Use of light and inorganic carbon acquisition by two morphotypes of Zostera noltii Hornem

www.elsevier.com/locate/jembe

Journal of Experimental Marine Biology and Ecology

297 (2003) 71–84

Use of light and inorganic carbon acquisition by two

morphotypes of Zostera noltii Hornem

Jesus M. Mercadoa,*, F.X. Niellb, Joao Silvac, Rui Santosc

aCentro Oceanografico de Malaga, Instituto Espanol de Oceanografıa, Puerto Pesquero s/n, Apdo. 285,

29640, Fuengirola, Malaga, SpainbDepartamento de Ecologıa, Universidad de Malaga, Campus Universitario de Teatinos 29071, Malaga, Spain

cCentro de Ciencias do Mar, CCMAR, Universidade do Algarve, Gambelas 8000, Faro, Portugal

Received 1 January 2003; received in revised form 16 July 2003; accepted 23 July 2003

Abstract

The affinity for dissolved inorganic carbon (DIC) and the mechanisms to use HCO3� as a source of

DIC for photosynthesis were investigated in two morphotypes of Zostera noltii Hornem. Both

morphotypes were collected at Ria Formosa lagoon (Southern Portugal) at two different levels in the

intertidal. Affinity for DIC at saturating photon fluence rate (PFR), estimated as photosynthetic

conductance for CO2 ( gp(CO2)), was reduced by 75% in the Z. noltii plants adapted to shade conditions

(lower intertidal) in comparison to the sun morphotype (45� 10� 6 and 182� 10� 6 m s� 1,

respectively), indicating that the plants acclimated to sun conditions (higher intertidal) had a higher

capacity to use HCO3� as DIC source for photosynthesis. Since external carbonic anhydrase activity

was negligible and a large inhibitory effect was produced by Tris buffer addition, this HCO3� use

was attributed to the operation of H+ ATPases creating low pH zones in periplasmic space. The

photosynthetic CO2-flux supported for this mechanism was calculated to be 53 Amol O2 m� 2 s� 1 in

sun morphotype, about 80% out of maximum photosynthesis rate. In order to determine the possible

photosynthetic energy cost of the HCO3� use, the effect of decreasing light on photosynthetic rates

and gp(CO2)was estimated. Photosynthetic conductance decreased in both morphotypes at non-

saturating PFR. This dependence of gp(CO2)on PFR indicated the existence of a positive interactive

effect between DIC and PFR which was more pronounced in the shade morphotype since the

ascending slope of O2 evolution vs. PFR curves at limiting PFRs was reduced from 7.2 to 2.3 mmol

O2 mol photon� 1 at 4 and 0.5 mol m� 3 of DIC, respectively.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Carbonic anhydrase; Carbon acquisition; Eutrophication; Intertidal system; Photosynthesis;

Seagrasses; Zostera

* Corresponding author. Tel.: +34-952476955; fax: +34-952463808.

0022-0981/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/S0022-0981(03)00368-X

E-mail address: [email protected] (J.M. Mercado).

J.M. Mercado et al. / J. Exp. Mar. Biol. Ecol. 297 (2003) 71–8472

1. Introduction

Seagrasses are the dominant macrophytes of many near-shore soft-bottom ecosystems

where they constitute an important and highly productive component of the benthic

environment (McRoy and McMillan, 1977). A decline in seagrass-dominated areas has

been observed worldwide (Denninson et al., 1993). Seagrasses are being replaced by other

macrophytes, particularly green macroalgae (Hernandez et al., 1997; Clavero et al., 1999).

It has been proposed that this replacement is a consequence of the great sensitivity of

seagrass ecosystems to human-induced perturbations. The eutrophication of coastal areas

results in a higher growth of the phytoplankton. The subsequent light limitation of benthic

communities reduces their primary production (Bondsorff et al., 1997) and eventually

decreases seagrasses cover (Short and Neckles, 1999). Changes in riverine fluxes of

organic matter and nutrients or suspended matter due to human activities can produce

similar effects (Carreira et al., 1995).

According to Moore et al. (1996), the long-term survival of Zostera marina is affected

by water turbidity rather than changes in the nutrient concentration or salinity. This fact

suggests that the photosynthetic performance of seagrasses could contribute to their high

sensitivity to anthropogenic inputs. Previous reports demonstrate that the photosynthesis

rates are limited by the availability of DIC under natural conditions in many seagrasses

species (Madsen and Sand-Jensen, 1991; Durako, 1993; Beer and Koch, 1996; James and

Larkum, 1996; Invers et al., 1999). However, Hellblom et al. (2001) suggested recently

that this apparent low affinity for DIC have been largely an artefact of the use of buffers in

the incubation medium. Accordingly, Schwarz et al. (2000) demonstrated that the seawater

DIC concentration was enough to saturate in situ photosynthesis of two tropical seagrass

species of the genera Cymodocea and Halophila, which inhabit the upper part of the

intertidal system (i.e. fully exposed to solar radiation). By contrast, photosynthesis by

shade-adapted forms of these tropical species was limited at the seawater DIC concen-

tration (Schwarz et al., 2000). These results suggest that acclimation to decreasing light

intensity could affect the DIC acquisition mechanisms. The capacity to use HCO3� by

seagrasses has been demonstrated from kinetic measurements in species of the genera

Thalassia and Zostera (Sand-Jensen and Gordon, 1984; Durako, 1993; Beer and

Rehnberg, 1997; Touchette and Burkholder, 2000). At least three different mechanisms

of HCO3� use for photosynthesis have been described in seagrasses: (1) its transformation

into CO2 performed by an external CA (Millhouse and Stroher, 1986; Bjork et al., 1997),

(2) its direct uptake by means of specific transport systems located at plasma membrane

(Touchette and Burkholder, 2000), and (3) more recently, Hellblom et al. (2001) suggested

the use of HCO3� was dependent on low pH zones at periplasmic space created by H+

ATPases pumping protons to the outside of the plasma membrane. The ability of seagrass

species to modify the activity of these mechanisms in response to changes on light

intensity remains unexplored.

The goal of the experiments described in this paper was to determine the affinity to DIC

and the presence of different mechanisms for using HCO3� in two morphotypes of Z. noltii

which grow under different light conditions in the field. For this proposal, the effect on the

photosynthesis of carbonic anhydrase inhibitors and inhibitors for the proton pump

dependant, HCO3� use mechanism (i.e. Tris buffer) was determined (Axelsson et al.,

J.M. Mercado et al. / J. Exp. Mar. Biol. Ecol. 297 (2003) 71–84 73

2000). Additionally, photosynthesis vs. photon fluence rate (PFR) curves were performed

at different DIC concentrations in order to determine the dependence of affinity for DIC on

incident PFR.

2. Materials and methods

2.1. Sampling sites and plant material

Plants were collected in January 2000 from Ria Formosa lagoon. This is a shallow

mesotidal lagoon, extending for about 55 km along the coast of southern Portugal, with an

average depth of about 2 m. The tidal amplitude varies from an average of 2.80 m in spring

tides to 1.30 m in neap tides. The total area of the lagoon is 170 km2 with an exposed

intertidal area of 67 km2 during spring tides (Andrade, 1990).

Zostera noltii morphology in the Ria Formosa varies with vertical distribution of the

species in the intertidal. At the upper limit the leaves are thinner and shorter in contrast

with the lower limit where they are larger and longer. The two morphotypes are referred to

in this work as sun-type and shade-type, and respectively correspond to the small (SM)

and large (LM) morphotypes described by Peralta et al. (2001). Environmental conditions

change significantly along the vertical distribution of species, particularly light availability.

The lower limit of this bed is never completely emersed, while the upper limit has an

average emersion between 6 and 8 h every day, depending on tidal amplitude. As a

consequence, during January, the shade-type receives an average daily PAR dose of 4 mol

photons m� 2, whereas the sun-type receives around 15 mol photons m� 2. Other variables

as the pH also are different at each position. Thus, the pH values at the site where the sun

morphotype was collected increased from 7.4 at low tide up to 8.8 during high tide. In

contrast, at the shade-morphotype site, the pH varies from 7.9 to 8.3.

Plants were collected from Ria Formosa and kept cooled and wet in the dark while they

were transported to the laboratory. In the laboratory, plants were maintained in natural,

nutrient-poor seawater. About 10 g of plant were incubated into Plexiglas cylinder of 4 l in

capacity. They were aerated vigorously (about 3 l air min� 1) at 15 jC under white

fluorescent lamps (F20W/CW lamps), at a photoperiod of 12 h light per day and a photon

fluence rate (PFR) of 100 Amol photons m� 2 s� 1. The PFR was determined with a

quantum spherical PAR sensor (LiCor 193SB) connected to a radiometer (Li-1000).

The experiments were performed using pieces of leaves of 2 cm in length, cut

approximately 2 cm above the sheath. Prior the experiments, 10 mg of this plant material

were frozen at � 20 jC until the analysis of its photosynthetic pigment content. For this

purpose, chlorophyll a and b were extracted in N,N-dimethylformamide and their

concentrations were calculated according to Inskeep and Bloom (1985).

2.2. Oxygen evolution and effects of the buffer and CA inhibitors

Oxygen evolution was measured in small (8–9 ml) temperature-controlled (15–16 jC)seawater chambers containing oxygen electrodes. The suitable agitation of the medium in

the chambers was obtained by a magnetic stirrer. The 2 cm leaf fragments were transferred


to the oxygen-evolution chambers containing natural seawater. A PFR of 600 Amol

photons m� 2 s� 1 was provided by a Duro-Test lighting (model F13DTT/55K) lamp. The

effect of Tris buffer on photosynthesis was tested at pH 8.2 as described by Axelsson et al.

(2000), prior to testing the effect of the inhibitors of carbonic anhydrase (CA) on oxygen

evolution. Different Tris biological buffer (Sigma) concentrations ranging from 0 to 45

mol m� 3 were produced into the chambers by adding small amounts of a 2 kmol m� 3 Tris

solution. The pH electrode and meter (Crison 52-08, Alella, Spain) were calibrated in

buffer solutions referred to the NBS scale (Crison).

Since Tris produced a strong inhibitory effect on photosynthesis, unbuffered seawater

was used to test the effect of CA inhibitors, 6-ethoxyzolamide (EZ) and acetazolamide

(AZ; Sigma, Madrid, Spain). It was assumed that AZ cannot penetrate into the cell and

inhibits only the extracellular CA (Palmqvist et al., 1990; Haglund et al., 1992; Axelsson

et al., 1995) while EZ penetrates into the cell and inhibits the external and internal CA.

Carbon dioxide-free stock solutions of the inhibitors were prepared in 0.05 N NaOH to a

final concentration of 50 mmol m� 3. The assays of inhibitors were started by introducing

fragments of plant into the oxygen evolution chamber containing unbuffered seawater.

When a constant O2 evolution rate was obtained, AZ or EZ were added. A complete effect

was obtained within 2–3 min.

2.3. Oxygen evolution rate vs. PFR curves

The response of photosynthesis to increasing PFR was tested under six DIC concen-

trations (250, 500, 1000, 2000, 3000 and 4000 mmol m� 3). Pieces of plant were

introduced into the oxygen evolution chambers containing Ci free synthetic seawater

(450 mol m� 3, NaCl, 30 mol m� 3 MgSO4, 10 mol m� 3 KCl, 10 mol m� 3 CaCl2; Beer et

al., 1990). The pH was adjusted to 8.2 with Tris 2 mol m� 3 final concentration (it

produced an inhibition of the photosynthesis rates lower than 5%). This Tris concentration

was enough to maintain the pH within the range 8.2–8.3 during the experiments. After

zero net O2 exchange rate was attained, small amounts of a 200 mol m� 3 NaHCO3

solution were injected into the chambers in order to create the required DIC concentration.

Nine different PFRs (10, 15, 30, 70, 130, 210, 270, 360 and 600 Amol photons m� 2 s� 1)

were used to perform the P-PFR curves. They were obtained with glass neutral density

filters. A total of 600 Amol photons m� 2 s� 1 did not produce apparent photoinhibiton in

the two morphotypes. PFR values were measured inside the chamber with a spherical

sensor (20HM33CM12KG, Zemoko, Holland). Respiration was measured in darkness

before switching the light on. Oxygen evolution was recorded for 10–15 min at each PFR.

Maximal photosynthetic rates (Pmax) and ascending slope at limiting PFRs (a) were

obtained after the fitting of the curves with the rectangular hyperbole equation (Henley,

1993):

P ¼ Pmax½aPFR=ðPmax þ aPFRÞ� þ Rd

where P is the photosynthetic rate and Rd is the respiration rate in darkness. The light

saturation parameter (Ek) was estimated as Pmax/a. Three independent curves (i.e. startingfrom different samples) were constructed for each DIC concentration tested. The curves


were analysed separately. Their goodness of fit to the model was tested by using least-

squares regression analysis. The average R2 value obtained from the least-squares

regression analysis was 0.99.

Oxygen evolution was expressed on a surface area basis. Fresh weight per unit area was

estimated by determining the surface area of samples that had previously been weighed.

Leaf surface area was determined after digitalisation using a computer video system as

described by Luning (1992).

2.4. Conductance for inorganic carbon ( gp)

The response of photosynthesis to increasing DIC concentration was determined at

different PFRs using the data of the P–PFRs curves. Photosynthesis vs. DIC concentration

curves were constructed at six selected PFRs (30, 70, 130, 270, 360 and 600 Amol photons

m� 2 s� 1). Conductance for DIC ( gp(DIC)) at each PFR was calculated as the initial slope

of these curves. Conductance for CO2 ( gp(CO2)) was obtained by multiplying gp(DIC) by the

CO2/DIC ratio estimated by using the apparent dissociation constants of carbonic acid in

seawater (Riley and Chester, 1977).

2.5. Assay of external CA activity

External CA activity was assayed using the potentiometric method. The time

required for a drop of 0.4 pH units within the pH range 8.4–7.4 was measured at

0–2 jC. About 50–200 mg FW were washed three times in a buffer (100 mol m� 3

Tris, 5 mol m� 3 EDTA-Na and 25 mol m� 3 ascorbic acid) before being placed in the

reaction vessel (Haglund et al., 1992). The reaction was started by rapidly introducing 1

ml of ice-cold CO2-saturated distilled water. One unit of enzyme activity (REA) was

defined as (to/tc)� 1 where to and tc are the times for pH change of the control (assay

buffer without sample) and sample, respectively. The activity was recalculated on a

fresh weight basis.

The external activity [termed CA (%)] was also determined by using the method

developed by Mercado et al. (1997). This method permits the relative importance of the

external CA for DIC acquisition to be assessed relative to direct uptake of HCO3�. 30 mg

of plant material was transferred to the O2 evolution chambers containing synthetic

seawater buffered at pH 8.7 with Tris 2 mol m� 3 final concentration. After zero net O2

exchange rate was attained, 200 Al of ice-cold CO2-saturated distilled water were added in

both the presence and absence of 50 mmol m� 3 AZ. CO2 and HCO3� dependent

photosynthetic rates were then calculated. Activity estimated according to Eq. (2) in

Mercado et al. (1997) was expressed in arbitrary units (ranging from 0 to 1, where a figure

of 1 represents the maximal detectable activity with this method).

2.6. Statistics

The results were expressed as the mean valuesF standard deviation (S.D.). Significant

differences among means were tested at p < 0.05 with a one-way ANOVA, followed by the

Fisher’s protected multirange test (Sokal and Rohlf, 1981).


3. Results

Differences between the two morphotypes were pronounced, especially for leaf width

which was higher in sun morphotype than in shade morphotype (see Peralta et al., 2001 for

a complete morphological description). Analysis of the morphometric characteristics of the

leaves utilized in our experiments (Table 1) revealed statistically significant differences in

fresh weight per unit area. The chlorophyll a and b contents expressed on a fresh weight

basis were similar in both morphotype although they expressed on an area surface basis

were higher in the shade morphotype. There were no significant differences in the Chl

b/Chl a ratio.

Photosynthetic oxygen production was strongly inhibited by the addition of Tris

without changing pH. The inhibition percentage was dependent on the buffer concentra-

tion (Fig. 1), reaching 80% inhibition at 45 mol m� 3 of Tris. Inhibition was lower than 5%

at 2 mol m� 3 of Tris, the concentration used in performing the P-PFR curves. This

concentration was shown to be adequate to maintain constant pH during the experiments

as tested by measuring pH after the incubations.

There was no agreement between the results of external CA activity obtained with the

two methods used. Thus, significant activity was detected by the potentiometric method

(Table 2), although it was very low in comparison with values reported for other

macrophytes (Mercado et al., 1998). In contrast, CA activity assayed using the method

described by Mercado et al. (1997) was not detected in three out of four samples, since the

criterion for determination of activity (i.e. a value of a/cV higher than 2 according to Eq.

(1) of Mercado et al., 1997) was not satisfied. It was due to the low inhibition of

photosynthesis obtained after acetazolamide (AZ) addition at pH 8.7 (the value used for

the assay). Since CA activity was detected potentiometrically, this low inhibition must

indicate that CO2 requirement of RUBISCO is fulfilled via mechanisms of HCO3� use

other that the external CA activity. Accordingly, the inhibitory effect of AZ at the normal

seawater pH was much less that it found after the addition of a high buffer concentration

(Fig. 2). In contrast to AZ, ethoxyzolamide inhibited photosynthesis by 80%. The

percentage inhibition of photosynthesis by AZ and EZ and the external CA activity were

similar in the two morphotypes.

Changes in the inorganic carbon concentration did not affect the respiration rates, which

were similar in the two morphotypes (9.73F 5.01 and 13.04F 4.86 Amol O2 m� 2 min� 1

for shade- and sun-morphotype, respectively). About 500 Amol photons m� 2 s� 1 were

Table 1

Fresh weight per area ratio (FW/area) and chlorophyll a and b contents in sun and shade morphotypes

Sun morphotype Shade morphotype

FW/area (g m� 2) 32.26F 9.12a (n= 10) 54.61F 20.83b (n= 11)

Chl a (mg m� 2) 63.55F 3.96a (n= 4) 85.74F 9.12b (n= 4)

Chl b (mg m� 2) 19.03F 1.07a (n= 4) 26.76F 1.94b (n= 4)

Chl b/a 0.31F 0.01a (n= 4) 0.30F 0.00a (n= 4)

Chl a/b ratio is also provided. The numbers are the meanF S.D. n indicates the numbers of independent samples

analysed.

Within each row, means with different superscripts are significantly different at p= 0.05.

Fig. 1. Effect of three different Tris concentrations on photosynthesis rates by sun and shade morphotypes of Z.

noltii. The experiments were performed at saturating PFR and pH 8.2. Oxygen evolution rates obtained before

adding Tris were used as the control. Data are meansF S.D. of four to five independent samples.


sufficient to saturate photosynthesis of both morphotypes. At PFR saturation and 2 mol

m� 3 of DIC (the usual concentration in seawater), photosynthesis in sun morphotype was

85% higher than in shade morphotype (66.4F 1.9 and 35.3F 0.8 Amol O2 m� 2 min� 1,

respectively). The difference in maximal photosynthesis was lower at 5 mol m� 3 of DIC

(77.0F 1.2 and 61.2F 7.9 Amol O2 m� 2 min� 1 for sun and shade morphotypes,

respectively). The light saturation parameter values obtained from the photosynthesis

vs. PFR curves (Ek, Fig. 3) were affected by changes on the DIC concentration although

there were non significant differences between the two morphotypes. The highest Ek value

was obtained at 3 mol m� 3 of DIC (276 and 258 Amol photons m� 2 s� 1 for shade and

sun morphotype, respectively). In contrast, much lower Ek values were obtained at 0.25

mol m� 3 of DIC. Fig. 4 shows a (ascending slope of O2 evolution vs. PFR curves at

limiting PFRs) as a function of the DIC concentration. There were no significant

differences in a through the DIC concentrations assayed in sun morphotype. In contrast,

in shade morphotype, a increased from 2.3F 0.3 to 7.2F 0.7 mmol O2/mol incident

photons at 0.5 and 4 mol m� 3 of DIC concentration, respectively. The mean value of a

Table 2

External carbonic anhydrase activity measured using the potentiometric method (units: REA gFW� 1; REA:

relative enzymatic activity) and the method described by Mercado et al. (1997) (units: %)

Sun morphotype Shade morphotype

Exter. CA (REA gFW� 1) 1.86F 0.55 (n= 6) 2.56F 1.22 (n= 6)

Exter. CA (%) ND-0.26 (n= 2) ND (n= 2)

n indicates the numbers of independent samples analysed. ND means no detectable according to the criteria used

by Mercado et al. (1997).

Fig. 3. Values of light saturation parameter (Ek) obtained at different DIC concentrations. Ek was estimated from

Pmax and a calculated by means of the fit of photosynthesis vs. PFR curves to equation provided by Henley

(1993). Three independent curves (i.e. starting from different samples) were constructed for each DIC

concentration tested.

Fig. 2. Effect of the carbonic anhydrase inhibitors (acetazolamide, AZ, and ethoxizolamide, EZ) on net

photosynthesis in sun and shade morphotypes of Z. noltii. Inhibitors were added at 50 mmol m� 3 final

concentration. Oxygen evolution rates obtained before adding inhibitors were used as the control. Data are

meansF S.D. of four to five independent samples.


Fig. 4. Ascending slope of photosynthesis vs. PFR curves at limiting PFRs (a) obtained at different DIC

concentrations. a was calculated by means of the fit of photosynthesis vs. PFR curves to equation provided by

Henley (1993). Three independent curves (i.e. starting from different samples) were constructed for each DIC

concentration tested.

Fig. 5. Photosynthetic conductance for CO2 as a function of the photon fluence rate in sun- and shade-

morphotypes of Z. noltii. The values of conductance for DIC ( gp(DIC)) at the different PFR assayed were

calculated as the initial slope of photosynthesis vs. DIC concentration curves. Data are the mean of three

independent samples.



was higher in sun morphotype (8.2F 0.7 mmol O2/mol photon) than in shade morphotype

(4.8F 1.8 mmol O2/mol photons).

The values of gp(CO2)showed a dependence on PFR (Fig. 5) with an asymptotic

relationship. The mean value of gp(CO2)increased by fivefold when PFR increased from 30

to 600 Amol photons m� 2 s� 1 for the two morphotypes. Higher gp(CO2)values in the sun

morphotype than in the shade morphotype were reached at each PFR used. In particular,

gp(CO2)at saturating PFR (600 Amol photons m� 2 s� 1) was fourfold higher in the sun

morphotype (182.3� 10� 6 m s� 1) than in the shade morphotype (45.1�10� 6 m s� 1).

4. Discussion

4.1. Inorganic carbon acquisition

The initial slope of photosynthesis rate vs. DIC curves (photosynthetic conductance for

CO2, gp(CO2)) at saturating irradiance has been proposed as an useful parameter for

determining the ability to extract Ci from seawater for photosynthesis (Johnston et al.,

1992). Thus, the different gp(CO2)values found for the two Zostera morphotypes at

saturating irradiance are indicative of a differential ability to use DIC because they were

fourfold lower in the shade morphotype when compared to the sun morphotype. The

possibility that gp(CO2)values are consistent with photosynthesis relying only on CO2

diffusive entry can be checked by calculating the minimum boundary layer thickness

which would permit these values (Johnston et al., 1992; Mercado et al., 1998). For this

proposal, it is assumed that no external transformation of HCO3� into CO2 is occurring.

The thickness of the diffusion boundary layer is calculated by multiplying CO2 diffusion

coefficient (1.3� 10� 9 m� 2 s� 1) to gp(CO2)(Johnston et al., 1992; Raven et al., 2002).

The calculated boundary layer thickness is 7 Am for sun morphotype and 29 Am for shade

morphotype. Different authors indicate that overall conductance for CO2 (including the

intracellular pathway) account for a diffusion boundary layer of 10–15 Am in macroalgae

without mechanisms for using HCO3� (Maberly et al., 1992; Raven et al., 2002). The value

obtained for shade morphotype is higher than it suggested by Maberly et al. (1992). Taking

into account the stirring conditions in the chamber where photosynthesis was measured, it

can be concluded that 29 Am is concordant with a use of DIC mainly based on CO2

diffusive entry. By contrast, the value of boundary layer thickness necessary to account for

measured gp(CO2)in sun morphotype is lower than the value calculated by Maberly et al.

(1992), therefore it indicates that use of HCO3� for photosynthesis is occurring in this

morphotype.

The reason for this difference in the ability to use HCO3� between the two morphotypes

could be the different activation state of the mechanisms involved in the HCO3� use.

Carbonic anhydrase-mediated indirect use of bicarbonate has been described for different

seagrasses species (James and Larkum, 1996; Invers et al., 1999), including two species of

Zostera, Z. muelleri (Millhouse and Stroher, 1986) and Z. marina (Beer and Rehnberg,

1997). In contrast, we have found that the role of the external CA on HCO3� indirect use in

Z. noltii must be very restricted since photosynthesis was only 20% inhibited with AZ and

no activity was detected by the method developed by Mercado et al. (1997). The findings


suggest that more efficient use of HCO3�, not dependent on external CA must be

occurring, at least in the sun morphotype. An alternative mechanism to explain the use

of HCO3� independently of external CA activity, is the one recently described by Hellblom

et al. (2001) in Z. marina. According to these authors, HCO3� use in this species is

facilitated by proton transport towards the outside of the cell membrane. These protons are

then used to facilitate the inward transport of inorganic carbon through the cell membrane

(Axelsson et al., 2000). Hellblom et al. (2001) based their model on the high sensitivity of

photosynthesis to proton buffers. We demonstrate that photosynthesis was inhibited almost

80% by a high Tris concentration (i.e. 45 mol m� 3) suggesting that such a mechanism is

the main component of HCO3� use in Z. noltii sun morphotype. According to the values of

photosynthesis rate obtained in the presence of Tris, the DIC flux supported by this

mechanism could count for 53 Amol O2 m� 2 min� 1 in sun morphotype. If this DIC-flux is

compared to the one theoretically supported by the spontaneous dehydration of CO2

(which can be calculated from the photosynthetic rates obtained in the presence of AZ plus

Tris), it can be calculated that the HCO3� use mechanism sensitive to Tris produces a CO2

concentration increase by 5 fold in the periplasmic space. It has been noted that the shade

morphotype, with a comparatively lower capacity for use HCO3�, also exhibits a high

sensitivity to buffer. Therefore, restricted capacity for use of HCO3� cannot be ruled out.

4.2. Use of light by shade and sun morphotypes

The reason for a higher Pmax at saturating light and DIC in the sun morphotype could be

its higher capacity to use HCO3� and/or a higher Rubisco content, as both lead to higher

carbon dioxide fixation rates (Sukenik et al., 1987). The lower a found in the shade

morphotype could also be related to DIC acquisition, since it was increased by twofold in

this morphotype when DIC concentration was increased from 0.5 to 4 mol m� 3. It is worth

noting that no effect was found in sun morphotype. In our experiments, the CO2/O2 ratio

in the medium was modified by changing the DIC concentration (Raven, 1984, 1997;

Kerby and Raven, 1985; Beardall, 1989; Mercado and Niell, 1999). Since the shade

morphotype exhibited a very low capacity to use HCO3� it can be speculated that

photorespiration could contribute to the lower a values observed at low DIC concentration

in comparison to the sun morphotype. Our results contrast to the ones obtained by Kubler

and Raven (1995) who showed that light-harvesting efficiency was inversely related to the

effectiveness of DIC acquisition in Palmaria palmata. According to those authors, the

interactive effect is a consequence of the trade-off between photosynthetic efficiency and

bicarbonate use based on the fact that the use of HCO3� requires energy. In our

experiments, the reliance on CO2 found in the shade morphotype would indicate that

the photon cost of photorespiration is higher than the possible cost of HCO3� use (Raven,

1984).

The dependence of gp(CO2)on PFR has been also described by other authors (Maberly,

1990; Johnston et al., 1992; Mercado et al., 2000, 2001) and demonstrates that a positive

interactive effect exists between light use and DIC acquisition. In both morphotypes, an

increase of DIC concentration would be necessary to support an equivalent photosynthetic

rate when incident light is reduced. Apparently, the photosynthetic rate of the sun

morphotype depends more on light quantity than on DIC concentration since it exhibited


a relative high capacity for HCO3� use. In contrast, both factors (availability of light and

DIC) limit the photosynthetic rates of the shade morphotype, as photosynthetic efficiency

was dependent on DIC concentration. In any case, the reduction of incident light leads to a

lower affinity to DIC in both morphotype, which may increase the susceptibility of Z.

noltii to eutrophication.

Acknowledgements

This work was financed by the Projects REN2002-00340/MAR and REN2002-04022-

C02-01 (CICYT) and ECOFITO (Instituto Espanol de Oceanografıa). J.M. Mercado is

supported from Ramon y Cajal Programme of the Ministerio de Ciencia y Tecnologıa from

Spain. J. Silva is supported by PRAXIS XXI/BD/15782/98 grant. [RW]

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