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Photoregulation of Nitrogen Metabolism and Protein Accumulation in the Red Alga Corallina elongata Ellis et SolandF. L. FigueroaDepartamento de Ecologia, Facultad de Ciencias, Universidad de Malaga, Campus Universi- tario de Teatinos s/n, E-29071 Malaga, SpainZ. Naturforsch. 48c, 788-794 (1993); received June 15/June 29, 1993Blue-Light Photoreceptor, Corallina elongata, Glutamine Synthetase, Light Quality, Nitrate Reductase

Red and blue light pulses of 5 min applied together with 45 (aM K N 0 3 stimulated the nitrate uptake and reduction and the assimilation of ammonium in darkness in the red alga Corallina elongata. Nitrate reductase and glutamine synthetase activities were increased in darkness after the application of both red and blue light pulses. Red light produced a dramatic increase in enzyme activities after the first hour in darkness but after 4 h the effect of blue light pulses was greater. The photostimulation of nitrogen metabolism was correlated with light-regulated accumulation of soluble proteins. Nitrogen incorporation, assimilation of ammonium, accu­mulation of total proteins and the increment in total intracellular nitrogen was greater in N-limited algae (C :N = 17.3) than in N-sufficient algae (C :N = 10.3) after the application of light pulses and nitrate. Because the stimulant effects of red and blue light on N-metabolism were partially reversed by far-red light, the possible involvement of the photoreversible red/ far-red photoreceptor, phytochrome, is proposed. In addition, the blue-light effect seems to be mediated by a specific B-light photoreceptor besides phytochrome due to the different time course of the response and the extent of the stimulation after blue compared to red light pulses.

Introduction

It has been proposed that chlorophyll, bili­protein and total protein synthesis are controlled by phytochrome, a blue-light photoreceptor and a green-light photoreceptor in various N-limited algae [1-5], The photoregulation of pigment and protein synthesis was related to the photocontrol of nitrogen metabolism [4, 6 ]. The steps of light regulation, however, have still not been com­pletely defined. Several steps could be regulated by light quality, namely, (a) uptake of nitrate, (b) nitrate reductase (NR) activity by activation of inactivated enzymes and/or by stimulation of de novo biosynthesis, and (c) assimilation of ammo­nium by the stimulation of glutamine synthetase (GS) and glutamate synthase activities (GOGAT).

Recently, it has been shown that the main step of photoregulation by phytochrome of soluble protein synthesis in Ulva rigida was the nitrate net uptake and the reduction of nitrate caused by the photostimulation of nitrate reductase activity [6 ]. Coaction of a blue-light photoreceptor and phyto­chrome has been shown in these processes as well

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as in protein accumulation [3, 6 ]. The greatest effect of red and blue light pulses on protein and amino acid accumulation corresponded to sub­strate level; more ammonia was available after red than after blue light pulses for protein synthesis. In the green algae Chlamydomonas reinhardii [7] and Monoraphidium braunii [8 ] blue light activated three key steps in the inorganic nitrogen metabo­lism, namely, (a) cell uptake in both nitrate of ni­trite, (b) nitrate reductase activity, and (c) biosyn­thesis of nitrite reductase. Furthermore, in Chlo­rella fusca blue light activated the nitrate specific permease [9],

The aim of this work is to investigate the effect of light quality on the inorganic nitrogen me­tabolism in algae previously incubated under N-limited (C :N =17.3) and N-sufficient condi­tions (C:N = 10.3) in order to relate the possible stimulation of N 0 3~ net uptake and reduction and ammonium assimilation with light-regulated accu­mulation of proteins.

Red and blue light pulses followed by far-red light were applied in order to define the possible action of phytochrome and a B-light photorecep­tor and to avoid the interference of photosynthe­sis. Biochemical evidence o f the existence of phyto­chrome-like protein but not of a B-light photore­ceptor has been reported in C. elongata [10, 11].

F. L. Figueroa • Control of N-Metabolism by Light Quality in Corallina 789

Materials and Methods

The red alga Corallina elongata Ellis et Soland was harvested in early spring and autumn from rocky shores along the coast o f Malaga (Mediter­ranean sea, Spain). Then, they were maintained in seawater containing a low level of nitrate ( 2 |im NCV) at a temperature of 16 °C and a cycle of16 h white light (photon fluence rate:80 |imol m -2

s_1): 8 h darkness. After 3—4 days in these condi­tions, the algae collected in spring had a C : N level of 10.3, corresponding 263 ± 27 mg C gDW ' 1 and 25.5 ± 2 .2 mg N gDW ' 1 (N-sufficient algae) and the algae collected in autumn reached a C : N level of 17.3 corresponding to 156 ± 13 mg C gDW“' and 9.0 mg N g D W 1 (N-limited algae).

Light treatments and light sources

After the pretreatment cited, the algae were transferred under green “safelight” into artificial seawater ASP-12 medium [12] and then 5 min red (R) and 5 min blue (B) light pulses together with 45 nm K N 0 3 (834 jig N gDW -1) were applied. In order to study the possible involvement of phyto­chrome, far-red (FR) light pulses were applied im­mediately after R and B light pulses. Red light was obtained using General Electric, 20 W R fluores­cent lamps filtered through a red plexiglass filter (PG 502) and blue light using General Electric, 20 W B fluorescent lamps fitted with two B plexi­glass filters (PG 627 and PG 602) (Fig. 1). Far-red

WAVELENGTH [ nm ]

Fig. 1. Spectral photon fluence rate (SPFR) of the differ­ent light qualities used. B = blue light, R = red light, and FR = far-red light. The photon fluence rate of R and B was 16.5 (imol m ~2 s_1 and of FR 25 |imol m ~2 s_l.

(FR) light was obtained from Linestra lamps (Os­ram, NU 4 20 W) filtered through a red plexiglass filter (PG 501) and 2 blue plexiglass filters (PC 627) (Fig. 1). The photon fluence rate for R and B light, measured with a Li-1800 UW spectro- radiometer, was 16.5 (imol m ~2 s“ 1 and of FR light was 25 ^mol n r 2 s"1. After the application of light pulses, the algae were incubated in darkness (D) for 4 h. The algae were also incubated in darkness after the application of 45 jim N 0 3~ and no light pulses (dark control).

N O f , NOi~ andN H 4+ measurements

All inorganic N-compounds were analyzed on a Technicon Autoanalyzer. The nitrate uptake was measured in darkness as compound disappearing from the medium containing 45 |im K N 0 3. N 0 2~ was determined colorimetrically from the medium by using sulfanilamide and N(l-naphthylethylene- diamine) HC1 method [13]. N 0 3~ plus NO,~ was measured as nitrite formed by reduction with a re- ductor Cadmium-column [13]. Nitrate was calcu­lated as the difference of these two values. NH4+ was measured colorimetrically from the medium by using sodium salicylate, sodium nitroprusside and C1“ in alkaline medium [14],

NR and GS assay

Nitrate reductase activity was measured in dark­ness by the in situ method [15] using 0.25 g FW al­gae per 5 ml of assay phosphate buffer pH 8.0 con­taining 0.05 m M EDTA-Na2, 0.1% 1-propanol 20 m M K N 0 3 and 0.01 m M Glucose. The enzyme activity expressed as imol NO,-produced gDW “ 1

h-1.Glutamine synthetase was measured by trans­

ferase assay [16] in vitro in the buffer described below for protein extraction. The GS activity is expressed by nmol of y-glutamyl hydroxamate produced per gDW -1 h_l.

Protein, carbon and nitrogen measurements

Soluble proteins were extracted at 4 °C in a buf­fer adjusted to pH 8.0 containing 50 m M Trizma hydrochloride (Tris [hydroxymethyl] aminometh- ane hydrochloride), 2 m M ethylenediaminetetra- acetic disodium salt (EDTA-Na2), 10 m M 2-mer- captoethanol, 10% (v/v) glycerol and 2 m M phen- ylmethylsulfonylfluoride (PMSF). After centrifu-

790 F. L. Figueroa ■ Control of N-Metabolism by Light Quality in Corallina

gation at 10,000 x g for 25 min, the soluble pro­teins were determined in the supernant spectro- photometrically by the method based on the principle of protein-dye binding by Comassie Blue G-250 [17].

At the end of the dark period the total intracel­lular C and N contents were estimated by the Per- kin-Elmer Autoanalyzer 240-C.

Statistics

The results are expressed as the mean value ± standard deviation (SD) from 8 samples. 4 repli­cate samples were collected on each occasion and the experiment was repeated twice. The signifi­cance of the means of all data were tested by Fischer’s protected LSD procedure followed by Model 1 one-way ANOVA.

Results and Discussion

Nitrate net uptake was clearly affected by light quality in Corallina elongata in algae previously in­

cubated under N-limited conditions (C:N = 17.3) (Fig. 2). Nitrate net uptake at all times was signifi­cantly different at 0.05 level in R and B light treat­ments related to FR light treatment and dark con­trol. FR light reversed partially the effects of R and B light. Throughout the period of darkness, si­multaneous uptake and excretion of nitrate were observed (Fig. 2): a rapid initial net uptake in the first 15 min, excretion between 3 0 m in - lh and net uptake in the last 2 h. In the dark control, 32 ± 3.5% of the applied nitrate (45 jim N 0 3 ) dis­appeared from the medium within 4 h, while, after R and B light pulses, the decrease in nitrate was far greater (65-75 ± 5% of the applied nitrate).

Ammonium net uptake did not occur in dark control and after R + FR and FR light pulses (Fig. 3). NH4+ uptake after 4 h in darkness was significantly greater (p<0.01) after B than after R light treatments. Both R and B effects were par­tially reversed by far-red light (Fig. 3).

Thus, net nitrate uptake was stimulated by R and B light pulses. Meanwhile, the incorporation

FR

B+FR

B

Fig. 3. Release and uptake of ammonium, expressed as |iM NH4+, in darkness after the same treatment as Fig. 2. The initial level of C : N was 17.3 (N-limited algae). Criti­cal difference values (Fisher PLSD) at 0.05 significance level at 0.15 h (0.57), 0.5 h (0.61), 1 h (0.44), 2 h (0.68), 3 h (0.46), 4 h (0.98). LP = Light pulses.

DarkR+FR

LP + 45 LlM nitrate , .Time m darkness

Fig. 2. Uptake of nitrate expressed as |iM N 0 3~ disap­pearing from the medium, in darkness, after the applica­tion of red (R), blue (B), far-red (FR), R + FR and B + FR light pulses (LP) of 5 min and 45 jim KNO,. The initial level of C :N was 17.3 (N-limited algae). Critical difference values (Fisher PLSD) at 0.05 difference level at 0.15 h (2.80), 0.5 h (2.64), 1 h (3.34), 2 h (2.93), 3 h (2.43), 4 h (2.53). LP = Light pulses.

LP + 45 uM nitrate , , , ,n Time in darkness [ h ]

^ 401 cno£ 30s =*• ™

B+FR

FR

F. L. Figueroa • Control of N-Metabolism by Light Quality in Corallina 791

of the ammonium previously released was mainly stimulated by B-light pulses. High initial net up­take of nitrate and ammonium in various algae, es­pecially after nitrogen starvation, has been report­ed [18]. In Corallina, nitrate and ammonium seemed to be used simultaneously as in other algae [19, 20]. Because R and B light effects on inorganic nitrogen incorporation were partially reversed, the possible involvement of phytochrome is proposed. The fact that NH4+ uptake in darkness was greater after B than after R light pulses, suggests the in­volvement of a specific B-light photoreceptor con­trolling ammonium uptake. The action of only phytochrome in B-light responses is discared be­cause theoretically the level of active phytochrome reached after R is greater (70%) than after B (2 0 %) and more stimulation would be expected after R than after B light pulses; precisely the reverse was found. Different time course after B than after R light is a result that support the pro­posal on the involvement of a specific B-light pho­toreceptor. The time course of the responses has been used a criterion to distinguish when the B effect is due to phytochrome or due to specific B-light photoreceptor [21]. In the green alga Ulva rigida R and B light stimulated the nitrate net up­take through phytochrome and blue-light photo­receptor systems [6 ],

In Corallina elongata, NR activity was clearly stimulated by R and B light pulses after the addi­tion of 45 |im N 0 3~ (Fig. 4). Red light produced a dramatic increase in NR activity but after 1 h in darkness the NR activity decreased. After B light pulses, however, no transient activity was ob­served and the NR activity increased throughout the time in darkness. Thus, in darkness, no limit­ing steps were observed after the application of B-light pulses. R and B light effects on NR activity were partially reversed by far-red light (Fig. 4). Thus, the involvement of two photoreceptors, phytochrome and a B-light photoreceptor is sug­gested based on the different time courses of NR activity after R compared to B light pulses. The transient increase in NR activity in Corallina after R light pulses has been observed in starved Por- phyra perforata after the addition o f nitrogen [2 2 ] and after the application of R light and nitrate in Ulva rigida [6 ], NR activity is photoregulated via a blue light photoreceptor in several algae [7, 23] and by blue-light photoreceptor and phytochrome

Fig. 4. Nitrate reductase activity, expressed as |imol N O r produced per gDW and h, in darkness after the same treatments as Fig. 2. LP = Light pulses.

in higher plants and in the green alga Ulva rigida [6 , 24]. In most cases, brief blue-light treatments resulted relatively ineffective compared to red light, but continuous blue light was more effective than continuous red light causing increases in ni­trate reduction [25].

The effect of light quality on GS activity was similar to that on NR activity (Fig. 5). A dramatic increase in GS activity after R light pulses and a slow increase after B light pulses were observed in the first hour in darkness (Fig. 5). However, after B light pulses and 4 h in darkness, GS activity was significantly greater (p<0.05) than that in the R-light treatment (Fig. 5). The stimulation of GS activity by light and nitrate was significantly (p<0.05) greater in N-limited algae than in N-sufficient algae (Fig. 5). GS activity was mainly stimulated by light and nitrate in algae with N-limitation. Regulation by light of GS during ni­trogen deficiency in microalgae has been reported [26, 27], but, instead of the phytochrome and B-light photoreceptor, activation of GS by the thioredoxin system was discovered [26, 27]. In higher plants, both GS and GOGAT activities were regulated by phytochrome [28]. The drastic

792 F. L. Figueroa Control of N-Metabolism by Light Quality in Corallina

Fig. 5. Glutamine synthetase activity in darkness after 5 min light pulses and the addition of nitrate (45 K NO j) in algae incubated previously under N-sufficient (C :N = 10 .3 ) and N-limited (C :N = 17 .3 ) conditions. LP = Light pulses.

decrease in GS activities after R light and 4 h in darkness could be explained by a rapid feedback inhibition caused by any or various of the several end products of nitrogen metabolism reported in other algae as the most effective inhibitors, e.g. alanine, glycine and serine, AMP and Gin [29, 30].

The accumulation of soluble proteins was also greater after light pulses and the addition of ni­trate to algae which had been previously starved (C : N = 17.3) (Table I). This accumulation was rel­atively very rapid (Table I), after 1 h dark incuba­tion R light pulses increased almost two times the level of protein reached in the dark control. Rapid accumulation of soluble protein and biliprotein in white light after the addition of nitrate has been re­ported in red algae cultivated previously, as in this work, under nutrient limitation i.e. Corallina elon- gata [31], Porphyridium purpureum [32] and Chon- drus crispus [5]. In N-sufficient algae, the accumu­lation of soluble protein was significantly (p < 0.05) greater after R light pulses than after B light pulses and in the dark control. However, in N-limited algae, the stimulation was similar after both light qualities. In both N-limited and

Table I. Increment of soluble protein per g algal DW (A mg soluble protein gDW -1) and the relation between the increment o f internal carbon related to the increment of internal nitrogen (AC: AN) in Corallina elongata after 1 h in darkness (1 h D) and 4 h in darkness (4 h D), and the application of light pulses (5 min) and 45 jaM nitrate. C : N proportions at the initial time (before light treatments) were 10.3 in N-sufficient algae and 17.3 in N-limited algae.

Light treatments Amg Protein gDW 1 A C: ANC :N C : N

10.3 17.3 10.3 17.3

Dark control (1 h D) Dark control (4 h D)

0.15 ±0.009 0.22 ± 0.01

0.65 ±0.04 0.76 ±0.06 25.0 ± 2 .0 6.5 ±0.5

5 min FR + 1 h D 5 min FR + 4 h D

0.26 ± 0.02 0.29 ±0.03

0.79 ±0.09 0.80 ±0.06 18.5 ± 1.6 4.5 ±0 .5

5 min R + 1 h D 5 min R + 4 h D

0.92 ±0.09 0.70 ±0.08

1.15 ± 0.10 1.20 ± 0.10 10.5 ± 1.3 3.0 ± 0 .4

5 min R + 5 min FR + 1 h D 5 min R + 5 min FR + 4 h D

0.26 ± 0.01 0.21 ± 0.01

0.90 ±0.07 0.83 ±0.08 22.7 ±3 .0 4.5 ± 0 .4

5 min B + 1 h D 5 min B + 4 h D

0.68 ±0.07 0.36 ±0.04

1.01 ± 0.10 1.10 ± 0.12 4.9 ±0.5 2.4 ± 0.3

5 min B + 5 min FR + 1 h D 5 min B + 5 min FR + 4 h D

0.26 ± 0.02 0.20 ± 0.01

0.65 ±0.05 0.81 ±0.08 15.8 ± 1.6 4.7 ± 0.5

F. L. Figueroa • Control o f N-M etabolism by Light Quality in Corallina 793

N-sufficient algae FR light pulses reversed partial­ly R and B light stimulant effects on protein syn­thesis. These results indicate that the action R and B photoreceptors is very complex and it could be affected by the intracellular content of nitrogen.

The relation between the increment of internal carbon related to the increment of internal nitro­gen was clearly affected by light quality after 4 h in darkness and the addition o f nitrate (Table I). In both, N-limited and N-sufficient algae, the incre­ment of nitrogen related to carbon was significant­ly (p < 0.1) greater after R and B light pulses (low AC:AN relation) than in the dark control and after FR light pulses. On the other hand, the rela­tion AC: AN was lower in N-limited algae than in N-sufficient algae in all light treatments (Table I). This indicates that the accumulation of nitrogen related to carbon was greater in algae previously starved than in repleted algae.

The relation between N 0 3~ uptake and reduc­tion and the ammonium assimilation with protein accumulation indicated that the photocontrol of the protein synthesis was produced as a conse­quence of the photoregulation o f N-metabolism as it has been reported in Ulva [6 ]. The greatest effect of R and B light pulses on protein accumulation

corresponded to substrate level; more ammonia was available after R and B light for protein syn­thesis than after FR light and in the dark control.

In summary, the involvement of at least two dif­ferent photoreceptors, namely, phytochrome and a B-light photoreceptor, controlling several steps of the inorganic nitrogen metabolism in the red alga C. elongata: (1) uptake of nitrate, nitrite and ammonium, (2) nitrate reductase activity and (3) glutamine synthetase activity is suggested. The participation of photosynthetic reactions in the control of N-metabolism is excluded because with the light treatments used (short pulses and 4 h in darkness) no differences in photosynthetic rates were observed [6 , 11]. Simultaneously to the action of red/far-red and blue photoreceptor the possible action of other photoreceptors is not discared. More investigations are necessary to define the complex interaction between the different photo­receptors that control N-metabolism.

A cknowledgemen ts

This work was financed by the Spanish Ministry of Education and Science (Project CICYT MAR- 90 0365).

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