IntroductionTop

Light is primarily used as a source of energy, enabling organisms to adapt to a steadily changing environment. The utilization of light by an organism is regulated by a process known as photoacclimation, or chromatic adaptation, including mechanisms to avoid an excess of light energy, such as photoprotection (Falkowski and La Roche 1991Falkowski P.G., LaRoche J. 1991. Acclimation to spectral irradiance in algae. J. Phycol. 27: 8-14.) and mechanisms to take advantage of low irradiances in any given ambient lighting. Light can also be used as an environmental signal to control metabolic and reproductive processes (Rüdiger and Figueroa 1992Rüdiger W., López-Figueroa F. 1992. Photoreceptors in algae. Ann. Rev. Photochem. Photobiol. 55: 949-954.). Monochromatic light can provide the signalling necessary to regulate algal metabolism, reproduction and growth (Dring 1988Dring M.J. 1988. Photocontrol of development in algae. Ann. Rev. Plant. Physiol. Plant. Mol. Biol. 39: 157-174.), prompting, in turn, a variety of photomorphogenetic strategies, such as the control of cell growth or induced enzymatic activation (Lüning 1992Lüning K. 1992. Day and night kinetics of growth rate in green, brown and red seaweeds. J. Phycol. 28: 794-803.). Additionally, light can induce shade-avoiding plasticity (Monro and Poore 2005Monro K., Poore A.G.B. 2005. Light quantity and quality induce shade-avoiding plasticity in marine macroalgae. J. Evol. Biol. 18: 426-435.).

Marine red algae occur in environments subjected to wide variations of irradiance and spectral distribution, and their pigment contents are regulated by irradiance and light qualities. These responses may be the result of photomorphogenetic signalling processes with photoreceptor control that regulate growth and metabolism (Talarico and Maranzana 2000Talarico L., Maranzana G. 2000. Light and adaptive responses in red macroalgae: an overview. J. Photochem. Photobiol. B. Biol. 56: 1-11.). Photosynthetic changes can result from an imbalance of photosystems, a difference in the rate at which photosystems turn over, a deficiency in the electron transport chain, or a major change in the spatial relationship between the two photosystem (Gantt 1990Gantt E. 1990. Pigmentation and photoacclimation. In: Cole K.M., Sheat R.G. (eds), Biology of the red algae. Cambridge University Press, Cambridge, pp 203-219.).

An increase in phycobiliprotein and chlorophyll a (Chl a) content was observed in Chondrus crispus cultivated under blue, red, and green light in comparison with white light controls (Franklin et al. 2001Franklin L.A., Kräbs G., Kuhlenkamp R. 2001. Blue light and UV-A radiation control the synthesis of mycosporine-like amino acids in Chondrus crispus (Florideophyceae). J. Phycol. 37: 257-270.). Halymenia floresii optimized its growth rates under green light, inducing Chl a and lutein synthesis, whereas blue and red light stimulated the accumulation of phycobiliproteins (Godínez-Ortega et al. 2008Godínez-Ortega J.L., Snoeijs P., Robledo D., et al. 2008. Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities. J. Appl. Phycol. 20: 253-260.). Blue light also stimulated nitrogen assimilation and the accumulation of such N-compounds as biliproteins (Figueroa et al. 1995aFigueroa F.L., Aguilera J., Niell F.X. 1995a. Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kützing (Bangiales, Rhodophyta). Eur. J. Phycol. 30: 11-18.). These alterations in red alga pigment content may be related to the size and amount of chromophores, as well as the number, structure or size of phycobilisomes, which are modified to optimize light absorption (Kursar et al. 1983bKursar T.A., Van Der Meer J., Alberte R.S. 1983b. Light-harvesting system of the red alga Gracilaria tikvahiae. II. Phycobilissome characteristics of pigment mutants. Plant Physiol. 73: 361-369., López-Figueroa and Niell 1989López-Figueroa F., Niell F.X. 1989. Red-light and blue-light photoreceptors controlling chlorophyll a synthesis in the red alga Porphyra umbilicalis and in the green alga Ulva rigida. Physiol. Plantarum. 76: 391-397., 1991López-Figueroa F., Niell F.X. 1991. Photocontrol of chlorophyll and biliprotein synthesis in seaweeds: possible photoreceptors and ecological considerations. Sci. Mar. 55: 519-527.). Furthermore, the synthesis of chlorophyll and biliprotein can be regulated by phytochrome (red/far-red light) and blue light photoreceptors (López-Figueroa and Niell 1991López-Figueroa F., Niell F.X. 1991. Photocontrol of chlorophyll and biliprotein synthesis in seaweeds: possible photoreceptors and ecological considerations. Sci. Mar. 55: 519-527.). Other responses in macroalgae have been assigned to photoreceptors, such as blue light control of reproduction in Laminaria saccharina gametophytes and hair differentiation in Scytosiphon lomentaria (Dring and Lüning 1973Dring M.J., Lüning K. 1973. Induction of two-dimensional growth and hair formation by blue light in the brown alga Scytosiphon lomentaria. Z. Pflanzenphysiol. Bd. 75: 107-117.). Blue light also induced reproductive responses of Petalonia fascia and P. zosterifolia and, in addition, erect axes were inducted by red light for these species (Lüning and Dring 1973Lüning K., Dring, M.J. 1973. The influence of light quality on the development of the brown algae Petalonia and Scytosiphon. Br. Phycol. J. 8: 333-338.). Lemanea germination was inhibited by blue light but this process was reverted by subsequent white light exposure (Thirb and Benson-Evans 1983Thirb H.H., Benson-Evans K. 1983. The effect of different light intensities and wavelengths on carpospore germination and the apical tips of the red alga Lemanea Bory (1808). Nova Hedwigia 37: 669-682.).

Gracilaria species are the main source of agar and, as such, drive a market worth millions of dollars around the world (Armisén 1995Armisén R. 1995. World-wide use and importance of Gracilaria. J. Appl. Phycol. 7: 231-243.). Gracilaria birdiae Plastino and E.C. Oliveira has shown higher potential for agar extraction, although only natural banks have been exploited (Plastino and Oliveira 2002Plastino E.M., Oliveira E.C. 2002. Gracilaria birdiae (Gracilariales, Rhodophyta), a new species from the tropical south American Atlantic with a terete frond and deep spermatangial conceptacles. Phycologia 41: 389-396.). The species has been harvested in northeast Brazil near coastal communities, and the short time interval between one harvest and the next is not sufficient to permit the complete recovery of seaweed beds (Carneiro et al. 2011Carneiro M.A.A., Marinho-Soriano E., Plastino E.M. 2011. Phenology of an agarophyte Gracilaria birdiae Plastino and E.C. Oliveira (Gracilariales, Rhodophyta) in Northeastern Brazil. Rev. Bras. Farmacogn. 21: 317-322.). This species occurs from the northeast (3°S) to the southeast (20°S) coast of Brazil (Plastino and Oliveira 2002Plastino E.M., Oliveira E.C. 2002. Gracilaria birdiae (Gracilariales, Rhodophyta), a new species from the tropical south American Atlantic with a terete frond and deep spermatangial conceptacles. Phycologia 41: 389-396.), and its extreme populations are in geographical areas 2500 km apart. Fertility is commonly observed, with abundance of tetrasporophytes in comparison with gametophytes (Carneiro et al. 2011Carneiro M.A.A., Marinho-Soriano E., Plastino E.M. 2011. Phenology of an agarophyte Gracilaria birdiae Plastino and E.C. Oliveira (Gracilariales, Rhodophyta) in Northeastern Brazil. Rev. Bras. Farmacogn. 21: 317-322.). Considering its geographic distribution and daily photoperiods (Brazilian National Institute for Space Research, http://sonda.ccst.inpe.br/basedados/index.html), G. birdiae may be exposed to photoperiods ranging from 10.8 to 13.9 h in the winter and summer solstice, respectively.

In accordance with the importance of G. birdiae, studies under controlled conditions are imperative to provide information about the biology of the species. In the laboratory, different strains of G. birdiae were isolated from different regions of the Brazilian coast, from southeastern to northeastern regions (Costa and Plastino 2001Costa V.L., Plastino E.M. 2001. Histórico de vida de espécimens selvagens e variantes cromáticas de Gracilaria sp. (Gracilariales, Rhodophyta). Rev. Brasil. Bot. 24: 491-500., 2011Costa V.L., Plastino E.M. 2011. Color inheritance and pigment characterization of red (wild-type), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta). J. Appl. Phycol. 23: 599-605., Plastino et al. 2004Plastino E.M., Ursi S., Fujii M.T. 2004. Color inheritance, pigment characterization, and growth of a rare light green strain of Gracilaria birdiae (Gracilariales, Rhodophyta). Phycol. Res. 52: 45-52.), and some studies have been performed to understand the phenotypic plasticity promoted by acclimation and adaptation processes (Ursi and Plastino 2001Ursi S., Plastino E.M. 2001. Crescimento in vitro de linhagens de coloração vermelha e verde clara de Gracilaria birdiae (Gracilariales, Rhodophyta) em dois meios de cultura: análise de diferentes estádios reprodutivos. Rev. Brasil. Bot. 24: 585-592., Ursi et al. 2003Ursi S., Pedersén M., Plastino E.M., et al. 2003. Intraspecific variation of photosynthesis, respiration and photoprotective carotenoids in Gracilaria birdiae (Gracilariales, Rhodophyta). Mar. Biol. 142: 997-1007., 2008Ursi S., Guimarães M., Plastino E.M. 2008. Deleterious effects of TRIS buffer on growth rates and pigment content of Gracilaria birdiae Plastino amd E.C. Oliveira (Gracilariales, Rhodophyta). Acta. Bot. Bras. 22: 891-896., Ayres-Ostrock and Plastino 2014aAyres-Ostrock L.M., Plastino E.M. 2014a. Effects of short-term exposure to ultraviolet-B radiation on photosynthesis and pigment content of red (wild types), greenish-brown, and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta). J. Appl. Phycol. 26: 867–879.,bAyres-Ostrock L.M., Plastino E.M. 2014b. Effects of UV-B radiation on growth rates, pigment content and ultrastructure of red (wild type), greenish-brown and green strains of Gracilaria birdiae (Gracilariales, Rhodophyta). Eur. J. Phycol. 49: 197-212.). Life history has been completed in the laboratory, but tetrasporangium differentiation was only observed with cultivation in short daylight (10L:14D) and irradiance of 100 µmol of photons m^–2 s^–1 (Costa and Plastino 2001Costa V.L., Plastino E.M. 2001. Histórico de vida de espécimens selvagens e variantes cromáticas de Gracilaria sp. (Gracilariales, Rhodophyta). Rev. Brasil. Bot. 24: 491-500.). While these studies give some indication of photoperiodic influence on the fertility of G. birdiae, tetrasporangium differentiation could be induced by variation of irradiance and specific light qualities. Therefore, since it is advantageous to understand and manage the reproduction of G. birdiae in the laboratory for future seedling production in mariculture, we designed light-controlled experiments to choose the best induction conditions of fertility in both gametophytes and tetrasporophytes. Fertility, growth and pigment content were all monitored. These parameters provided information about the relationship between fertility and vegetative performance. Moreover, a comparative analysis between the isomorphic stages of life history of the species is presented.

Materials and methodsTop

Biological material

Non-reproductive apical segments of tetrasporophytes and male and female gametophytes of Gracilaria birdiae Plastino and E.C. Oliveira were obtained from algal collections of the “Édison J. Paula” marine seaweed laboratory at the University of São Paulo, Brazil (Lourenço and Vieira 2004Lourenço S.O., Vieira A.A.H. 2004. Culture collections of microalgae in Brazil: progress and constraints. Nova Hedwigia 79: 149-173., Costa et al. 2012Costa E.S., Plastino E.M., Petti R., et al. 2012. The Gracilariaceae germoplasm bank of the University of São Paulo, Brazil a DNA barcoding approach. J. Appl. Phycol. 1: 1-11.). Different individuals from the red wild type were utilized in this study. These algae were derived from a natural population of Paracuru (3°25’31”S and 39°01’29”W), Ceará State, Brazil.

General culture conditions

Unialgal cultures were established from non-reproductive apical segments of tetrasporophytes and gametophytes of Gracilaria birdiae, as described by Plastino and Oliveira (1990)Plastino E.M., Oliveira E.C. 1990. Crossing experiments as an aid to the taxonomic recognition of the agarophyte alga Gracilaria (Rhodophyta, Gigartinales). In: Oliveira E.C., Kautsky N. (eds) Cultivation of Seaweeds in Latin American. Universidade de São Paulo, São Paulo, pp. 127-133.. Standard culture conditions were as follows: 25±2°C, alternating aeration periods of 30 minutes, 400 ml Erlenmeyer flasks, containing sterile seawater (salinity of 32) enriched with von Stosch medium (Ursi and Plastino 2001Ursi S., Plastino E.M. 2001. Crescimento in vitro de linhagens de coloração vermelha e verde clara de Gracilaria birdiae (Gracilariales, Rhodophyta) em dois meios de cultura: análise de diferentes estádios reprodutivos. Rev. Brasil. Bot. 24: 585-592.) diluted to 25%. This enriched seawater was changed weekly. Each flask contained four apices (all tetrasporophytes or two male plus two female gametophytes). The recognition of sexual stage of gametophytes was possible because these algal sections were derived from original fertile individuals, male and female gametophytes (with cystocarps). After sexual recognition, they were cultivated in different flasks for two months to avoid fertilization and to assure absence of cystocarps. In general, initial mean fresh weight biomass per flask (average±standard deviation) was 14.6±1.2 mg; if each reproductive stage was considered separately, there was 14.3±1.2 for tetrasporophytes, and 14.9±1.2 mg of fresh weight for gametophytes. Three replicates were utilized.

Experimental light treatments

Experiments were conducted over the course of nine weeks in biochemical oxygen demand culture chambers (Fanem, 347 G), with tubes positioned vertically in the door. Two light: dark cycles (L:D) were tested: 14L:10D and 8L:16D. Four light qualities were utilized: white (WH), green (GR), red (RD) and blue (BL). The different light qualities were obtained from Daylight Osram 40W tubes with four acrylic plate filters (3 mm width): transparent for WH, green for GR, red for RD, and blue for BL. Each chamber was prepared to receive only one type of acrylic plate. These plate filters were inserted at the biochemical oxygen demand culture chamber door, between the tubes and the internal environment of the culture chamber. These plates were positioned in a way that the white light emitted by Daylight tubes was filtered and only specific wavelengths could achieve the Erlenmeyer flasks where algae were cultivated. These flasks were distributed in different positions of chamber, so as to receive similar amounts of irradiance. Light transmittances of these filtering acrylic systems were obtained in a spectrophotometer (HP 8452A) by positioning a piece of each acrylic plate in the place of the cuvette. These curves are shown in Figure 1.

Irradiances were measured with a Li-COR spherical sensor (Model Li-193) connected to a Li-COR quantameter (Model Li-185). The photon fluence rate in all treatments was 350 mmol photons m^–2 s^–1. Photosynthetic responses of the species were not inhibited, as previously reported by Ursi et al. (2003)Ursi S., Pedersén M., Plastino E.M., et al. 2003. Intraspecific variation of photosynthesis, respiration and photoprotective carotenoids in Gracilaria birdiae (Gracilariales, Rhodophyta). Mar. Biol. 142: 997-1007.. To achieve this photon fluence rate, different quantities of tubes were utilized at each light quality (4 tubes for WH, 14 for GR, 6 for RD and 16 for BL).

Fertility and growth rate analysis

Branch morphology and reproductive occurrences of tetrasporangia, spermatangial conceptacles or cystocarps were evaluated weekly throughout the experimental period. The differentiation of reproductive structure was assessed with a stereomicroscope, adding light quality filters to ensure uniform exposure of algae to light quality similar to that of the experimental chamber in order to avoid contamination of any white light flash throughout the experimental period. At the end of nine weeks, total (T) and fertile (F) lengths of tetrasporophytes and male gametophytes were measured in centimetres, starting from the basal portion of the branch. Considering each flask as a replicate, T and F were obtained from the sum of the lengths of four apices (for tetrasporophytes) and two apices (for male gametophytes) contained in each flask, totalizing one value of T, F, and fertility per flask, and this value was considered as replicate. Percentage of fertility data (F%) were considered as F%=(F/T)100 to obtain a percentage. The number of cystocarps on female gametophyte thalli was also evaluated after nine weeks.

Fresh biomass was recorded weekly for nine weeks, and the growth rates (GRT) were estimated according to Lignell and Pedersén (1989)Lignell A., Pedersén M. 1989. Agar composition as a function of morphology and growth rate. Studies on some morphological strains of Gracilaria secundata and Gracilaria verrucosa (Rhodophyta). Bot. Mar. 32: 219-227.: GRT=[(W_f/W_i)^1/t–1]100, where W_f is the final measure, W_i is the initial value of fresh weight, and t is the time. GRT unit was % day^–1. Data for statistical analyses and comparisons were selected considering periods where these values remained constant regardless of the time. These were observed between the third and sixth weeks for all conditions and included the same quantity of days.

Pigment analysis

Pigment analysis was performed upon completion of the experimental period. Two hundred mg of tissue from tetrasporophytes and gametophytes cultivated in different treatments were ground with liquid nitrogen, followed by addition of 50 mM phosphate buffer, pH 5.5. Crude extracts were centrifuged at 36000 g for 25 min to obtain phycobiliproteins. Chl a was extracted after dissolving the pellet in 90% acetone and centrifuging at 12000 g for 15 min (Kursar et al. 1983aKursar T.A., Van Der Meer J., Alberte R.S. 1983a. Light-harvesting system of the red alga Gracilaria tikvahiae. I. Biochemical analyses of pigment mutations. Plant Physiol. 73: 353-360., modified by Plastino and Guimarães 2001Plastino E.M., Guimarães M. 2001. Diversidad intraespecífica. In: Alveal K., Antezana T. (eds) Sustentabilidad de la biodiversidad. Universidad de Concepción, Concepción, pp. 19-27.).

Pigments were quantified in a spectrophotometer (HP 8452A), and concentrations of phycobiliproteins (allophycocyanin [APC], phycocyanin [PC] and phycoerythrin [PE]) and Chl a were calculated according to Kursar et al. (1983a)Kursar T.A., Van Der Meer J., Alberte R.S. 1983a. Light-harvesting system of the red alga Gracilaria tikvahiae. I. Biochemical analyses of pigment mutations. Plant Physiol. 73: 353-360. and Jeffrey and Humphrey (1975)Jeffrey W., Humphrey G.F. 1975. New spectrophotometric equations for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural phytoplankton. Biochem. Physiol. Pflanzen. (BPP) Bd. 167: 191-194., respectively.

Statistical analysis

Three-way analyses of variance (ANOVA) were performed on all data (independent variables: light quality, reproductive stage, and photoperiod). Homogeneity of variances (Cochran test) and normality were evaluated previously for ANOVA procedures. Where significant differences were detected, post hoc multiple comparisons were made using the Newman-Keuls test. Confidence interval (CI) utilized was at 95% (p<0.05) (Zar 1999). Fertility percent data were adjusted with arcsine data transformation. A Pearson correlation analysis was performed between growth rates and fertility values.

ResultsTop

Apices of tetrasporophytes and gametophytes of Gracilaria birdiae cultivated in RD light became flattened after nine weeks (Fig. 2). Otherwise, those cultivated in WH, GR, and BL light remained filled, irrespective of photoperiod. In general, algae cultivated in WH and GR showed greater size and became more branched than those cultivated in RD or BL light (data not shown).

Fertility

Tetrasporangium differentiation was observed in thalli exposed to GR, RD, and BL light, irrespective of photoperiod. Otherwise, tetrasporangia were only observed in WH light when algae were cultivated in 8L:16D (Fig. 3). Under this photoperiod, the number of fertile tetrasporophytes among a total of 12 for each condition was 11 in WH, 8 in GR, 12 in RD, and 10 in BL. For the 14L:10D photoperiod, there were 10 in GR, 12 in RD, and 4 in BL.

Neither spermatangial conceptacles nor cystocarps were observed in gametophytes cultivated in WH light, but they could be seen in other conditions (Fig. 3). Under a photoperiod of 8L:16D, the number of fertile male individuals among a total of six for each condition was 1 in GR and 3 in the BL; for the 14L:10D photoperiod, there were 5 in GR, 4 in RD, and 6 in BL. Under a photoperiod of 8L:16D, the number of individuals with cystocarps among a total of 6 for each condition was one individual with only one cystocarp in BL; for the 14L:10D photoperiod, there were 4 in GR with a total of 10 cystocarps and 2 in RD with a total of 8 cystocarps.

Fertility percentage was significantly influenced by light quality and reproductive stage, while no effects were observed as a consequence of photoperiodic treatments (Table 1). Tetrasporophytes cultivated in RD showed a higher fertility percentage than those cultivated in WH, GR, and BL light, irrespective of photoperiod (Fig. 3). After nine weeks, tetrasporangia occupied the thalli in greater numbers than spermatangial conceptacles in all culture conditions (Fig. 3), with the exception of three conditions in which the occupation was similar: BL (both photoperiods) and GR (14L:10D).

Growth rates

Growth rates were influenced by all analysed factors and all of their interactions. However, the main factor causing differences in this parameter was photoperiod (50.73% of variance), followed by light quality (30.56% of variance). Although significant, interactive effects represented less than 8% of growth rate variations (Table 1). Tetrasporophytes showed higher GRT than gametophytes when cultivated under 14L:10D photoperiod in WH and GR light (Fig. 4). However, no differences were observed in other conditions. Algae cultivated in WH and GR light showed higher GRT than those cultivated in RD and BL, irrespective of photoperiod (Fig. 4). Compared with algae cultivated in WH and GR light, the growth rate in 14L:10D was higher than that observed in 8L:16D, whereas algae cultivated in RD and BL showed a similar GRT in both photoperiods (Fig. 4). In 14L:10D, the GRTs of algae cultivated in WH and GR light were similar, whereas in 8L:16D, the GRTs were higher in WH than in GR light.

Tetrasporangium differentiation and growth rates showed a significant negative correlation (p<0.05) in algae cultivated in GR and RD light (8L:16D: GR, r=–0.67; RD, r=–0.78; and 14L:10D: GR, r=–0.63; RD, r=–0.82). However, differentiation of spermatangial conceptacles or cystocarps showed no correlation with growth rates in any condition (p>0.05).

Pigment content

Light quality was the main factor that promoted variation in the content of accessory pigments in G. birdiae. More than 50% of variations in APC, PE and PC content were observed as a result of these treatments (Table 1). PE and PC contents were also influenced by the isolated photoperiod factor. Algae cultivated in different light qualities showed higher PC and PE contents in the 8L:16D photoperiod than algae cultivated in 14L:10D (Fig. 5). Considering each photoperiod, algae cultivated in BL showed higher PC and PE contents than those of algae exposed to WH, GR, and RD light. In 14L:10D and 8L:16D, PC and PE contents of algae cultivated in GR were higher than those under WH and RD. Furthermore, in 8L:16D, tetrasporophytes exposed to GR and RD showed higher PC content than tetrasporophytes cultivated in WH light. In general, APC content was also higher in BL and GR-cultivated samples. The highest amount was recorded for gametophytes cultivated in 8L:16D and tetrasporophytes cultivated in 14L:10D, both of which showed higher APC content under BL light than either gametophytes or tetrasporophytes exposed to WH, GR, and RD under both photoperiods (Fig. 5).

Photoperiod was the principal factor affecting Chl a content, although this pigment content was also influenced by light quality (Table 1). Algae cultivated in 8L:16D showed a higher Chl a content than algae cultivated in 14L:10D, irrespective of light quality and reproductive stage, with the exception of tetrasporophytes exposed to WH, which showed similar Chl a contents in both photoperiods (Fig. 5). Individuals cultivated in 8L:16D showed a higher content of Chl a when cultivated in GR and BL than when cultivated in WH and RD, whereas in 14L:10D only tetrasporophytes cultivated in GR had a higher Chl a content than those cultivated in WH and RD.

Reproductive stages did not seem to cause many significant variations in the pigment content of G. birdiae. This factor caused only a few variations in APC, PC and PE concentrations, i.e. only 5.05%, 3.38% and 0.41% of variation (Table 1). With few exceptions, detailed analyses showed the absence of pigment content differences between tetrasporophytes and gametophytes cultivated in the same conditions: i) in GR, tetrasporophytes had higher Chl a content than gametophytes (14L:10D) and ii) in BL, tetrasporophytes had higher APC, PC and PE contents than gametophytes (14L:10D), while gametophytes had higher APC, PC and PE contents than tetrasporophytes (8L:16D) (Fig. 5). Furthermore, interaction of factors was also significant for causing pigment content variations, although the highest percentage of variance was observed for interaction of photoperiod and reproductive stage (4.81%). Other factor interactions were reported, mainly involving APC, PE and Chl a contents (Table 1).

DiscussionTop

In this study, it is shown that both light quality and photoperiod influenced the differentiation of reproductive structures in Gracilaria birdiae. A stimulatory effect was mainly observed under RD light, since all tetrasporophytes showed changes in apical tips, as well as a higher percentage of fertility. Tetrasporangium development, as well as apical tip changes, could be regulated by red/far-red light phytochrome-like photoreceptors because these modifications were observed with RD light, although to confirm this hypothesis, it is necessary to show a reversion of the response by far-red light. Phytochrome-like photoreceptors have been identified in different algal species, including red macroalgae (López-Figueroa et al. 1989López-Figueroa F., Lindemann P., Braslavsky S.E., et al. 1989. Detection of a phytochrome -like protein in macroalgae. Bot. Acta. 102: 178-180.). These possible photoreceptors in G. birdiae can also be more active in short-day conditions (8L:16D), in which the highest percentage of tetrasporangium occurred. Considering its geographical distribution, G. birdiae occurs under different photoperiods, within a narrow range of 10.8 to 13.9 hours of light by day, depending on the latitudinal position. Thus, the photoperiod can influence reproductive status of tetrasporophytic thalli, even if not exposed exactly to 8L:16D in nature. Under these circumstances, the photoperiod could explain the frequency variation of reproductive stages observed in natural populations of G. birdiae by Carneiro et al. (2011)Carneiro M.A.A., Marinho-Soriano E., Plastino E.M. 2011. Phenology of an agarophyte Gracilaria birdiae Plastino and E.C. Oliveira (Gracilariales, Rhodophyta) in Northeastern Brazil. Rev. Bras. Farmacogn. 21: 317-322.. These authors observed higher tetrasporophyte percentages during the winter months when photoperiods are shorter than in summer months.

The high occurrence of tetrasporangia in G. birdiae was negatively correlated with GRT, as previously observed in other Gracilaria species, in which lower GRTs were reported in both fertile tetrasporophytes (Santelices and Varela 1995Santelices B., Varela D. 1995. Regenerative capacity of Gracilaria fragments: effects of size, reproductive state and position along the axis. J. Appl. Phycol. 7: 501-506.) and cystocarpic plants (Guimarães et al. 1999Guimarães M., Plastino E.M., Oliveira E.C. 1999. Life history, reproduction and growth of Gracilaria domingensis (Gracilariales, Rhodophyta) from Brazil. Bot. Mar. 42: 481-486.). These results could be related to an energy transfer of growth to reproductive structure differentiation, and in G. birdiae it was essentially stimulated by RD light. This light quality also promoted sorus differentiation in Dictyota dichotoma, as well as lower GRT, as reported by Müller and Clauss (1976)Müller S., Clauss H. 1976. Aspects of photomorphogenesis in the brown alga Dictyota dichotoma. Zeitschrift. fur Pflanzen. 78: 461-465.. These authors suggested that these responses could have also occurred due to the absence of BL spectra, resulting in a reduction in photosynthetic rates. Moreover, according to the action spectra for photosynthesis in red algae, photosynthetic activity is lower under BL than under RD, owing to the low efficiency of electron transport in photosystem II under BL light because most chlorophyll (about 85%) in red algae is located in photosystem I (Lüning and Dring 1985Lüning K., Dring M.J. 1985. Action spectra and spectral quantum yield of photosynthesis in marine macroalgae with thin and thick thalli. Mar. Biol. 87: 119-129., Grzymski et al. 1997Grzymski J., Johnsen G., Sakshug E. 1997. The significance of intracellular self-shading on the bio-optical properties of brown, red and green macroalgae. J. Phycol. 33: 408-414.). By this logic, the absence of BL spectra could stimulate the investment of light energy for reproduction instead of photosynthesis and, consequently, growth in tetrasporophytes of G. birdiae. Otherwise, cystocarps and spermatangial conceptacles were not differentiated in sufficient amounts to promote the decrease of GRT, and their differentiation could not be related to any specific wavelength. The higher fertility of tetrasporophytes in comparison with gametophytes agrees with the phenology of G. birdiae (Carneiro et al. 2011Carneiro M.A.A., Marinho-Soriano E., Plastino E.M. 2011. Phenology of an agarophyte Gracilaria birdiae Plastino and E.C. Oliveira (Gracilariales, Rhodophyta) in Northeastern Brazil. Rev. Bras. Farmacogn. 21: 317-322.). Moreover, tetrasporophytes were much more abundant and efficient at occupying environmental niches than gametophytes, indicating a greater ability to respond to environmental signals as inductors of fertility, in turn resulting in higher propagation of the species.

Growth rates of Gracilaria birdiae cultivated in GR light were similar to those observed in algae subjected to WH light and 14L:10D. This response could have resulted from the absorption of green light by phycobiliproteins, which transferred the collected energy to essential pigments, thereby sustaining the performance of the photosynthetic apparatus (Lüning and Dring 1985Lüning K., Dring M.J. 1985. Action spectra and spectral quantum yield of photosynthesis in marine macroalgae with thin and thick thalli. Mar. Biol. 87: 119-129.). Similar GRTs in GR and WH lights were also observed in Gracilaria sp. (Beer and Levy 1983Beer S., Levy I. 1983. Effects of photon fluence rate and light spectrum composition on growth, photosynthesis and pigment relations in Gracilaria sp. J. Phycol. 19: 516-522.).

The response of photoacclimation in Gracilaria birdiae to GR light can also be related to a change in phycobilisomal structure, as reported in GR-acclimated Phormidium sp., in which phycobilisomes were twice those observed in RD light (Ohki and Fujita 1992Ohki K., Fujita Y. 1992. Photoregulation of phycobilisome structure during complementary chromatic adaptation in the marine cyanophyte Phormidium sp. C86. J. Phycol. 28: 803-808.). Phycobiliproteins in phycobilisomes are usually connected by photosystem II, and they can carry energy to photosystem I, thus optimizing energy absorption and increasing photosynthetic rates. In photosystem I, an elevated content of carotenoids can play a similar role (Figueroa et al. 1995aFigueroa F.L., Aguilera J., Niell F.X. 1995a. Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kützing (Bangiales, Rhodophyta). Eur. J. Phycol. 30: 11-18.). The increase of phycobiliproteins that occurred in G. birdiae cultivated under BL and GR light could be related to improved energy absorption, which is transferred to Chl a to make up for the absence of other light qualities that are usually absorbed by chlorophyll. The same observation was shown in Porphyra leucosticta (Tsekos et al. 2002Tsekos I., Niell F.X., Aguilera J., et al. 2002. Ultrastructure of the thallus stage of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light. Phycol. Res. 50: 251-264.). Additional evidence of this optimization of light energy and possible rearrangement of phycobilisome structure in G. birdiae was achieved by the same proportion of pigment content that could be seen in algae cultivated in WH, GR, and RD light, irrespective of photoperiod, although the amounts of these pigments were higher in GR-cultivated samples. This aspect shows an evident induction of increasing phycobiliproteins and Chl a in G. birdiae cultivated in GR light.

While GR light was efficiently utilized in G. birdiae, BL light hindered its optimal development, but this light quality was also essential for maximal productivity of other alga species, such as brown algae, stimulating saturated photosynthesis as a result of the transport of inorganic carbon from seawater to chloroplasts (Forster and Dring 1994Forster R.M., Dring M.J. 1994. Influence of blue light on the photosynthetic capacity of marine plants from different taxonomic, ecological and morphological groups. Eur. J. Phycol. 29: 21-27.). For G. birdiae, however, any kind of response related to the positive effects of BL could be compromised by continuous exposure, and the absence of other essential light qualities minimized any positive effect. Higher GRTs under GR light than under RD or BL light were previously reported in Palmaria palmata (Sagert and Schubert 1995Sagert S., Schubert H. 1995. Acclimation of the photosynthetic apparatus of Palmaria palmata (Rhodophyta) to light qualities that preferentially excite photosystem I or photosystem II. J. Phycol. 31: 547-554.) and Halymenia floresii (Godínez-Ortega et al. 2008Godínez-Ortega J.L., Snoeijs P., Robledo D., et al. 2008. Growth and pigment composition in the red alga Halymenia floresii cultured under different light qualities. J. Appl. Phycol. 20: 253-260.). In the latter case, it was suggested that the activity of Chl a, which accumulated under GR, increased the efficiency of light absorption. These types of response to BL and GR light could be considered as physiological acclimation in G. birdiae, similarly to Mazzaella laminarioides (Marquardt et al. 2010Marquardt R., Schubert H., Varela D.A., et al. 2010. Light acclimation strategies of three commercially important red algal species. Aquaculture 299: 140-148.). These authors showed that macroalgae can follow physiological versus morphological photoacclimation strategies in response to variations in both irradiance and light quality (Marquardt et al. 2010Marquardt R., Schubert H., Varela D.A., et al. 2010. Light acclimation strategies of three commercially important red algal species. Aquaculture 299: 140-148.).

The accessory pigment synthesis in Gracilaria birdiae was stimulated by BL light. This response could be controlled by cryptochrome-like photoreceptors, in a manner similar to that suggested for the inhibition of erect axis development in Scytosiphon lomentaria (Dring and Lüning 1975Dring M.J., Lüning K. 1975. A photoperiodic response mediated by blue light in the brown algae Scytosiphon lomentaria. Planta (Berl.) 125: 25-32.). Moreover, G. birdiae could have photoacclimated under BL, resulting in a package effect, as reported in Porphyra umbilicalis (Figueroa et al. 1995aFigueroa F.L., Aguilera J., Niell F.X. 1995a. Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kützing (Bangiales, Rhodophyta). Eur. J. Phycol. 30: 11-18.). The package effect is related to the absorption of Chl a content and can be attributed to four factors: (1) changes in cell size, shape, or morphology; (2) changes in chloroplast structure; (3) changes in the degree of stacking of thylakoid membranes; and (4) changes in optical properties in thylakoid membranes (Berner et al. 1989Berner T., Dubinsky Z., Wyman K., et al. 1989. Photoadaptation and the “package” effect in Dunaliella tertiolecta (Chlorophyceae). J. Phycol. 25: 70-78.). For G. birdiae, we have suggested a proportionally greater increase of the external part of phycobilisome in BL in contrast to RD, thus increasing the potential for a possible package effect, much like P. umbilicalis under BL light. Considering the high PE and PC contents in G. birdiae, a higher BL-enhanced production in pigment proteins is suggested, similar to Chondrus crispus (López-Figueroa 1991López-Figueroa F. 1991. Red, blue and green photoreceptors controlling chlorophyll, biliprotein and total protein synthesis in the red alga Chondrus crispus. Br. Phycol. J. 26: 383-392.) and P. umbilicalis (Figueroa et al. 1995aFigueroa F.L., Aguilera J., Niell F.X. 1995a. Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kützing (Bangiales, Rhodophyta). Eur. J. Phycol. 30: 11-18.). BL could also stimulate the accumulation of nitrogen compounds in G. birdiae, such as PE and PC, and the resultant higher density of phycobilisomes under BL than that observed under RD and GR, as reported in P. leucosticta (Tsekos et al. 2002Tsekos I., Niell F.X., Aguilera J., et al. 2002. Ultrastructure of the thallus stage of Porphyra leucosticta (Rhodophyta) grown in red, blue and green light. Phycol. Res. 50: 251-264.). Because of the relatively rapid accumulation of PE, this photosynthetic pigment has been suggested as an N-store compound, as in cyanobacteria and red macroalgae (Lapointe et al. 1984Lapointe B.E., Dawes C.J., Tenore K.R. 1984. Interactions between light and temperature on the physiological ecology of Gracilaria tikvahiae (Gracilariales, Rhodophyta). II. Nitrate uptake and levels of pigments and chemical constituents. Mar. Biol. 80: 171-178., Figueroa et al. 1995aFigueroa F.L., Aguilera J., Niell F.X. 1995a. Red and blue light regulation of growth and photosynthetic metabolism in Porphyra umbilicalis (L.) Kützing (Bangiales, Rhodophyta). Eur. J. Phycol. 30: 11-18.).

Since the condition of constant light quality is unusual to its development, Gracilaria birdiae could have utilized its possible photoprotection mechanisms to survive under these conditions. Two examples can be given. First, the high amount of PE in G. birdiae cultivated under BL could act as a system of energy dissipation, together with a possible xanthophyll cycle, a phenomenon which has been related to energy dissipation (Demmig-Adams 2003Demmig-Adams B. 2003. Linking the xanthophyll cycle with thermal energy dissipation. Photosynt. Res. 76: 73-80.). The occurrence of this cycle in G. birdiae was suggested by Ursi et al. (2003)Ursi S., Pedersén M., Plastino E.M., et al. 2003. Intraspecific variation of photosynthesis, respiration and photoprotective carotenoids in Gracilaria birdiae (Gracilariales, Rhodophyta). Mar. Biol. 142: 997-1007., and an incomplete xanthophyll cycle was proposed for G. tenuistipitata by Barufi et al. (2011)Barufi J.B., Korbee N., Oliveira M.C., et al. 2011. Effects of N supply on the accumulation of photosynthetic pigments and photoprotectors in Gracilaria tenuistipitata (Rhodophyta) cultured under UV radiation. J. Appl. Phycol. 23: 457-466.. A second photoprotective mechanism involves the position of different phycobiliproteins in the phycobilisome. G. birdiae, in general, showed a lower variation of APC content than that of other phycobiliproteins (PC and PE), indicating that APC is located in the innermost part of the phycobilisome, in accordance with phycobilisomal models suggested by Gantt (1990)Gantt E. 1990. Pigmentation and photoacclimation. In: Cole K.M., Sheat R.G. (eds), Biology of the red algae. Cambridge University Press, Cambridge, pp 203-219.. Thus, whereas APC is protected against degradation by any specific wavelength, the other more externally located phycobiliproteins are at higher risk for degradation by any given wavelength.

The changes in apical tips of Gracilaria birdiae were probably a consequence of continuous RD. This is a typical case of morphological photoacclimation, such as those presented by Marquardt et al. (2010)Marquardt R., Schubert H., Varela D.A., et al. 2010. Light acclimation strategies of three commercially important red algal species. Aquaculture 299: 140-148. for Gracilaria chilensis. However, we cannot directly associate these changes with RD because this condition also increased tetrasporangium development. These modifications in apical portions of the thalli could be an effect of interaction between radiation and reproductive status. In addition, a decrease in GRT was observed in this condition. It is not possible to ascribe a deleterious effect to RD because no alga mortality was observed, and mortality was considered imperative to characterize a deleterious effect (Gerard 1997Gerard V.A. 1997. Environmental stress during early development of kelp sporophytes (Laminaria saccharina): how long do effects persist? J. Appl. Phycol. 9: 5-9.). Nonetheless, further studies are required to clarify this issue, as well as any kind of reversibility of morphological changes, such as the transfer to algae to less stressful conditions after treatment under RD light.

Based on the light qualities tested, we provide indications of physiological mechanisms which allow improvements of possible mariculture and commercial exploitation approaches of G. birdiae. Although the photoperiod 8L:16D is not achieved in environmental distribution of the species, it is interesting to test the physiological capacity in broader photoperiodic conditions than those in nature to evaluate the potential capacity for a possible biotechnological application. Therefore, we suggest to test also usage of 10L:14D following the algal distribution pattern in future experiments. Our results for 8L:16D can be analysed as indications of short-day regulated responses. In addition, we have indicated RD light quality as a possible inductor of fertility to produce tetraspores, which could be used as seedlings. Otherwise, we have shown the negative effects of BL light exposure on GRT of G. birdiae. However, if the aim is to achieve high phycoerythrin content, the exposure to this light quality could be a good alternative. Furthermore, a combination of different wavelengths could allow better economic resource exploitation.

AcknowledgementsTop

This research was supported by the São Paulo Research Foundation (FAPESP, 98/11943-1; 02/04987-0) and the Brazilian National Council for Scientific and Technological Development (CNPq; 300148/93-3). We thank Rosário Petti for providing technical support.

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