Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatifida (Phaeophyceae)

Phycologia (2006) Volume 45 (6), 642–647

Published 1 November 2006

Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatifida (Phaeophyceae) XU ZHANG1, HANHUA HU2* 1

AND

TIANWEI TAN1

Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, P. R. China 2 State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, P. R. China X. ZHANG, H. HU AND T. TAN. 2006. Photosynthetic inorganic carbon utilization of gametophytes and sporophytes of Undaria pinnatifida (Phaeophyceae). Phycologia 45: 642–647. DOI: 10.2216/05-28.1 The characteristics of inorganic carbon assimilation by photosynthesis were investigated in male and female gametophytes and juvenile sporophytes of Undaria pinnatifida. Gametophytes and sporophytes have detectable extracellular and intracellular carbonic anhydrase (CA) activity, and the CA inhibitor, acetazolamide (AZ), significantly inhibited their photosynthesis O2 evolution. In pH-drift experiments, it was found that gametophytes did not raise the final pH of seawater above 9.00 (CO2 concentrations of about 2.2 ␮M), indicating a low ability to utilize inorganic carbon. In contrast, sporophytes rapidly raised pH to over 9.53 and depleted the free CO2 concentration to less than 0.16 ␮M. The apparent photosynthetic affinity for CO2 was almost the same for gametophytes and sporophytes, whereas gametophytes had a much lower affinity for HCO3⫺ than sporophytes. Two inhibitors of band 3 anion exchange protein (DIDS and SITS) inhibited the photosynthesis of gametophytes but not that of sporophytes. It was indicated that both gametophytes and sporophytes were capable of using HCO3⫺, which involved the external CA activity, and a direct HCO3⫺ use also occurred in the former, but the latter showed a greater capacity of HCO3⫺ use than the former. In addition, male and female gametophytes did not show great differences in the inorganic carbon uptake mechanism underlying photosynthesis. KEY WORDS: Carbonic anhydrase, Gametophytes, Inorganic carbon, Photosynthesis, Sporophytes, Undaria pinnatifida

INTRODUCTION Undaria pinnatifida (Harvey) Suringar is a brown macroalga, originally endemic to Japan. Used mainly as seafood, U. pinnatifida is economically important and has been commercially cultivated in China, Japan and Korea. U. pinnatifida has been accidentally introduced in Australia, New Zealand and Europe via ballast water discharged from ships and has become a marine pest in the natural ecosystems of some areas (Hay & Luckens 1987; Fletcher & Manfredi 1995). Undaria pinnatifida has an annual life history that alternates between microscopic stages (spores and gametophytes) and the visible kelp stage (sporophytes). Within its native range, U. pinnatifida exhibits a strongly defined annual growth cycle. The sporophytes grow through winter and mature in early to midspring. As sea temperatures increase, the sporophytes produce haploid spores by meiosis that germinate and grow into microscopic haploid gametophytes (male and female). As seawater temperatures drop, fusion of eggs and sperm produced by the dioecious gametophytes gives rise to the next season’s sporophytes (Li 1995). The total amount of dissolved inorganic carbon (DIC) in seawater includes dissolved CO2 (CO2(aq)), HCO3⫺ and CO32⫺. Within the normal pH range of seawater (8.0 to 8.3) and at equilibrium with atmospheric CO2, the bulk of DIC is HCO3⫺. Its concentration is approximately 2 mM, whereas the level of CO2(aq) is 12 to 15 ␮M, at 25⬚C (Sukenik et al. 1997). Johnston (1991) concluded that marine macroalgae display at * Corresponding author ([email protected]).

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least three mechanisms of DIC assimilation: (1) diffusive CO2(aq) uptake; (2) CO2(aq) uptake dependent on extracellular CA catalyzed dehydration of HCO3⫺; and (3) direct transport of HCO3⫺ into the cell. The mechanism of DIC utilization is species-dependent. Many marine macroalgae are able to utilize HCO3⫺ in addition to CO2(aq) (Sand-Jensen & Gordon 1984; Beer & Shragge 1987; Surif & Raven 1989; Maberly 1990; Mercado et al. 1997; Israel et al. 1999). On the other hand, CO2(aq) is the only source of DIC for some macroalgae (Maberly 1990). The ability to deplete DIC is linked with the ecology or taxonomy of the species (Surif & Raven 1989; Maberly 1990). However, the influence of life history phase on DIC acquisition has not been investigated in many brown macroalgae. The aims of this work were to investigate and distinguish the DIC acquisition mechanisms of gametophytes (including male and female) and juvenile sporophytes of U. pinnatifida, which might help to increase the production of the economic seaweed through the appropriate supply of carbon source in different developmental stages.

MATERIAL AND METHODS Organisms and growth conditions Gametophytes of U. pinnatifida were obtained as an axenic culture from the Institute of Oceanology, Chinese Academy of Sciences, and were cultured as previously described (Pang & Wu 1996). The filamentous male (5–10 ␮m broad) and

Zhang et al.: Inorganic carbon utilization in Undaria female (10–20 ␮m broad) gametophytes were fragmented by sonication at 100 W for 1 min, and were harvested by centrifugation at 1000 ⫻ g for 3 min at 25⬚C. The sonicated gametophytes were observed under microscope, and it was found that they did not burst and were able to grow into juvenile sporophytes. Then the gametophytes were washed and resuspended in the solution to be used in the subsequent experiments. Juvenile sporophytes (1–2 mm) of U. pinnatifida were obtained by mixing male and female gametophytes together in a container with a rough surface glass plate as substratum. The culture conditions proposed by Pang & Wu (1996) were adopted: 17⬚C, 60 ␮mol photons m⫺2 s⫺1, with a 12 : 12 h light/dark cycle. Measurement of pH change in external solution About 0.5 g fresh weight (FW) of male or female gametophytes, or juvenile sporophyte blades of U. pinnatifida were placed in 50 ml glass bottles, which were filled with sterilized seawater, fitted with a pH electrode and stirred with a magnetic bar to measure the pH drift. After sealing to prevent CO2 exchange with the atmosphere, bottles were incubated at 25⬚C and 40 ␮mol photons m⫺2 s⫺1 for gametophytes, and 17⬚C and 60 ␮mol photons m⫺2 s⫺1 for sporophytes. The pH was recorded continuously until no increase was observed for at least 1 h. Measurement of net photosynthetic rates Net photosynthetic rates were measured as O2 evolution with a DW2/2 oxygen electrode (Hansatech Ltd, King’s Lynn, Norfolk, UK) under the same conditions as for pH drift experiments with red LED as light sources. The measuring chamber contained 2.0 ml sterilized seawater or artificial seawater (ASW: 400 mM NaCl, 50 mM MgCl2, 30 mM Na2SO4, 10 mM CaCl2, 10 mM KCl) (Lyman & Fleming 1940) and 0.05 g FW male or female gametophytes, or juvenile sporophyte blades of U. pinnatifida. Photosynthetic rates were expressed as micromoles of O2 per milligram of chlorophyll a per hour. Chlorophyll a was measured by centrifuging the algae at 3000 ⫻ g for 10 min at 4⬚C. The pellet was resuspended in 2 ml 90% (v/v) acetone, extracted for 24 h at 4⬚C, and then centrifuged at 10,000 ⫻ g for 10 min at 4⬚C to remove debris. Chlorophyll a in the extract was measured using the equations of Jeffrey & Humphrey (1975). Photosynthetic response to pH change To determine photosynthetic response to pH change, algae were transferred to the O2 electrode chamber containing natural seawater (at 2.3 mM DIC) at different pH, using 25 mM MES, TRIS or CAPS (Sigma Co., St. Louis, USA) as buffers. Photosynthetic response to external inorganic carbon To determine the photosynthetic response to external inorganic carbon, algae were immersed in inorganic carbon–free ASW buffered with 25 mM TRIS for pH 8.3 or 25 mM CAPS for pH 9.3. After sealing the system and providing illumination, the algae were allowed to consume any residual DIC, until no net O2 evolution was observed. Increasing levels of freshly prepared NaHCO3 solution were then injected into the

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chamber and O2 evolution was recorded. Apparent half-saturation values (K1/2) were estimated for DIC and CO2(aq) using the Michaelis–Menten equation. Measurement of carbonic anhydrase activity The activity of intra- and extracellular CA was measured as the ability of a sample to catalyze the hydration rate of CO2(aq) to HCO3⫺. About 0.5 g FW male or female gametophytes, or pieces of sporophyte thallus were sampled for extracellular CA determinations, and the algal homogenates (using a tissue homogenizer at 4⬚C) were used to measure total (intra- and extracellular) CA. According to the method of Israel et al. (1999), the samples were immersed in 50 ml CO2(aq)–free ASW at pH 8.2 (5 mM TRIS-HCl) at 4⬚C. The reaction was started by introducing 10 ml ice-cold CO2(aq)–saturated distilled water into the mixture. Enzyme units (EU) were calculated from the time taken to lower the pH from 8.2 to 6.3 using the following equation: EU ⫽ 10(t0/tc ⫺ 1)/mg chlorophyll a where t0 is the time for the uncatalyzed reaction and tc is the time for the catalyzed reaction. Measurement of alkalinity and total DIC, and the calculation of CO2(aq) and HCO3⫺ Alkalinity was measured as described by Parsons et al. (1989). Total DIC was calculated from carbonate alkalinity (Calk) and pH according to Stumm & Morgan (1970). [DIC] ⫽ (Calk ⫹ [H⫹ ] ⫺ [OH⫺ ])/(a1 ⫹ 2a2 ) a 0 ⫽ (1 ⫹ K1 /[H⫹ ] ⫹ K1K2 /[H⫹ ] 2 )⫺1 a1 ⫽ (1 ⫹ [H⫹ ]/K1 ⫹ K2 /[H⫹ ])⫺1 a2 ⫽ (1 ⫹ [H⫹ ]/K1 /K2 ⫹ [H⫹ ]/K2 )⫺1 [CO2(aq) ] ⫽ a 0 [DIC]

[HCO3⫺ ] ⫽ a1 [DIC]

where K1 and K2 are the first and second apparent dissociation constants of H2CO3, according to Goyet & Poisson (1989): pK1 ⫽ 812.27/T ⫹ 3.356 ⫺ (0.0017 ⫻ S ⫻ ln T) ⫹ (0.000091 ⫻ S 2) pK2 ⫽ 1450.87/T ⫹ 4.604 ⫺ (0.00385 ⫻ S ⫻ ln T) ⫹ (0.000182 ⫻ S 2) where T is absolute temperature and S is salinity. Inhibitors Three inhibitors (Sigma Co., St. Louis, USA) were used: AZ; 4⬘,4⬘-diisothiocyanatosilbene-2,2-disulphonic acid (DIDS); and 4-acetamido-4⬘-isothiocyanostilbene-2,2⬘-disulphonic acid (SITS). Changes of the net photosynthetic rates of gametophytes or sporophytes of U. pinnatifida submerged in seawater were compared before and after the exposure to 200 ␮M AZ, 300 ␮M DIDS or 600 ␮M SITS. Statistics The data were expressed as the mean values ⫾ standard deviation (s). Statistical significance of the means was tested

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Fig. 1. The pH drift for gametophytes and sporophytes of Undaria pinnatifida in natural seawater (pH 8.28) at 25⬚C and 40 ␮mol photons m⫺2 s⫺1 for gametophytes, and 17⬚C and 60 ␮mol photons m⫺2 s⫺1 for sporophytes. Mean ⫾ s, n ⫽ 5.

with one-way analysis of variance (ANOVA) followed by a Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons using the commercial software Statistica 6.0 (StatSoft Inc, Tulsa, Oklahoma, USA). The significance level was set at 0.05.

RESULTS Drifts of pH in a closed system For male and female gametophytes of U. pinnatifida, the pH of seawater increased steadily from 8.28 to maximal values of 8.82 and 8.81, respectively, after illumination for 10 h in a closed system (Fig. 1). No significant difference was observed between male and female gametophytes in the rate of alkalization (P ⬎ 0.05). Over the same time period, juvenile sporophytes increased the pH of seawater from 8.28 to 9.53 (Fig. 1). The final inorganic carbon concentrations were calculated from the pH values. For male and female gametophytes, the concentration of CO2(aq) approached 2.20 ␮M and HCO3⫺ decreased by only 30%, from 2.00 mM to 1.36–1.40 mM. The residual inorganic carbon concentrations at the end of the pH drift experiments with sporophytes were significantly lower than for gametophytes, with CO2(aq) at 0.16 ␮M (F2,12 ⫽ 11,724.60, P ⬍ 0.0001) and HCO3⫺ at 0.52 mM (F2,12 ⫽ 639.59, P ⬍ 0.0001). Net photosynthetic rate response to pH variations Rates of photosynthesis of both gametophytes and sporophytes were maximal at pH 7.2 and 8.3, and approached zero at pH 10.3 (Fig. 2). From pH 8.3 to 9.3, the photosynthesis of gametophytes decreased but that of sporophytes did not change. The effects of low pH (⬍ 7.2) on photosynthesis were greater for female gametophytes than for males. Dark respiration rates of sporophytes did not vary significantly between

Fig. 2. Rates of net photosynthesis (NPS) and dark respiration (RES) of gametophytes and sporophytes of Undaria pinnatifida in natural seawater [2.3 mM dissolved inorganic carbon (DIC)] at different pH. Mean ⫾ s, n ⫽ 6.

pH 5.5 and 9.3 (F4,25 ⫽ 0.48, P ⫽ 0.7529), but increased by about 40% at pH 10.3. For gametophytes, dark respiration increased at pH above and below that of seawater. Photosynthetic response to external inorganic carbon The apparent half-saturation values for DIC were similar for male and female gametophytes (Table 1), but juvenile sporophytes had a higher affinity (lower K1/2) for DIC than gametophytes (for pH 8.3: F2,12 ⫽ 14.42, P ⫽ 0.0006; for pH 9.3: F2,12 ⫽ 145.12, P ⬍ 0.0001). Gametophytes tested at pH 8.3 had a higher affinity for DIC than those tested at pH 9.3, but the affinity for DIC of sporophytes was not affected by pH. The affinity for dissolved CO2 did not differ between gametophytes and sporophytes (F2,12 ⫽ 1.62, P ⫽ 0.2389).

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Table 1. Apparent half-saturation values (K1/2) of dissolved inorganic carbon (DIC) at pH 8.3 and 9.3 and CO2 at pH 8.3 for gametophytes and sporophytes of Undaria pinnatifida. Mean ⫾ s, n ⫽ 5. K1/2 (DIC) (mM)

1,2

pH 9.3

pH 8.3

1.522 ⫾ 0.282 1.254 ⫾ 0.0521 0.955 ⫾ 0.0402 F2,12 ⫽ 14.42, P ⫽ 0.0006

2.133 ⫾ 0.183 1.950 ⫾ 0.0491 1.015 ⫾ 0.0372 F2,12 ⫽ 145.12, P ⬍ 0.0001

18 ⫾ 7 15 ⫾ 51 11 ⫾ 61 F2,12 ⫽ 1.62, P ⫽ 0.2389

1

Male gametophytes Female gametophytes Juvenile sporophytes ANOVA, followed by Tukey’s HSD

K1/2(CO2(aq)) (␮M)

pH 8.3

1

1

Within each column, different superscripts indicate means that are significantly different.

Carbonic anhydrase activity

DISCUSSION

Gametophytes and sporophytes of U. pinnatifida were tested for carbonic anhydrase activity in crude extracts (total) and in intact cells (extracellular). Both extracellular and intracellular CA were detected in U. pinnatifida (Table 2). Sporophytes had higher internal and external CA activities than gametophytes, and a smaller proportion of the total CA activity was external. The CA activities of male and female gametophytes were similar.

The results indicate that, while both gametophytes and sporophytes of U. pinnatifida can utilize HCO3⫺ in addition to

Effects of inhibitors When the membrane-impermeable CA inhibitor, AZ, was added to seawater in the pH drift experiments, the maximal pH of seawater after 60 min was not greatly reduced relative to controls in male and female gametophytes (from 8.65 to 8.52 and from 8.62 to 8.54, respectively; Fig. 3). For juvenile sporophytes, however, the addition of AZ reduced the maximal pH after 60 min from 8.72 to 8.41. DIDS and SITS are inhibitors of the band 3 anion exchange protein, which is the most common HCO3⫺ transporting protein in biological systems (Smith 1988), and their effects (and that of AZ) on net photosynthetic O2 evolution rates were compared for gametophytes and sporophytes of Undaria pinnatifida (Table 3). All three inhibitors had similar effects on male and female gametophytes, but their effects on juvenile sporophytes were significantly lower than on gametophytes (for AZ: F2,15 ⫽ 11.59, P ⫽ 0.0009; for DIDS: F2,15 ⫽ 35.75, P ⬍ 0.0001; for SITS: F2,15 ⫽ 43.09, P ⬍ 0.0001). Addition of DIDS and SITS did not inhibit the O2 evolution rate of sporophytes significantly (P ⬎ 0.05).

Table 2. External and total carbonic anhydrase (CA) activity of gametophytes and sporophytes of Undaria pinnatifida. Mean ⫾ s, n ⫽ 5.

CA activity (EU mg⫺1 chlorophyll a)1

Male gametophytes Female gametophytes Juvenile sporophytes ANOVA, followed by Tukey’s HSD 1

External

Total

22.6 ⫾ 5.4 26.8 ⫾ 7.22 45.3 ⫾ 6.73 F2,12 ⫽ 17.48, P ⫽ 0.0003

72 ⫾ 162 67 ⫾ 122 215 ⫾ 563 F2,12 ⫽ 30.03, P ⬍ 0.0001

2

External CA as a percentage of total CA 31.4% 40.0% 21.1%

EU, enzyme units. Within each column, different superscripts indicate means that are significantly different. 2,3

Fig. 3. The pH drift for gametophytes and sporophytes of Undaria pinnatifida in natural seawater (pH 8.28) without acetazolamide (AZ) (䡵) or with 200 ␮M AZ (䉱). Mean ⫾ s, n ⫽ 4⬃6.

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Table 3. Percentage of photosynthetic activity of gametophytes and sporophytes of Undaria pinnatifida that remains after 10 min in natural seawater containing one of three inhibitors (200 ␮M AZ, 300 ␮M DIDS or 600 ␮M SITS). Mean ⫾ s, n ⫽ 6. Remaining photosynthetic activity (%) Male Female Juvenile Inhibitor1 gametophytes gametophytes sporophytes AZ

66.5 ⫾ 6.72

58.6 ⫾ 5.72

77.4 ⫾ 7.83

DIDS

63.6 ⫾ 7.02

68.4 ⫾ 6.92

92.8 ⫾ 5.23

SITS

76.0 ⫾ 4.92

67.4 ⫾ 8.22

99.8 ⫾ 5.13

ANOVA, followed by Tukey’s HSD F2,15 ⫽ 11.59, P ⫽ 0.0009 F2,15 ⫽ 35.75, P ⬍ 0.0001 F2,15 ⫽ 43.09, P ⬍ 0.0001

1 AZ, acetazolamide; DIDS, 4⬘,4⬘-diisothiocyanatosilbene-2,2-disulphonic acid; SITS, 4-acetamido-4⬘-isothiocyanostilbene-2,2⬘-disulphonic acid. 2,3 Within each row, different superscripts indicate means that are significantly different.

CO2(aq) for photosynthesis, there are some differences in the photosynthetic carbon metabolism of the life history phases. U. pinnatifida is a member of Laminariales and usually grows in intertidal rock-pools down to the subtidal zone. Maberly (1990) showed that, based on the pH-drift technique, Laminaria saccharina, L. digitata and L. hyperborea could use HCO3⫺. The final pH recorded for these macroalgae were 9.20–9.76, and the concentrations of DIC, CO2(aq) and HCO3⫺ were 1.56–1.28 mM, 0.61–0.06 ␮M and 0.84–0.31 mM, respectively. For sporophytes of U. pinnatifida, the final concentrations of DIC, CO2(aq) and HCO3⫺ were 1.36 mM, 0.16 ␮M and 0.52 mM. The ability of sporophytes of U. pinnatifida to use HCO3⫺ in photosynthesis and the extent of the operation of CO2(aq) concentrating mechanism, seems therefore similar to other members of the Laminariales. However, the affinity for DIC of sporophytes of U. pinnatifida, was greater than that reported for other Laminariales, whose apparent half-saturation values for DIC all exceed 1.50 mM (Maberly 1990). The pH around seaweeds with efficient HCO3⫺ uptake systems increases rapidly to pH 9.0 or higher (Maberly 1990). However, in the present study pH did not reach 9.0 in closed systems containing gametophytes of U. pinnatifida, which indicated that gametophytes had much lower abilities to use HCO3⫺. On the other hand, at pH 8.0 and above, the principal species of inorganic carbon in seawater was HCO3⫺, with CO2 concentration decreasing from 15 ␮M to nearly zero at pH 10.0. Parallel with the increase of pH from 8.3 to 9.3 and the decrease of CO2 concentration, photosynthetic rate was reduced drastically, suggesting that gametophytes counted firstly on free CO2 concentration to drive photosynthesis. It was evident that gametophytes had a limited capacity for HCO3⫺ utilization. It is well recognized that the activity of carbonic anhydrase is involved in the utilization of HCO3⫺ during the period of photosynthesis (Johnston 1991; Haglund et al. 1992; Mercado et al. 1998). The extracellular CA catalyzes the conversion of HCO3⫺ to CO2, which is taken up through the plasma membrane and then fixed in photosynthesis. The present results showed that both gametophytes and sporophytes of U. pinnatifida exhibited CA activities detected potentiometically, and the CA inhibitor, AZ, significantly inhibited their photosynthesis O2 evolution and alkalization rate. This suggested

that both gametophytes and sporophytes of U. pinnatifida were capable of using HCO3⫺, which involved the external CA activity. The ability of using HCO3⫺ by means of this mechanism is generally reduced sharply by increased pH, and is essentially very poor at above pH 9.5 (Axelsson et al. 1995, 2000). The use of HCO3⫺ following its direct uptake has been described in some green macroalgae and red algae (Johnston et al. 1992; Axelsson et al. 1995; Larsson et al. 1997; Andria et al. 1999). The photosynthetic O2 evolution by gametophytes of U. pinnatifida was depressed remarkably by the anion exchanger inhibitor, DIDS and/or SITS, implying that the mechanism of U. pinnatifida by direct uptake played an important role in the inorganic carbon utilization in the gametophytes of the brown macroalgae, U. pinnatifida. However, the photosynthesis of sporophytes was not affected by putative anionexchange inhibitor DIDS or SITS, which suggested that a DIDS- or SITS-sensitive anion-exchange-type HCO3⫺ transporter was unlikely to be present in DIC acquisition by sporophytes. On the other hand, the effects of DIDS upon indirect measures of DIC acquisition should be interpreted cautiously as DIDS may have nonspecific effects upon whole cell function, and affect transport processes not directly related to HCO3⫺ uptake (Young et al. 2001). HCO3⫺ can be transported into the cells against an electrochemical gradient, which requires the energy provided via a plasmalemma-associated ATPase (Axelsson & Beer 2001). ATPases show negligible DIDS sensitivity, so a DIDS-susceptible component of DIC acquisition may be regarded as distinct from ATPase-mediated transport (Young et al. 2001). The HCO3⫺ transport system identified in sporophytes of U. pinnatifida in our study could be further catalyzed by extracellular CA activity, which might be the primary way of utilizing DIC for sporophytes. In general, algae possessing direct HCO3⫺ uptake mechanism exhibit a greater affinity for DIC than the algae with only the indirect HCO3⫺ utilization catalysed by CA activity (Axelsson et al. 1995, 1999). However, our present experiments showed this is not necessarily so in such macroalga as U. pinnatifida. While gametophytes of U. pinnatifida had a lower affinity for DIC than sporophytes, the former could take up HCO3⫺ directly and the latter hardly possessed the mechanism of direct HCO3⫺ uptake. Perhaps the mechanism of direct HCO3⫺ use occurring in gametophytes was too poor to accumulate DIC intracellularly and thereby to raise the DIC affinity. The final pH values of over 9.2 (equivalent to 0.6 ␮M CO2 in seawater) have been viewed as an indicator of HCO3⫺ use in macroalgae. The higher final pH values, the greater the ability of HCO3⫺ using (Maberly 1990; Johnston et al. 1992). Thus, the higher final pH values in sporophytes also suggested that sporophytes had a greater ability of HCO3⫺ use than gametophytes. Such a higher HCO3⫺ use capacity in juvenile sporophytes might be related with their greater demands for DIC to mature.

ACKNOWLEDGEMENTS This study was supported by the Key Laboratory of Bioprocess of Beijing, P.R. China (SYS100100421).

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Received 2 May 2005; accepted 1 May 2006 Associate editor: C. Amsler