Light Quality Affects Morphology and Polysaccharide Yield and Composition of Gelidium Sesquipedale (Rhodophyceae)

Light quality affects morphology and polysaccharide yield and composition of Gelidium sesquipedale (Rhodophyceae)

Raquel Carmona*, Juan J. Vergara, Marc Lahaye & F. X. Niell 1Departamento de Ecolog´ıa, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain 2Departamento de Ecolog´ıa, Facultad de Ciencias del Mar, Universidad de Cádiz, 11510 Puerto Real, Cádiz, Spain 3Institut National de la Recherche Agronomique, Unite de Recherche sur les Polysaccharides, leurs Organisations et Interactions, BP 71627, 44316 Nantes, France

(∗Author for correspondence; e-mail: [email protected])

Received 1 February 1998; revised 30 June 1998; accepted 13 July 1998

Key words: Gelidium sesquipedale, light quality, branch proliferation, agar, starch

Abstract Morphology and polysaccharide characterization of Gelidium sesquipedale (Clem.) Bornet et Thuret were studied in cultures grown under various light qualities. White light (WL), blue light (BL) and red light (RL) (all at photon ﬂuence rate of 40 µmol m−2 s−1) were used for the study of morphological characteristics, and in addition yellow light (YL) for polysaccharide characterization. RL and BL induced a proliferating growth, which resulted in bushy plants under RL. Cortical cells of BL-grown plants were smaller and presented a higher density per unit area, whereas those of WL- and RL-grown alga were larger. Medullary cells followed the inverse pattern. Light quality also affected polysaccharide yield and composition, with the yield being higher under BL, RL or YL than WL. Most of the polysaccharide was extracted in distilled water at 100 ◦C, while a low amount was solubilized at 22 ◦C and 120 ◦C. Extracts from BL-grown alga presented the highest galactan content. The starch concentration was lower in extracts from RL-, BL- and YL-cultivated alga than in those from the initial plants. The degree of substitution with methoxyl groups and precursor was very low in all the agar fractions, but fractions extracted from BL- and WL-grown alga were more substituted by precursor. The highest sulfate content was reached under BL (about 9% w/w) and the highest 2-O-methyl-3,6-anhydro-L-galactose and 6-O-methyl-D-galactose content were found in extracts from alga grown under YL.

Introduction However, external appearance of the alga changes in response to environmental conditions such as nutri- Gelidium sesquipedale (Clem.) Bornet et Thuret is ent supply (Haglund & Pedersén, 1992), seasonality the most important raw material for the agar indus- (Mouradi-Givernaud et al., 1992), stage in the on- try in Spain. Previous studies on this species dealt togeny (Rodríguez Vargas & Collado-Vides, 1996), with photosynthesis in relation to temperature, pig- salinity (Rebello et al., 1996), water agitation (San- ment concentration and light quality, with nitrogen telices, 1978) and light regime (Baghdadli et al., metabolism and with cell wall structure and compo- 1994). sition (Torres et al., 1991, 1995; Vergara et al., 1993; There are numerous reports on light quality ef- Vignon et al., 1994a, b; Carmona et al., 1996). fects on red alga (see Gantt, 1990) and most of the Differences in the morphology of the Gelidiaceae studies relating growth conditions with agar charac- have usually been considered as a taxonomic char- teristics have been carried out on Gracilaria (Lahaye acter (Dixon, 1958; Santelices, 1976; Rico, 1992). & Rochas, 1991; Murano, 1995). However, there has

article: japh576; pips nr 186100 (japhkap:bio2fam) v.1.1 japh576.tex; 13/11/1998; 1:25; p.1 324 been no investigation on the relationships of the light were dehydrated in a graded ethanol series and sub- spectral composition with the morphology of Gelid- sequently infiltrated in Historesin 7100. Cross- and ium sesquipedale and the composition and structure of longitudinal sections (4 µm thick) were cut in an au- its cell wall polysaccharides. tomatic microtome Leica Supercut 2065 and stained The objectives of the present work are to study with 1% toluidine blue. the effect of light quality on Gelidium sesquipedale with respect to: (1) external and cellular morphol- Agar extraction ogy; (2) agar yield and composition of this species, The available plant material was scarce, since it came developing a micromethod for agar extraction. from cultures in chemostats to maintain a constant flow of nutrients, and parallel samples were also taken for biochemical and physiological analysis. There- Material and methods fore, a method to extract agar from small amounts of samples was adapted from the sequential extrac- Plant material and culture conditions tion described by Lahaye et al. (1986). This method consists of extracting the polysaccharides at different Thalli of Gelidium sesquipedale were collected on the temperatures based on the differing solubilities of the rocky shore of Punta Carnero (Algeciras, Southern ◦ 0 ◦ 0 agar compounds. Sequential extraction of agar has Spain: 6 05 N, 5 26 W), where the alga occurs in been interpreted to reflect the solubility and associa- crevices at low irradiance, and were transferred to the tion of polysaccharides in the algal cell-wall (Lahaye laboratory for culture in chemostat (Carmona et al., et al., 1986). 1996). Culture was carried out in artificial seawater ◦ The modified method is shown in Figure 1. The (Woelkerling et al., 1983) at 17 C with 12 h light:12 h alga was dried at 60 ◦C for 48 h, milled to a flour dark. The volume of medium was 2.5 L. Agitation was in a liquid N2-grinder and washed with 80% ethanol obtained by bubbling the medium with air at 4.4 L −1 several times until the supematant was colourless. The min . The alga was cultured for the first phase under algal powder (50 mg) was suspended in 2 mL distilled µ −2 −1 white light of 100 mol photon m s for 10 days. water in test tubes for 1 h at 22 ◦C and centrifuged Subsequently, plants were transferred to different light for 20 min at 1000 g with a benchtop centrifuge. The qualities (white, blue, red, yellow) at the same value of supematant was pipetted and placed in a round-bottom photosynthetic active radiation (PAR) (40 µmol m−2 −1 flask; this extraction was repeated twice (the last time s ) for 14 days; this value was of a similar order to with 1 mL distilled water). The three supernatants that measured in crevices on the rocky shore. were combined, freeze-dried and are subsequently termed the cold extract. The algal residues were then Morphometric measurements extracted three times with distilled water at 100 ◦C and 120 ◦C as for the cold extract. The last extraction The degree of branching of the plants was measured at 120 ◦C was performed with 1 mL for 30 min and on fronds photocopied with a 2 × magnification by the combined respective extracts were freeze-dried. counting the number of secondary branches per unit The final pellet had the appearance of a fine powder, length (number of branches cm−1) during the period indicating that the agar extraction was very effective. of culture. The main axis was taken as the branch of Two extractions and subsequent chemical analysis order 0, primary branches as order 1 and secondary were made from two replicates coming from alga sam- branches as order 2. Measurements were also made ples of each light treatment. Plants were taken out the on micropinnulae of all branches. Microscopy obser- chemostats for this polysaccharide study after the first vations were made on pieces of secondary branches phase of culture (initial sample) and at the end of the immersed in Eukitt (Vitromed-Basel) as a mounting light quality treatments. agent by means of a Fluovert FV Leitz inverted mi- croscope connected to a Leica Quantimet 500 image Chemical analyses analysis system. The following parameters were determined in surface view: volume and density (cells Sugars were identified and quantified in extracts by mm−2) of cortical cells from four optical fields. Mea- gas chromatography after reductive acid hydrolysis surements of medullary cells were made on fronds with trifluoroacetic acid (2N, 120 ◦C, 1 h) and 4- fixed in 4% formaldehyde solution in seawater. They methylmorpholineborane (MMB), following the pro-

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Figure 1. Scheme of the micromethod for agar extraction. The second and third extract were treated as detailed for the first extract. cedure described by Stevenson and Furneaux (1991), 1H nuclear magnetic resonance spectroscopy and conversion into alditol acetates (Blakeney et al., 1983). Samples containing myo-inositol as standard, Samples (10 mg) were dissolved in deuterium oxide were injected on a fused silica capillary column DB- (D20) (99.9% Aldrich) and then freeze-dried. This 225 (25 m × 0.32 mm, 0.25 µm thick, Scientific Glass ◦ operation was repeated twice to eliminate water and Engineering, France) eluted at 220 C with hydrogen. substitute hydroxyl protons with deuterium. Finally, Quantification was achieved using weight response 0.5 mL of 100% D2O (Sigma) were added to dissolve factors determined from standard sugars. Sulfate con- the dried sample and the solution was then transferred tent was measured by HPLC according to the fol- to a 5 mm 1H NMR tube. 1H nuclear magnetic res- lowing procedure: 10 mg polysaccharide sample was ◦ ◦ onance (NMR) spectra were recorded at 60 Cona hydrolysed with 1 mL of TFA (2N, 120 C, 2 h). After Bruker ARX 400 operating at 400.13 mHz. The 1H cooling to room temperature, the solution was cen- NMR chemical shifts were measured in ppm from the trifuged and 0.5 mL was taken from the supernatant, HOD resonance set to 4.1 ppm. evaporated in vacuo, washed twice with 0.5 mL dis- The degree of substitution with methoxyl groups tilled water and diluted with distilled water. HPLC was i.e. number of methoxyl groups per disaccharide re- performed with an Ionpac AS 11 column and Ionpac × × peating unit (DS) was calculated from the ratio of AS 11 guard column (4 250 mm and 4 50 mm, the integral value, divided by 3, for specific sig- respectively; Dionex, Sunnyvale, CA, USA) using −1 nals at 3.28 and 3.19 ppm, attributed to the methyl 10 mM NaOH as eluant at a flow rate of 1 mL min . protons located on C2 of 3,6-anhydrogalactose (2- The eluate was monitored by conductimetry. Solutions Me) and C6 of galactose (6-Me), respectively, to containing 10–100 ppm of standard sodium sulfate twice the mean integral value for anomeric signal of were used for quantification. Values are expressed in 4-linked galactose [L-galactose-6-sulfate (A1∗)and weight percent of sulfate per dry weight of agar. anhydrogalactose (A1) at 5.06 and 4.92 ppm, respectively]; [(Me/3)/(A1 + A1∗) × 2] on the spectra.

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The degree of substitution with L-galactose-6-sulfate being those with the lowest density (Figure 3A). Tak- (considered as the agarobiose precursor) was calcu- ing into account the cell volume, it can be observed lated from the integral of this sugar anomeric proton that cortical cells of BL-grown alga were significantly and the mean integral value for 4-linked galactose smaller than the other ones (Figure 3B). Medullary (A1∗ × 100/A1∗ + A1). The starch content was cal- cells showed the opposite, being larger under WL and culated from the ratio of the integral of this sugar BL than under RL (Figure 3C). anomeric proton at 5.13 ppm (S) and the mean integral value for the anomeric proton of the 4-linked Agar yield and composition galactose [S × 100/S + (A1∗ +A1)×2]. For all these calculations, an equimolarity of 4-linked to 3-linked The yield of polysaccharides solubilized in water at galactose was assumed. different temperatures and calculated as milligrams of crude extract per gram of algal dry weight is shown Statistics in Table 2. There was no significant difference among the treatments and the mean values ranged from 66% The results are expressed as mean standard error to 71% of the algal DW. Since starch contributed as (SE). The standard error of the mean was calculated a large weight percentage of the extracts, it was nec- according to Sokal and Rohlf (1981). Statistical signif- essary to substract its content from the raw yields in icance of the means was tested with a model 1 one-way order to determine the agar content. After such correc- ANOVA followed by a multirange test Fisher’s pro- tion, the polysaccharide yield from the plants grown tected least significance difference (LSD) (Sokal & under RL, BL and YL was higher than that from those Rohlf, 1981). grown under WL. The latter displayed a similar yield to that obtained from the initial algal sample, before transferring the plants to the different light qualities Results (Table 2). (All the chemical components are expressed as percentage of polysaccharide dry weight = crude ex- Morphology tract yield corrected for starch content.) Most of the polysaccharides were extracted in the 100 ◦C water The degree of branching at the level of primary ramifi- fraction (Figure 4), regardless of the light treatment, cation did not change during culture, but the secondary reaching a mean value of 60% of the total solubilized ramifications increased markedly in RL-grown alga, agar. and to a lesser extent in BL-grown alga, whereas The highest galactan content in the raw fractions they remained almost constant in WL-grown plants. was measured in BL-grown plants (Table 2), followed The WL-grown alga was taken as a control to check by RL-grown alga. The richest galactan fractions were changes in branching related to culture conditions, as extracted at 100 ◦C. it remained similar to the wild plants (Figure 2). A high correlation was found between glucose, The density of micropinnulae upon the branches measured by chemical analysis of the neutral sug- was much higher under RL than under BL (twenty ars, and starch, measured by 1H NMR spectroscopy times) and about sixty times higher than under WL (Figure 5). The starch content measured by the lat- (Table 1). These differences existed in the micropinnu- ter method yielded similar values in the total extract lae that were bome by the branches of order 1, while from the WL- grown plants compared to that of the there was no significant difference in the number of initial alga (Table 2). In contrast, all the other light micropinnulae on the main axis (branch of order 0). treatments induced a decrease in starch content, espe- Regardless of the light treatment, there were more cially RL. The starch content, presented in Figure 6 in micropinnulae per unit length with increasing branch a qualitatively way, corresponds to the content within order (Table 1). Light quality makes the plants visually the fraction without correction for the extraction yield. different from each other. Red light gave the plants a Two main pools of starch can be seen in all the al- typical bushy appearance compared to plants grown gal samples, one extracted at 22 ◦C and the other at under blue light that have some proliferations and to 120 ◦C. WL-grown ones that lack these micropinnulae. Corti- The molar proportion of precursor repeating units cal cells were more abundant per unit area in the plants of the crude extracts increased in all the light treat- cultured under BL than under RL; the WL-grown alga ments from a degree of substitution of 0.04 in the

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Table 1. Morphometric characteristics of Gelidium sesquipedale cultured at − − 40 µmol m 2 s 1 of different light qualities. Number of micropinnulae and micropinnulae density upon each branch.

Branch order Number of micropinnulae Micropinnulae density − (number of micropinnulae cm 1) White Blue Red White Blue Red Light Light Light Light Light Light

0 14 16 15 2.5 2.5 2.1 1 129 243.5 710 4.8 5.6 12.5 2 5 172.8 646 0.5 9.6 29.4

2.5 -1 White light

2.0 Blue light Red light

1.5

1.0 Number of branches mm 0.5 -10 -5 0 5 10 15 Time (day) − − Figure 2. Time course of the branching degree of thalli of G. sesquipedale cultured under 100 µmol m 2 s 1 of white light and under 40 µmol − − m 2 s 1 of white light, blue light and red light. Data are the mean of two replicates SE. initial plants, to 0.08 in BL and WL and to a lower Table 2. Effect of light quality on total crude extract yield (% algal dry wt), total crude extract yield corrected for the starch value in RL and YL-grown alga (Table 3). The sul- content (% algal dry wt), galactan yield (% polysaccharide dry fate content in the extracts was low, except for those wt) and starch content (% polysaccharide dry wt) in Gelidium of the BL-grown alga for which it reached about 9% sesquipedale. Standard errors in parenthesis (n =2). w/w. However, in all the cases, the sulfate content Light Yield Yield Galactans Starch was higher than that of the fractions from the initial treatment (-starch) alga (Table 3). On the other hand, most of this sulfate was extracted at 22 ◦C in all the plants, excepting BL- Initial 68.6 (0.95) 54.6 (0.26) 41.0 (2.79) 20.6 (1.2) grown alga that also showed a signiﬁcant content in White light 66.1 (1.38) 52.9 (0.9) 37.9 (2.02) 20.2 (0.24) the 120 ◦C fraction. Red light 65.7 (1.66) 59.8 (1.51) 44.1 (2.31) 9.06 (0.2) The agarobiose repeating unit of the agar in Blue light 68.7 59.1 49.9 (0.61) 13.9 G. sesquipedale cultured in our experiments showed Yellow light 71.1 (3.27) 57.4 (2.48) 40.8 (2.34) 13.5 (0.37) a very low concentration of 2-O-methyl-3,6-anhydro- L-galactose and 6-O-methyl-D-galactose (Table 3), measured by 1H NMR spectroscopy, the degree of substitution for both being below 0.05 in most sam- (0.04), except for that obtained from YL-grown plants ples. The degree of substitution by 2-O-methyl-L- with a value of 0.1. The distribution of 6-O-methyl- galactose was quite low in agar from all the treatments D-galactose in the agar exhibited some variations with

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Table 3. Sulfate content and degree of substitution with precursor (L-galactose-6-sulfate), with 2-O-Methyl 3,6-anhydro-L-galactose and with 6-O-Methyl D-galactose containing repeating units of the crude extract, expressed on % polysaccharide dry wt, in Gelidium sesquipedale cultured under different light qualities. Standard errors in parenthesis (n =2).

Light Sulfate Precursor 2-O-Methyl 6-O-Methyl treatment

Initial 1.38 (0.05) 0.039 (0.001) 0.05 (0.004) 0.03 (0.00? White light 2.5 (0.16) 0.075 (0.001) 0.05 (0.005) 0.04 (0) Red light 3.18 (0.15) 0.059 (0.002) 0.04 (0) 0.03 (0) Bluelight 8.93 (0.06) 0.076 (0) 0.039 0.04 Yellow light 4.22 (0.13) 0.05 (0) 0.104 (0.006) 0.08 (0.0?

wavelength of light used for the algal growth. While modifications have not been described in the natural the highest content was again measured in the YL sam- habit of this species. ple (DS 0.08), the methylation in the RL-grown plants Although G. sesquipedale has a slow metabolism did not increase, opposite to the WL- and BL-grown and thus demonstrates smaller effects linked to eco- alga. physiological growth conditions than fast-growing alga, its thalli pigment, C and N contents were also affected by light quality (Carmona et al., 1996). The Discussion well known BL effect on protein accumulation in plants (Kowallik, 1987; Senger, 1987) was also ob- The effects of ecophysiological conditions on growth, served in G. sesquipedale by Carmona et al. (1996) for morphology and biochemical composition of alga do which a higher insoluble protein content can be related not follow a single pattern in alga and thus, cannot with the increase in cortical cells density per unit area be generalized. In the case of G. sesquipedale, light observed in this study. quality not only affects photosynthesis and chemical Light quality also affects G. sesquipedale polysac- composition, but also has remarkable effects on the charide biosynthesis, as previously shown. morphology of the plant. For this slow-growing alga, The overall polysaccharide yields did not markedly that showed growth rates of 1.86, 1.78 and 1.33% d−1 differ among the algal samples but were all quite under RL, BL and WL, respectively (Carmona et al., high compared to those described in the literature 1996), RL and BL induced a proliferative growth, (Whyte & Englar, 1980; Craigie & Wen, 1984; Lahaye yielding bushy plants in the case of RL. This latter et al., 1986; Bird, 1988; Lignell & Pedersén, 1989), morphology was equivalent of the thallus expansion probably due to the efficiency of the extraction proce- of Porphyra leucosticata Thuret in Le Jolis grown un- dure. However, when the starch content is subtracted der RL, but the growth of this alga is inhibited by BL from these yields, differences appeared, suggesting (Figueroa et al., 1995). The growth and morphologi- that this compound plays an important role in the re- cal behaviour of G. sesquipedale under experimental sponse of the polysaccharide pool to light treatments. light qualities differed from those of the agarophyte The sequential extraction described herein provides Gracilaria tenuistipitata Zhang et Xia var. liui which information on the distribution of agar according to has a higher growth rate under RL and YL that un- their structure and possible linkages and associations der BL (authors, unpublished data). Other divergent in the cell wall. The pattern of agar extraction in effects of light quality on growth and morphology of G. sesquipedale is different from other described pre- alga were reported from the brown alga Cystoseira viously (Lahaye et al., 1986; Lignell & Pedersén, 1989), since most of it is extracted at 100 ◦C, the yield barbata C. Ag. fo. aurantia (Kütz.) Giaccone. This ◦ species grew faster under BL, which also induced pri- of the 22 C fraction being very low, but, as expected, mary and secondary ramifications, than under RL and rich in sulfated compunds. WL (Baghdadli et al., 1994). These morphological

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Figure 4. Effect of light quality on crude extract yield in the dif- ◦ ◦ ferent fractions, extracted in distilled water at 22 C, 100 Cand ◦ 120 C. Bars represent standard errors (n = 2). I = Initial sample; W = White light; R = Red light; B = Blue light; Y = Yellow light.

Figure 5. Correlation between starch and glucose measured by different means. Mol% of glucose refers to the starch in the polysaccharide extracts determined by NMR and sugar analysis by chemical means. The regression line and coefﬁcient are shown (n = 23).

Torres et al. (1995) studied the inﬂuence of light quality on cell-wall polysaccharide content in G. sesquipedale at short term-scale (hours), RL in- ducing the highest increase in these compunds. The differences between this and our study suggest that the mechanisms of synthesis and mobilization of polysaccharides are dependent on time-scale. Besides, the analytical method used by these authors was less accurate than present one. Figure 3. Effect of light quality on cellular characteristics of G. The drop in the starch content of the alga grown sesquipedale: (A) cortical cell density, (B) cortical cell volume and under RL may reﬂect the mobilization of this pho- (C) medullary cell volume. Bars represent standard errors (n =4); tosynthate to support the algal growth. Therefore, in W = White light; B = Blue light; R = Red light. G. sesquipedale, starch and polysaccharide contents are inversely related under BL, RL and YL and this

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Wen, 1984), but our results for G. sesquipedale and 60 G. tenuistipitata (authors, unpublished data) do not support such a correlation. 50 The total sulfate was markedly high in BL-grown 40 alga (8.9%). Part of this sulfate must be associated to other compounds than agar precursor repeating 30 units, since this value cannot be explained totally by the L-galactose-6-sulfate content. On the other hand, 20 RL and YL also raised the sulfate content of the aextracts to values higher than those found for Ge- 10 lidium latifolium (Grev.) Thur. et Bornet (2.08–2.6%; Mouradi-Givernaud, 1992) or for Gelidium chilense 0 (Mont.) Santelices et Montalva (2.8%; Matsuhiro & I W R B Y I W R B Y I W R B Y Starch (Molar% of polysaccharides) Urzúa, 1991) agars. In this case, the correlation between sulfate and growth rate or young tissue could Figure 6. Starch distribution (measured by NMR) in extracted fractions from G. sequipedale cultured under different light qualities. not be applied, although it has to be taken into account I = Initial sample; W = White light; R = Red light; B = Blue light; that these values represent the total sulfate content, in- Y = Yellow light. cluding the so-called ‘alkali-labile’ and ‘alkali-stable’ sulfate. Methylation of the agarobiose unit has been re- could mean that starch is involved in the biosynthetic lated to tissue age in Gracilaria tikvahiae McLachlan pathway of agar. Degradation of floridean starch has (Craigie & Wen, 1984), with an early stage during been observed in Gelidium coulteri Harv. with the biosynthesis in G. eucheumoides Harv. (Lahaye et al., + 1986) and with growth rate in G. tenuistipitata (au- addition of NH4 (Macler, 1986), in Gracilaria sordida (Harv.) Nelson in N-limited cultures and under thors, unpublished data). However, there seems to be salinity stress (Ekman et al., 1991) and in Gracilariop- no general trend as in Gelidium sesquipedale: the de- sis lemaneiformis (Bory) Dawson, Acleto et Foldvik, gree of substitution is very low, and, on the other hand, grown in the dark (Rincones et al., 1993). In the lat- under YL that produces the slowest growth of the ter case, the authors proposed that the carbon derived plants, substitution by methoxyl groups is favoured. from floridean starch degradation may be used for agar In general, our data do not show an obvious relation- synthesis. Differences in starch extraction with water ship between the chemical composition of galactans at 22 ◦C and 120 ◦C suggests the existence of two and thallus morphology in this alga. different starch pools. The one soluble at 22 ◦Cmay Algae growing at low light levels in nature are sub- consist of small fragments with a short ‘storage life- ject to a light spectrum impoverished in the red part time’, perhaps being recruited for active growth of the of the spectrum, so the laboratory data suggest that alga (and/or osmorregulation). The other one, solu- they may have a higher starch content and an agar ble at 120 ◦C, may consist of high molecular weight more sulfated and substituted with charged groups polymers and serve as a true reserve. than plants growing at higher light levels. This is im- Conversion of L-galactose-6-sulfate into 3,6- portant for economic purposes, as the light treatment anhydro-L-galactose by alkali treatment is known to applied could improve the quality and the yield of the improve the gelling properties of agar (Duckworth agar extracted. et al., 1971; Craigie et al., 1984). In the present study In conclusion, light quality affects the morphology we characterized the composition of the unmodified and the agar composition of Gelidium sesquipedale agar, both because we were interested in its native state cultured in chemostats. Further research is required in the alga and on whether a good quality agar can to determine how the spectral composition of light be obtained without alkali treatment. The degree of controls agar biosynthesis pathways. substitution of agar by L-galactose-6- sulfate, considered as a marker for the agarobiose precursor repeating unit, was higher under WL and BL than under RL and YL. The precursor content has been considered to be related to growth rate or young tissue (Craigie &

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Acknowledgments Macler BA (1986) Regulation of carbon flow by nitrogen and light in the red alga Gelidium coulteri. Plant Physiol. 82: 136–141. This work was financed by the Ministry of Educa- Matsuhiro B, Urzua CC (1991) Agars from Chilean Gelidiaceae. Hydrobiologia 221: 149–156. tion and Sciences (CYCYT projects AMB93-1211 Mouradi-Givernaud A, Givernaud T, Morvan H, Cosson J (1992) and AMB94-0684). R. Carmona is supported by a Agar from Gelidium latifolium (Rhodophyceae, Gelidiales): bio- grant from Junta de Andalucía. The authors thank chemical composition and seasonal variations. Bot. mar. 35: 153–159. Dr B. Quemener for help with sulfate analysis and Murano E (1995) Chemical structure and quality of agars from Dr A. Medina and A. Alvarez for assistance with algal Gracilaria. J. appl. Phycol. 7: 245–254. tissue inclusions. Rebello J, Ohno M, Critchley AT, Sawamura M (1996) Growth rates and agar quality of Gracilaria gracilis (Stackhouse) Steentoft from Namibia, Southern Africa. 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