Light Quality Affects Morphology and Polysaccharide Yield and Composition of Gelidium Sesquipedale (Rhodophyceae)
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Journal of Applied Phycology 10: 323–331, 1998. 323 © 1998 Kluwer Academic Publishers. Printed in the Netherlands. 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 M´alaga, 29071 M´alaga, Spain 2Departamento de Ecolog´ıa, Facultad de Ciencias del Mar, Universidad de C´adiz, 11510 Puerto Real, C´adiz, 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 de- termined 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- japh576.tex; 13/11/1998; 1:25; p.2 325 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.