloroplasts (Bisalputra et al., 1971). In the Fucales, similar experiments with eggs, sperm, zygotes, and embryos of Fucus seratus have recently been reported (McLachlan and Bidwell, 1978).

Material and Methods

Plant Material

Cultures of male and female gametophytesof Laminaria saecharina (L.) Lamour. (Laminariales,Phaeophyta) were grown in unisexual, unialgal cultures under continuous red light in the laboratory. Reproduction of the gametophytes was induced by breaking down the gametophyte clumps into few-celledfragments with a mortar and pestle, mixing the fragmented male and female gametophytes, and placing them under white light (light: dark= 16:8; 2,5001x) (Markham et al., 1978). Material used for the experimentsincluded unisexual vegetative male and female gametophytes, female gametes (=extruded eggs, slightly contaminated by fragments of gametophyte cells), germinating zygotes, and young sporophytes of less than 3 mm total length. Determination of ~4C02 Fixation Rates

Abbreviations: RuBP-C=ribulose-l,5-bisphosphate carboxylase;

PEP-CK=phosphoenolpyruvate carboxykinase; PEP=phosphoenolpyruvate; PS=photosynthesis; DF=dark fixation

For an evaluation of the rates of photosyntheticand light independent (dark) carbon assimilation the plant material (about 0.1 g

0032-0935/79/0144/0497/$01.00

B.P. Kremer and J.W. Markham: Carbon Assimilation in Laminaria

498 fresh weight or an equivalent suspension of gametes and zygotes) was incubated in a [H~4CO~]seawater medium in 1.5 ml microtubes containing 10 gCi NaH14CO3 at 10~ C. Photosynthetic experiments were run under white light of 15,000lx at the sample surface. The specimens were harvested by centrifugation, repeatedly washed, deep frozen in liquid N2, and finally lyophilized. Fixation rates were calculated as total amount of organically bound 14C on a dry weight basis.

Carboxylating Enzymes For a further characterization of the carboxylating potentials of the different life history stages of Laminaria saccharina the activities of ribulose-1,5-bisphosphate carboxylase (RuBP-C; EC4.1.1.39) and phosphoenolpyruvate carboxykinase (PEP-CK; EC4.1.1.32) were measured as in vitro acid stable 14C fixation by a crude enzyme extract. Because of the relatively small quantities of plant material the extraction was reduced to 0.1 of those quantities employed earlier (Kremer and Kfippers, 1977"). For the individual tests, 0.5 of all reaction mixtures (Kremer and Kfippers, 1977; Kiippers and Kremer, 1978) was used.

Analytical The assimilatory products after 30 min photosynthesis or dark carbon assimilation were analyzed by two dimensional thin-layer chromatography on cellulose (MN 300 or Avicel) according to Feige et al. (1969) and further analyzed as described earlier (Kremer, 1978).

Table 1. Rates of photosynthesis (PS) and dark fixation (DF) by

different stages of the life cycle of Laminaria saccharina on a dry weight basis Specimen

PS

DF

DF in %

(nmol CO2/10mg/hr) of PS Gametophyte Male Female Gametes (9) Sporophyte Zygote 0.3 cm plant 5.0 cm plant 200 cm plant" intercalary growing zone distal part of the blade

256 277 249

23 24 35

9.2 8.7 14.3

190 239 318

25 26 36

13.4 10.8 11.6

261 803

46 37

17.6 4.6

ValuesfromKfippersandKremer (1978)includedfor comparison

seen that, o n a dry weight basis, photosynthesis is o p e r a t i o n a l in all stages i n almost the same order of m a g n i t u d e . The p h o t o s y n t h e t i c c a r b o n r e d u c t i o n is a c c o m p a n i e d , however, by a light i n d e p e n d e n t , n o n - p h o t o s y n t h e t i c c a r b o n dioxide assimilation of noticeable efficiancy. O n the average, a b o u t 11% of the total c a r b o n fixation p o t e n t i a l is c o n f i n e d to dark assimilation.

Results

Activities o f Carboxylating Enzymes Rates o f Carbon Assimilation The highly active dark c a r b o n fixation requires a further characterization by rate d e t e r m i n a t i o n s of the c a r b o x y l a t i n g enzymes involved. Since earlier work showed that only R u B P - C a n d P E P - C K operate in Laminaria spp., whereas p h o s p h o e n o l p y r u v a t e carb-

All stages in the life cycle of Laminaria saccharina i n c l u d e d in this study proved to be capable of p h o t o synthetic as well as of light i n d e p e n d e n t (dark) c a r b o n fixation. T a b l e 1 s u m m a r i z e s the results. It m a y be

Table 2. In vitro enzymatic incorporation of ~4C02 by Laminaria saccharina RuBP-C and PEP-CK.

Values in dmin- t 14C per total enzyme extract (1.0 ml) Specimen

RuBP-C

PEP-CK

RuBP-C PEP-CK (RuBP-C + PEP-CK = 100%)

Gametophyte Male Female Gamete (9)

15,055 12,100 8,709

3,200 2,690 4,320

78.5 77.7 50.4

21.5 22.3 49.6

13,407 9,120 14,218

4,845 1,843 2,835

63.8 79.8 81.1

36.2 20.2 19.9

8,004 11,805

6,225 1,272

22.3 89.6

77.7 10.4

Sporophyte Zygote 0.3 cm plant 5.0 cm plant 200 cm plant a intercalary growing zone distal part of the blade "

Valuesfrom Kfippers and Kremer (1978) included for comparison

B.P. Kremer and J.W. Markham: Carbon Assimilation in Laminaria

499

oxylase (PEP-C; EC4.1.1.31) is only present in the range of detection limits (Kremer and Kfippers, 1977), only RuBP-C and PEP-CK were considered further. Table 2 presents the activities of both enzymes, which are expressed as 14C activity incorporated in vitro by a crude enzyme extract into acid stable organic binding. Because of the small quantities of plant material available, the data are not based on one of the reference systems usually employed. Since the enzyme activities of RuBP-C and PEP-CK were determined simultaneously from the same preparations, they are strictly comparable only for the same specimen. The data of Table 2 reflect a similar situation to that encountered for the in vivo carbon fixation in the light and in the dark. Besides high amounts of RuBP-C-activity, which is to be expected in a photosynthesizing organism, appreciable activities of PEPCK are detectable which are hence regarded as being the enzyme system responsible for the high dark fixation rates (cf. Akagawa et al., 1972). The carboxylating potential via PEP-CK is significantly higher in the female gametes, in the zygotes, and in the young sporophytes than in the gametophytes taken directly from red light culture. This, too, is consistent with the results from in vivo experiments on carbon fixation with the same stages (Table 1).

Assimilate Pattern and Distribution o f 14C

Figure 1 shows autoradiographs prepared from twodimensional thin-layer chromatograms of ethanolic extracts representing the typical pattern of single assimilates after 30 min incubation. Qualitatively there are no essential differences from earlier findings with mature stages of the same species (Kremer and Willenbrink, 1972). Even the early developmental stages show the same spectrum of intermediary or accumulated photosynthetic products that are normally encountered (cf. Kremer, 1978). Including the main compounds listed in Table 3, more than 60 photoassimilatory products have been traced on the autoradiographs. Some variations are observed with regard to the percentage of 1r recovered from single assimilates of the different stages included for comparison. According to Table 3, mannitol is the most strongly ~4C-labelled compound throughout. In addition, substantial 14C-labelling of certain amino acids is found. In the female gametophytes, zygotes, and the young sporophytes, aspartate and glutamate incorporated relatively more radiocarbon than the other stages. This special feature is suggested as being due to intense/%carboxylation and is thus generally consistent with the rates presented in Table 2.

Fig. 1a andb. ZygotesofLaminaria saccharina. Patterns of assimilates after 30 min photosynthesis(a) and 30 min dark fixation (b) : autoradiographs prepared from two-dimensional thin-layer chromatograms of l~C-labelled soluble compounds. 1 origin area, 2 organic phosphates, 3 aspartate, 4 glutamate, 5 citrate, 6 malate, 7 fumarate, 8 succinate, 9 mannitol, 10 glycine+serine, 11 alanine

The assimilate pattern after dark carbon assimilation is composed of considerably less compounds than the variety of assimilates produced by photos?~lhetic carbon fixation. Organic phosphates and mannitol are not ~4C-labelled in the dark (Fig. 1). The distribution of radiocarbon among the main dark assimilates is compiled in Table 4. Rather intense ~4C-labelling is obtained for aspartate, glutamate, malate.

500

B.P. Kremer and J.W. M a r k h a m : Carbon Assimilation in Laminaria

Table 3. Distribution of 14C a m o n g various photoassimilatory products after 30 min incubation as percentage of total ~4C-labelling of the soluble fraction Specimen

Origin area

Organic phosphates

Aspartate

Glutamate

Glycine + Serine

Alanine

Mannitol

Organic acids

Gametophyte Male Female Gamete (9)

1.7 1.8 1.5

2.3 3.4 2.1

2.8 2.3 3.1

3.2 2.4 11.3

20.2 21.9 4.0

16.7 19.8 27.7

48.9 42.3 35.0

4.2 3.6 6.3

Sporophyte Zygote 0.3 cm plant 5.0 cm plant

1.8 1.1 0.4

2.0 2.9 1.4

3.2 2.2 2.3

8.0 1.4 1.9

2.8 21.9 16.8

26.3 20.4 24.3

41.2 47.5 50.4

5.6 2.9 2.5

Table 4. Distribution of 14C a m o n g the main dark assimilates after 30 min incubation as percentage of total 14C-labelling of the soluble fraction

Specimen

Organic phosphates

Aspartate

Glutamate

Alanine

Malate

Citrate

Others

Gametophyte Male Female Gametes (9)

< 1