Journal of Photochemistry and Photodiology, B: Biology, 4 (1989) 185 - 193 185

EFFECTS OF RED AND FAR-RED LIGHT PULSES ON THE CHLOROPHYLL AND BILIPROTEIN ACCUMULATION IN THE RED ALGA CORALLINA ELONGATA

FELIX LOPEZ-FIGUEROA+, ROSA PEREZ and F. XAVIER NIELL

Departamento de Ecologia, Facultad de Ciencias, Universidad de Mblaga, Mdlaga, 29071 (Spain)

(Received February 1,1989; accepted March 21,1989)

Keywords. Corallina elongata, chlorophyll a, phycobiliprotein, phytochrome, Rhodophyta.

Abbreviations. APC, allophycocyanin; Chl a, chlorophyll a; DW, dry weight of algae; FR, far-red light; P, photosynthetic pigments; PC, phycocyanin; PE, phycoerythrin; Pfr, far-red-absorbing form of phytochrome; Pls, light pulses; R, red light; SD, standard deviation.

Summary

The effects of red and far-red light pulses on the synthesis of photo- synthetic pigments (chlorophyll and phycobiliprotein) in the red alga Coral- lina elongata are examined. Chlorophyll a, phycocyanin and allophycocyanin synthesis is induced by red light pulses. In contrast, phycoerythrin synthesis is not induced by red light. To investigate the involvement of phytochrome successive red and far-red light pulses were applied. Since the effect of red light shows far-red reversibility, the involvement of phytochrome is pro- posed. The existence of other photoreceptors controlling chlorophyll and biliprotein synthesis is not discarded.

1. Introduction

The effect of different light qualities on photosynthetic pigment syn- thesis has been studied in algae in relation with chromatic adaptation [ 1, 21. The chromatic adaptation has been rigorously demonstrated to occur in blue-green algae [3, 41; red light (R) stimulated the phycocyanin synthesis and green light (G) stimulated the phycoerythryn synthesis [2]. A control

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186

by R and G of biliprotein accumulation in different red algae, namely Corallina elongata, Porphyra umbilicalis, and Plocamium cartilagineum has also been observed [5, 61. Bjiirn and Bjijrn [7] indicated that some red and green photoreversible photochromic pigments (termed phycochromes) should be involved in light perception in blue-green algae. However, the role of these phycochromes in the biological phenomena of blue-green algae was questioned [ 8 - 111.

However, the photocontrol of chlorophyl synthesis in red algae has not been extensively studied. Ramus et al. [12] showed that various macro- algae changed the total pigment concentration of their photosynthetic antennae and also the relative proportion of various pigments as a function of both the colour and the intensity of light. The photoregulation of Chl a synthesis has been better studied in green and brown algae than in red algae [13]. A control by blue light of Chl a synthesis in algae has been reported [ 13 - 151. While the photoreceptors controlling chlorophyll synthesis in higher plants are phytochrome and cryptochrome [ 161, the photoregulation systems in algae are not well identified [17, 181. Senger [19] proposed a control of the chlorophyll synthesis in green algae by a specific blue light photoreceptor. However, a control by both phytochrome and a blue light photoreceptor of chlorophyll synthesis has been also reported [ 201. In this work we describe the effect of R and far-red light (FR) pulses on the chloro- phyll and biliprotein accumulation in the red alga Corallina elongata.

2. Materials and methods

Adult plants of similar size to the red alga Corallina elongata Ellis et Soland were collected from rocky shores on the coast of Malaga (South of Spain) during the winter period. The pigment content of the algae did not significantly vary (P < 0.1). Thus, the material was considered to be homo- geneous. Algae were kept in sea water in the laboratory with an aquarium air system at 16 “C, in the dark for 12 h (preliminary treatment).

2.1. Light sources and radiation measurement R was provided by two red-fluorescent lamps (Silvania F 20W/R, X,,,

625 nm and HW 60 nm). FR was from a 300 W tubular halogen lamp behind a water filter (10 cm thick) to eliminate excess heat, combined with an FR glass filter (Schott SFK 17 a, X,,, 720 nm and HW 55 nm). In each case the fluence rate was 14 W m-*. Radiation was measured with a spectro- radiometer (Licor, Li-1800 VW). The relative spectral photon fluence rate of R and FR is shown in Fig. 1.

2.2. Experimental design and light treatments After the preliminary treatment indicated above, the plants (2.5 g of

fresh weight) were transferred under dim green light to vessels with 500 ml of filtered (Whatman filter GF/C, 1.2 pm in diameter) sea water. KNOs

187

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(5 pM) was added to the water before every light t rea tment since a depletion in the nitrate concentra t ion during the experimentat ion time causes a decrease in the pigment concentrat ion [5]. After transfer, R, FR and R following FR (R + FR} pulses were applied. The exposures of R and FR were 30 min and 5 min, respectively. After these pulses the plants were kept in the dark for 4 h. The pigment concentra t ion was determined before all light t reatments (at the initial time), just after light pulses and during the period in the dark (tx) at 30, 60, 120, 180 and 240 min intervals. In addi- t ion, a programme of successive R and FR pulses was applied, i.e. 5 min FR, 30 min FR, 30 min R, 30 min R + 5 min FR, 30 min R + 5 min FR + 15 min R, 30 min R + 5 min FR + 15 min R + 5 min FR (see Table 2). The sec- ond R pulse was of 15 min durat ion instead of 30 min because with this time programme (30R + 5FR + 15FR) no significant differences in the photo- synthesis rate with respect to 30R and 30R + 5FR were observed (data not shown). At least three pieces of algae longitudinally cut were taken at each time and the average pigment content was estimated. The pigment concen- trat ion was also calculated for the dark control. All t reatments were conduc- ted at 16 + 2 °C. All data are mean values of 3 - 4 replicates at each time. The standard deviation (SD) is shown in Tables 1 and 2. The range of SD was between 6% and 12%.

2.3. Pigment determination Chlorophyll a (Chl a) was determined spectrophotometr ical ly in a

Beckman DU-7 spec t rophotometer following the method proposed by Jeffrey and Humphrey [21]. This assay does no t discriminate between Chl a and Chlorophyllide a. The extract ion was made from fresh tissue with ace- tone {90%; Merck) and neutralized with Na2CO3 {Sigma) in an extract ion mortar . Phycoerythr in (PE), phycocyanin (PC) and al lophycocyanin (APC) were determined spectrophotometr ical ly using the equations introduced by

188

TABLE 1

Concentration of accumulated Chl a, PE, PC and APC, inductive effect (IE) and per cent

of reversibility (R%) in Corallina elongata after different light pulses and 4 h in the dark

Pigments Light treatment mg P (g DW)-i IE R%

Chl a 30 min R 0.5 f 0.025 0.46 f 0.04 80 f 7.2 30 min R + 5 min FR 0.13 + 0.01

5 min FR 0.04 + 0.0026

PE 30 min R 0.07 f 0.006 0.03 f 0.0025 66 + 5.7 30 min R + 5 min FR 0.05 + 0.004

5 min FR 0.04 f 0.0035

PC 30 min R 0.48 f 0.045 0.43 + 0.08 73 f 5.5 30 min R + 5 min FR 0.165 f 0.015

5 min FR 0.045 f 0.004

APC 30 min R 0.36 f 0.035 0.31 ?z 0.028 71 + 6.4 30 min R + 5 min FR 0.14 f 0.015

5 min FR 0.05 + 0.004

Rosenberg [22]. For PE X = 565,615 and 650 nm, for PC h = 615 and 650 nm, and for APC h = 650 and 615 nm were used. X = 750 nm was used as base line. The extraction was carried out with phosphate buffer (0.1 M, pH 6.5) at 4 “C. The amount of pigment is expressed in milligrams per gram of dry weight (DW) of algae.

The concentration of accumulated pigments (C) was expressed accord- ing to the following formula:

c= [P]x,t, - [P]d,t,

where [P]x,t, is the pigment concentration (chlorophyll or biliprotein) after any light treatment at time t, in the dark and [P] c&t, is the pigment concentration at the same time in the dark control.

The inductive effect of R and the per cent of reversibility of the res- ponse after FR were calculated according to Mohr [ 231.

3. Results

R pulses induced Chl a, PC and APC synthesis in a relatively short period of time in Corallina elongata (Fig. 2). The pigment concentration after R pulses was higher than after FR pulses and much higher than in the dark control (Fig. 2). However, PE is not induced by R although a partial reversibility by FR was also observed. A saturation of the Chl a concentra- tion was observed after 2 h in the dark. However, the maximum PE and APC concentration was observed after only 1 h in the dark and the maxi- mum PC concentration at 4 h. After light pulses no continuous white light seems to be necessary since a relatively rapid synthesis is observed in the dark. The induction of Chl a and PC synthesis was similar (Table 1). The

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Fig. 2. Concentration of Chl a and hiliprotein (PE, PC, APC) expressed in mg g-l of dry weight before the light treatment (at the initial time) and in the dark after different light pulses (Pls): 30 min R (full circles), 30 min R + 5 min FR (open circles), 5 min FR (open triangles). Concentration of Chl a and biliprotein in a dark control (full triangles). Fluence rate is 14 W mp2. All data are mean values of 3 - 4 replicates. The range of SD is between 6% and 12%.

per cent of reversibility after FR was greater for Chl a than for PC. The reversion was similar for PC and for APC (Table 1). For treatment with successive R and FR pulses a partial reversibility was also observed (Table 2). The concentration of accumulated pigments was greater when the last pulses applied were R than when the last pulses were FR (Table 2). Pigment concentration was higher after two red light pulses (R + FR + R) than after just one R.

4. Discussion

Our results clearly show an action of red and far-red light on the chlorophyll and biliprotein synthesis in Cordina elongatu. In addition to the red and green light regulation of phycobiliprotein synthesis observed by Lopez-Figueroa [6], a red light induction and a partial reversion by far-red light are now detected. Since phytochrome is the only red-light-absorbing sensor pigment described that mediates red and far-red photoreversible responses, phytochrome or a phytochrome-like photoreceptor should be involved in the induction of photosynthetic pigment synthesis in this red alga. This does not imply that exclusively this photoreceptor is involved. The coaction between phytochrome and a blue light photoreceptor in the Chl a induction was proposed for Corullinu elongutu [20] and a control by red and green light of biliprotein synthesis was already observed [6]. The system of reduction of protochlorophyllide cannot be responsible for this red and far-red effect in the accumulation of pigments because the protochloro-

191

phyll-chlorophyll photoconversion is irreversible [24]. The reversibility by far-red light is not complete. A small amount of Pfr could promote pigment synthesis after far-red light pulses, since it is possible to detect a small amount of Pfr after FR [25]. In addition, a rapid synthesis of effecters could induce pigment synthesis during R pulses resulting in a low level of reversion. This rapid effect is observed with successive light pulses. The pigment concentration was greater after two red pulses than after only one. In the green alga Ulva rigida and in the red alga Porphyra umbilicalis the extent of the response was dependent on the length of the light pulses [6, 261. In Corallina, the induction of chlorophyll synthesis was higher after 30 min R pulses than after 5 min R pulses and the reversion after FR pulses was greater with 5 min R than with 30 min R [6]. Thus, a fast escape from reversibility in this response has been observed [6]. Future experiments including fluence-response curves and action spectra will elucidate unequi- vocally if phytochrome is involved in this response.

The control of physiological responses in algae by phytochrome has been reported [18, 26 - 301 but at this moment the accepted idea is that chlorophyll synthesis is controlled by a specific blue light photoreceptor [4, 191 and biliprotein synthesis is stimulated by a specific green-red photo- receptor [17]. We propose that at least phytochrome could be operating. The existence of other photoreceptors controlling the pigment synthesis is not discarded. The coaction of phytochrome and a specific blue light photo- receptor in several macroalgae has already been demonstrated [ 6,20,26]. In addition to this physiological experiment, a protein of about 130 KDa from Corallina elongata has been detected immunochemically by monoclonal antibodies directed to phytochrome from etiolated Avena (ACC5) and Zea (Z3B1, Z2B3, Z4B5) [31]. Future physiological experiments in addition to biochemical and immunochemical studies should determine if the phyto- chrome-like protein detected by Lopez-Figueroa et al. [31] controls the photosynthetic pigment synthesis in red algae.

Acknowledgements

This work was supported by the Education and Science Ministry of Spain (Investigation project PR-1063/85). We thank Prof. Dr. S. E. Braslavsky for critically reading the manuscript.

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