The role of external carbonic anhydrase in the photosynthetic use of inorganic carbon in the deep-water alga Phyllariopsis purpurascens (Laminariales, Phaeophyta)

Mechanisms of inorganic carbon assimilation were investigated in the deep-water alga Phyllariopsis purpurascens (C. Agardh) Henry et South (Laminariales, Phaeophyta). The gross photosynthetic rate as a function of external pH, at a constant concentration of 2 mM dissolved inorganic carbon (DIC), decreased sharply from pH 7.0 to 9.0, and was not substantially different from 0 above pH 9.0. These data indicate that P. purpurascens is inefficient in the use of external HCO₃ ⁻ as a carbon source in photosynthesis. Moreover, the photosynthetic rate as a function of external DIC and the highest pH (9.01 ± 0.07) that this species can achieve in a closed system were consistent with a low capacity to use HCO₃ ⁻, in comparison to many other species of seaweeds. The role of external carbonic anhydrase (CA; EC 4.2.1.1) on carbon uptake was investigated by measuring both the HCO₃ ⁻-dependent O₂ evolution and the CO₂ uptake, at pH 5.5 and 8.0, and the rate of pH change in the external medium, in the presence of selected inhibitors of extra- and intracellular CA. Photosynthetic DIC-dependent O₂ evolution was higher at pH 5.5 (where CO₂ is the predominant form of DIC) than at pH 8.0 (where the predominant chemical species is HCO₃ ⁻). Both intra- and extracellular CA activity was detected. Dextran-bound sulfonamide (DBS; a specific inhibitor of extracellular CA) reduced the photosynthetic O₂ evolution and CO₂ uptake at pH 8.0, but there was no effect at pH 5.5. The pH-change rate of the medium, under saturating irradiance, was reduced by DBS. Phyllariopsis purpurascens has a low efficiency in the use of HCO₃ ⁻ as carbon source in photosynthesis; nevertheless, the ion can be used after dehydration, in the external medium, catalyzed by extracellular CA. This mechanism could explain why the photosynthetic rate in situ was higher than that supported solely by the diffusion of CO₂ from seawater. Received: 6 March 1998 / Accepted: 22 June 1998     

Different mechanisms of inorganic carbon acquisition in red macroalgae (Rhodophyta) revealed by the use of TRIS buffer

The effects on photosynthesis of acetazolamide (AZ, an inhibitor of the external carbonic anhydrase) and TRIS buffer at pH 8.7 were assessed in 24 species of red macroalgae. Only Palmaria palmata was unaffected by both substances. The rest of species were classified into three groups according to their sensitivity to TRIS and AZ. Photosynthesis of fourteen species was significantly inhibited by both TRIS and AZ. Inhibition by TRIS varied from almost 100% to 25% while AZ produced similar effects. Inhibition by TRIS was completely reverted by increasing the dissolved inorganic carbon concentration (DIC). This species group had half-saturation constants for photosynthesis (K_m(DIC)) ranging from 0.5 to 1.1 mM of DIC. TRIS produced a significant increase of K_m(DIC). Altogether, these results indicate that the algae sensitive to TRIS are capable of using HCO₃⁻ efficiently at pH 8.7. Furthermore, the buffering capacity of TRIS was responsible for its inhibitory effect on photosynthesis suggesting that HCO₃⁻ use was facilitated by excretion of protons outside the plasma membrane, which creates regions of low pH resulting in a higher-than-ambient CO₂ concentration. In contrast, photosynthesis by two Porphyra species analysed was slightly stimulated by TRIS and completely inhibited by AZ, suggesting that the mechanism was different. In a third group of seaweeds, photosynthesis was insensitive to TRIS but it was significantly inhibited by AZ. These species had relatively high values of K_m(DIC) indicating that they relied on purely diffusive entry of CO₂ generated by external carbonic anhydrase activity. Consequently, the results demonstrate that external carbonic anhydrase is widespread among red macroalgae since only P. palmata was insensitive to AZ. The functional significance of this enzyme was quite variable among the tested species.     

Effect of inorganic carbon supply on the photosynthetic physiology of Gracilaria tenuistipitata

Gracilaria tenuistipitata Zhang et Xia was cultured for 15 d at low, normal and high inorganic carbon concentrations under constant light, temperature and nutrient conditons. Carbonic anhydrase (CA; EC 4.2.1.1.) activity, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco; EC 4.1.1.39) content, pigment content and C/N ratio were measured, and also the photosynthesis and growth rates. Both Rubisco content and CA activity increased under conditions of low inorganic carbon (C_i) but decreased at high C_i with respect to the control. The amount of pigments declined considerably at high C_i and was slightly higher at low C_i. The maximum rate of photosynthesis and the photosynthetic efficiency increased in low C_i and the opposite was found at high C_i concentration. The effects of C_i concentration on maximum rate of photosynthesis and photosynthetic efficiency are discussed in relation to the variation in pigment and Rubisco contents and CA activity. The data indicate that C_i may be an important factor controlling the photosynthetic physiology of G. tenuistipitata with regard, not only to the enzymes of C_i metabolism, but also to the pigment content.Abbreviations APS_max maximum apparent photosynthetic rate - CA carbonic anhydrase - Chl chlorophyll - C_i inorganic carbon - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This work has been supported by grants No. PB91-0962 and No. MAR90-0365 from Spanish Direction for Science and Technology (DIGICYT). M.J. G-S holds a fellowship from the DIGICYT.     

Distribution of carbonic anhydrase in British marine macroalgae

Summary Thirty-four species of marine macroalgae from around St. Andrews, Scotland, have been assayed for their external activity and thirty-three species for their total activity of carbonic anhydrase. Activity was detected in all the Rhodophyta tested apart from Chondrus crispus, but was absent in Codium fragile, Enteromorpha sp. and Monostroma fuscum (Chlorophyta), and Alaria esculenta, Laminaria digitata, L. saccharina and L. hyperborea (Phaeophyta). Total activity of carbonic anhydrase per unit fresh weight tended to be higher in the Rhodophyta than in the Chlorophyta or Phaeophyta. External activity was present in two of the six Chlorophyta, four of the twelve Phaeophyta and four of the sixteen Rhodophyta tested. On average, when present, external carbonic anhydrase activity represented 2.7% of the total activity. A relationship was found between total carbonic anhydrase activity and habitat. Species from the high intertidal and the low-light subtidal habitats had significantly higher activity than species from the mid and low intertidal, rockpools, or high-light region of the subtidal. External carbonic anhydrase activity did not vary significantly with habitat. There appeared to be no strong relationship between carbonic anhydrase activity and the ability of a species to use HCO _- ³ in photosynthesis under water.     

氮、磷对小新月菱形藻无机碳利用与碳酸酐酶活性的影响

在实验室条件下研究了氮磷浓度变化对小新月菱形藻无机碳利用与碳酸酐酶活性的影响,结果显示小新月菱形藻随培养液中氮、磷浓度的升高比生长速率明显提高。低氮浓度导致胞外碳酸酐酶活性丧失,但胞内碳酸酐酶活性依然存在。高氮浓度下胞内、外碳酸酐酶活性均明显升高。胞内碳酸酐酶活性在高磷浓度下明显升高,但胞外碳酸酐酶活性并没有受到磷浓度变化的影响。高氮、磷浓度培养下的小新月菱形藻的最大光合作用速率(Vmax)、对CO2亲和力(K0.5(CO2))和光系统II最大光化学效率(Fv/Fm)均明显提高。以上结果表明小新月菱形藻可以通过改变胞内、外碳酸酐酶活性调节无机碳利用以适应不同氮磷浓度的环境。     

Regulation of the mechanism for HCO 3 − use by the inorganic carbon level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)

The capacity for HCO₃ ⁻ use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (C_i) was investigated. The use of HCO₃ ⁻ at alkaline pH by P. leucosticta was␣demonstrated by comparing the O₂ evolution rates measured with the O₂ evolution rates theoretically supported by the CO₂ spontaneously formed from HCO₃ ⁻ . Both external and internal carbonic anhydrase (CA; EC 4.2.1.1) were implied in HCO₃ ⁻ use during photosynthesis because O₂ evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and ethoxyzolamide (inhibitors for external and total CA respectively). Both external and internal CA were regulated by the C_i level at which the algae were grown. A high C_i level produced a reduction in total CA activity and a low C_i level produced an increase in total CA activity. In contrast, external CA was increased at low C_i although it was not affected at high C_i . Parallel to the reduction in total CA activity at high C_i is a reduction in the affinity for C_i, as estimated from photosynthesis versus C_i curves, was found. However, there was no evident relationship between external CA activity and the capacity for HCO₃ ⁻ use because the presence of external CA became redundant when P. leucosticta was cultivated at high C_i. Our results suggest that the system for HCO₃ ⁻ use in P. leucosticta is composed of different elements that can be activated or inactivated separately. Two complementary hypotheses are postulated: (i) internal CA is an absolute requirement for a functioning C_i-accumulation mechanism; (ii) there is a CO₂ transporter that works in association with external CA. Received: 20 April 1996 / Accepted: 5 August 1996     

Exogenous inorganic carbon sources for photosynthesis in seawater by members of the Fucales and the Laminariales (Phaeophyta): ecological and taxonomic implications

Summary Characteristics of inorganic carbon assimilation by photosynthesis in seawater were investigated in six species of the Fucales (five Fucaceae, one Cystoseiraceae) and four species of the Laminariales (three Laminariaceae, one Alariaceae) from Arbroath, Scotland. All of the algae tested could photosynthesise faster at high external pH values than the uncatalysed conversion of HCO ₃ ^- to CO₂ can occur, i.e. can use external HCO ₃ ^- . They all had detectable extracellular carbonic anhydrase activity, suggesting that HCO ₃ ^- use could involve catalysis of external CO₂ production, a view supported to some extent by experiments with an inhibitor of carbonic anhydrase. All of the algae tested had CO₂ compensation concentrations at pH 8 which were lower than would be expected from diffusive entry of CO₂ supplying RUBISCO as the initial carboxylase, consistent with the operation of energized entry of HCO ₃ ^- and / or CO₂ acting as a CO₂ concentrating mechanism. Quantitative differences among the algae examined were noted with respect to characteristics of inorganic C assimilation. The most obvious distinction was between the eulittoral Fucaceae, which are emersed for part of, or most of, the tidal cycle, and the other three families (Cystoseiraceae, Laminariaceae, Alariaceae) whose representatives are essentially continually submersed. The Fucaceae examined are able to photosynthesise at high pH values, and have lower CO₂ compensation concentrations, and lower K_1/2 values for inorganic C use in photosynthesis, at pH 8, than the other algae tested. Furthermore, the Fucaceae are essentially saturated with inorganic C for photosynthesis at the normal seawater concentration at pH 8 and 10°C. These characteristics are consistent with the dominant role of a CO₂ concentrating mechanism in CO₂ acquisition by these plants. Other species tested have characteristcs which suggest a less effective HCO ₃ ^- use and CO₂ concentrating mechanism, with the Laminariaceae being the least effective; unlike the Fucaceae, photosynthesis by these algae is not saturated with inorganic C in normal seawater. Taxonomic and ecological implications of these results are considered in relation to related data in the literature.     

Responses of the macroalga Gracilaria tenuistipitata var. liui (Rhodophyta) to iron stress

Chlorophyll (Chl), phycoerythrin (PE), total nitrogen (TN% dw) and Fein tissues were measured in Fe-deficient cultures of Gracilariatenuistipitata var. liui over a period of 60 days. ⁵⁵Fe uptakeand photosynthetic carbon fixation (NaH¹⁴CO³) werecompared in Fe-rich and Fe-deficient cultures and analyzed the effects ofFe-deficiency on the ultrastructure. The maximum carbon fixationdecreased significantly (p < 0.01) under Fe-deficiency. Thechlorophyll and phycoerythrin contents also declined with decreasing tissueiron content, falling, respectively, to 7.9 and 33.8% of their originallevel. Photosynthesis in Fe-deficient cells became light-saturated at lowerirradiance than the control. Total N in tissue decreased from 3.65 to2.49%. ⁵⁵Fe uptake rate for cultures grown on NO₃ ^-was measured following resuspension in either NH₄ ⁺ orNO₃ ^- as N source. Enhanced Fe uptake developedunder Fe stress, especially with cells resuspended in NH⁺ ₄-N medium. The Vmaxfor Fe uptake was higher with NH₄ ⁺ thanNO₃ ^- (62.8 versus 12.1 pmol mg dw^-1 h^-1). The requirement for N accelerates further Fe uptake. Ultrastructuralobservations of Fe-deficient cells showed reductions in chloroplast number,degeneration of lamellar organization, decrease in mitochondrial matrixdensity and variation in accumulation body number and morphology. During Fe-deficiency, the growth rate continued to decline and after 40days of iron deficiency, no further growth was detectable, and eventuallyiron deficiency resulted in chlorosis. The results suggest that the lowergrowth rate of Gracilaria tenuistipitata var. liui underFe-deficiency may result from largely from inhibition of photosynthesis andnitrogen utilization.     

Affinity for inorganic carbon of Gracilaria tenuistipitata cultured at low and high irradiance

Regulation by irradiance level of the mechanism for dissolved inorganic carbon (DIC) acquisition was examined in the red macroalga Gracilaria tenuistipitata Zhang et Xia. For this purpose, affinity for external DIC, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli grown at 45 and 500 μmol photons m⁻² s⁻¹. Oxygen evolution rates declined by 50% when the medium pH was changed from 8.1 to 8.7, and the pH compensation point attained was ca. 9.2. These characteristics were unaffected by the light treatments. In contrast, photosynthetic conductance for DIC at pH 8.7 was doubled in thalli grown at high irradiance compared with those grown at low irradiance (to 0.74 × 10⁻⁶ from 0.33 × 10⁻⁶ m s⁻¹). Photosynthetic rates at saturating DIC concentration were also higher by 60% in thalli grown at high irradiance. These differences could not be attributed to changes in the use of external DIC, since external CA activity did not vary. Although the irradiance level did not modify the pool size of Rubisco, Rubisco content expressed on a chlorophyll a basis was almost doubled at high irradiance. These results likely indicate that the internal transport of DIC towards the active-site of Rubisco, rather than the external use of DIC, is enhanced in the thalli grown at high irradiance. Received: 7 June 1999 / Accepted: 16 October 1999     

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

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 (g_p(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 μmol 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 g_p(CO2) was estimated. Photosynthetic conductance decreased in both morphotypes at non-saturating PFR. This dependence of g_p(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.     

Sustained high yields of Gracilaria (Rhodophyta) grown in intensive large-scale culture

Gracilaria ferox J. Agardh was grown continuously in large, outdoor tanks under a pulse-fed nutrient regime for four years. Productivity ranged from 21.4 to 59.2 g d. wt m⁻² d⁻¹ with a mean of 39.7 g d. wt m⁻² d⁻¹ over the entire study period. Because the cultures were maintained under non-nutrient limiting conditions, productivity was regulated primarily by seasonal changes in light and temperature, which accounted for 75% of the variability of growth in algal yields. Salinity ranged from 31.0 to 36.5‰ and had insignificant effects on growth within this range. The original vegetative strain was maintained over the entire study without the need for additional supplementation from field-collected stock. Because of the pulse-fed nutrient supply, epiphytic growth on the target species was negligible (< 3% total biomass) throughout the study. The yields attained in this study rank among the highest reported for any intensively managed photosynthetic crop and demonstrate the feasibility of growing red macroalgae like Gracilaria at a sustained high yield in a large-scale, land-based culture system. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of carbonic anhydrase in photosynthesis and inorganic-carbon assimilation in the red alga Gracilaria tenuistipitata

The mechanism of inorganic-carbon (C_i) accumulation in the red seaweed Gracilaria tenuistipitata Zhang et Xia has been investigated. Extracellular and intracellular carbonic-anhydrase (CA) activities have been detected. Photosynthetic O₂ evolution in thalli and protoplasts of G. tenuistipitata were higher at pH 6.5 than at pH 8.6, where HCO ₃ ^– is the predominant form of C_i. Dextran-bound sulfonamide (DBS), a specific inhibitor of extracellular CA, reduced photosynthetic O₂ evolution at pH 8.6 and did not have any effect at pH 6.5. After inhibition with DBS, O₂ evolution was similar to the rate that could be supported by CO₂ from spontaneous dehydration of HCO ₃ ^– . The rate of photosynthetic alkalization of the surrounding medium by the algal thallus was dependent on the concentration of C_i and inhibited by DBS. We suggest that the general form of C_i that enters through the plasma membrane of G. tenuistipitata is CO₂. Bicarbonate is utilized mainly by an indirect mechanism after dehydration to CO₂, and this mechanism involves extracellular CA.Abbreviations C_i inorganic carbon (CO₂ + HCO ₃ ^– ) - CA carbonic anhydrase - DIC dissolved inorganic carbon (total) - DBS dextran-bound sulfonamide - EZ ethoxyzolamide - NSW natural seawater - PPFD photosynthetic photon flux density - REA relative enzyme activity - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase This research was supported by the Deutsche Forschungsgemeinschaft (Bonn) as a programme of the Sonderforschungsbereich 251 der Universität Würzburg and by the Fonds der Chemischen Industrie (Frankfurt). Joint work in Würzburg was possible thanks to travel grants from the Chancellor of the University of Würzburg, Professor R. Günther, from the Australian National University under the auspices of its Overseas Studies Programme, and from the New Zealand — Federal Republic of Germany Scientific and Technological Exchange Programme, which are gratefully acknowledged. We thank Dr. A. Meyer and Ms. E. Kilian for untiringly conducting part of the experimental work, Ms. G. Theumer and Ms. D. Faltenbacher-Werner for their valuable assistance, and Mr. H. Walz (Walz Company, Effeltrich, FRG) for his skilled help with the calibration of our gas-exchange system for measurements with helox. The Department of Conservation, New Zealand, is thanked for permission to collect lichens.     

The acquisition of inorganic carbon by four red macroalgae

Photosynthesis was studied in four species of red marine macroalgae: Palmaria palmata, Laurencia pinnatifida, Lomentaria articulata and Delesseria sanguinea. The rate of O₂ evolution for submersed photosynthesis was measured as a function of incident photon flux density at normal pH and inorganic carbon concentration (pH 8.0, 2 mol m^–3), and as a function of inorganic carbon concentration at pH 8.0 at saturating and at limiting photon flux density. The rate of CO₂ uptake was measured for emersed photosynthesis as a function of CO₂ partial pressure at saturating photon flux density. Previous pH-drift results suggest that Palmaria and Laurencia are able to use HCO _inf3 ^sup– as well as CO₂ whereas Lomentaria and Delesseria are restricted to CO₂. None of the algae are saturated by 2 mol m^–3 inorganic carbon at high light (400 mol m^–2 s^–1) but are saturated at low light (35 mol m^–2 s^–1). The inorganic C concentration at which half the light-saturated rate of O₂ evolution is achieved is higher for Palmaria and Laurencia (1.51 and 1.85 mol m^–3) than for Lomentaria and Delesseria (0.772 and 0.841 mol m^–3). The lower values for the latter two species could reflect their putative restriction to CO₂. If expressed in terms of CO₂, the half-saturation values yield 7.2 and 7.8 mmol m^–3 respectively, which are very similar to values obtained previously during pH-drift experiments but at lower concentrations of HCO _inf3 ^sup– , consistent with restriction to CO₂. The photosynthetic conductance (m s^–1), calculated from the initial slope for photosynthesis at low concentrations of inorganic carbon, correlates with the suggested ability to extract inorganic carbon based on pH-drift results. Calculations made assuming that CO₂ is the only species diffusing across the boundary layer are consistent with boundary layer thicknesses of 20 and 19 m for Lomentaria and Delesseria respectively, which is feasible given the rapid water movement in the experiments. For Laurencia however, an unreasonably small boundary layer thickness of 6 m is necessary to explain the flux, which indicates co-diffusion by HCO _inf3 ^sup– . In the apparent absence of external carbonic anhydrase, direct uptake of HCO _inf3 ^sup– , rather than external conversion to CO₂ is indicated in this species. In air, the CO₂ concentration at which photosynthesis is half-maximal increases in the same order as the ability to raise pH in drift experiments. At 21 kPa the CO₂ compensation partial pressures for Palmaria and Laurencia at 0.56 and 1.3 Pa are low enough to suggest a carbon-concentrating mechanism is operating, while those of Lomentaria at 1.8 Pa and particularly that of Delesseria at 4.5 Pa could be explained without a carbon-concentrating mechanism. The algae tested (all except Delesseria) showed more O₂ evolution than could be accounted for with a photosynthetic quotient of 1.0 and uncatalysed conversion of HCO _inf3 ^sup– to CO₂ outside the cell in high light at pH 8.0 when high algal fresh weight per unit medium was used. These results are concordant with other data suggesting use of HCO _inf3 ^sup– by Palmaria and Laurencia, but discordant with the rest of the available information in indicating use of HCO _inf3 ^sup– by Lomentaria. The reason for this is unclear. The lightsaturated rate of O₂ evolution on an algal area basis and the photon flux density needed to saturate photosynthesis were related partly to the habitat from which the seaweeds were collected, but more strongly to the ability to use HCO _inf3 ^sup– . Values for the two users of HCO _inf3 ^sup– , Palmaria (population used was intertidal; also occurs subtidally) and Laurencia (intertidal/shaded intertidal), were greater than for Lomentaria (shaded intertidal), which was greater than Delesseria (subtidal), both of which are believed to be restricted to CO₂. In accordance with earlier ¹³C data and, for Delesseria, estimates of the achieved growth rates in situ, carbon is likely to be saturating and use of HCO _inf3 ^sup– is unlikely to occur in the normal low-light habitats of Lomentaria and Delesseria. Analysis of N-use efficiencies show that they are closer to the low-CO₂-affinity Laminariales than the high-CO₂-affinity Fucaceae.     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Strain selection and genetic variation in Gracilaria chilensis (Gracilariales, Rhodophyta)

Strain selection processes in seaweed often have assumed that sterile clones could be maintained for long periods in a diversity of environments without major genetic changes. However, clonal species such as Gracilaria chilensis exhibit intra-clonal variation in performance and ongoing studies suggest such changes may be due to rapid changes in DNA composition associated with growth, via mitotic recombinations. Therefore performance of a given ramet in this type of seaweed should be understood as the dynamic outcome of rapid reactions between the environment and the changing genotype of the selected strain. To evaluate this idea, we measured changes in genetic variability, as detected by DNA-fragment polymorphism using RAPDs-PCR, exhibited by clones of G. chilensis after two transfers to different environmental conditions (from field to controlled laboratory conditions and from the laboratory to large-scale tank culture). The transfer to laboratory conditions reduced the frequency of low similarity values and increased the frequency of intermediate similarity values in DNA banding patterns, suggesting the branchlets produced under controlled laboratory conditions have less genetic variability (evaluated as total DNA polymorphism) than plants recently collected in the field. Tank incubation reduced the total range of similarity and significantly increased the frequency of high similarity values. Results thus suggest the dynamic of genetic changes in vegetative clones of Gracilaria chilensis that is fast and strongly affected by the external environment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure of reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis (Gracilariaceae,Rhodophyta)

Reproductive apparatus of Gracilaria/Gracilariopsis lemaneiformis collected from Qingdao city were studied with a light and a transmission electron microscope. The special superficial arrangement of spermatangium for this species was clearly observed, and the ultrastructure of spermatangial devel-opment revealed the similar cytodynamic pattern followed by all the Gracilariaceae members devel-oped from spermatangial mother cells to spermatangium. The female reproductive apparatus before fertilization was also observed and trichogyne was found protruding above the cortex, contrary to the earlier reports. Tetrasporangium was formed by an outer cortical cell and the tetraspores became spherical and expended after being released.     

Galactans from Gracilaria millardetii and G textorii (Gracilariales,Rhodophyta) of Indian waters

Galactans containing methylated galactan moieties were extracted from Indian agarophytes, namely, Gracilaria millardetii and Gracilaria textorii growing naturally along the west coast of India. The galactans were treated with α‐amylase to remove floridean starch. These were characterized by Fourier Transform‐Infrared (FT IR), ¹³C‐Nuclear Magnetic Resonance (C NMR), Gas Chromatography‐Mass Spectrometry (GC MS), Inductively Coupled Plasma spectroscopy (ICP) and Gel Permeation Chromatography (GPC) and were found to be composed of d ‐galactose, 6‐O‐methyl‐D‐galactose and 3,6‐anhydro‐L‐galactose. Methylation analyses revealed that both the polysaccharides consisted of 3‐, 2,3‐, 4,6‐linked galactose as well as four‐linked 3,6‐anhydro galactose residues. Both the Gracilaria species produced low gelling (<100 g cm^?2) and highly sulfated (2.1% to 4.8%) galactans containing very low heavy metal contents (ICP). These galactans may be of potential utility in food and biological applications.     

Unusual cell structures in tumor-like formations of Gracilaria (Rhodophyta)

This paper deals with electron microscopic observations on cultivated plants of the marine red alga Gracilaria verrucosa which developed simple galls; also sea collected material, without galls, had been studied. The galls showed unusual but characteristic cell structures, caterpillar-like bodies, containing rows of fusiform bodies. These were found mostly in the cytoplasm near the plastids, in one case connected with the endoplasmic reticulum, occasionally even inside the nucleus, and are described here, as far as we know, for the first time. It does not seem probable that the caterpillar-like bodies represent mitochondria or bacteria, but the hypothesis that fusiform bodies are related to virus-like structures is discussed. The normal tissues as well as the gall tissue of the laboratory plants contained, besides plastids typical for the red algae, another type of plastids characterized by tubular thylakoids.This work was supported by grants from Consiglio Nazionale delle Ricerche (Rome) and Ministero dell'Agricoltura e delle Foreste of Italy.