Effect of artificial UV radiation on carbon and nitrogen ...atarazanas.sci.uma.es/docs/tesisuma/16603734.pdfPrimary Research Paper Effect of artificial UV radiation on carbon and

Primary Research Paper

Effect of artificial UV radiation on carbon and nitrogen metabolism

in the macroalgae Fucus spiralis L. and Ulva olivascens Dangeard

Benjamın Vinegla1,2,*, Marıa Segovia1 & Felix L. Figueroa11Departamento de Ecologıa y Geologıa, Facultad de Ciencias, Universidad de Malaga, Campus Universitario de Teatinoss/n, E-29071, Malaga, Spain2Departamento de Biologıa Animal, Biologıa Vegetal y Ecologıa, Facultad de Ciencias Experimentales, CampusUniversitario Paraje las Lagunillas s/n, E-23071, Jaen, Spain (*Author for correspondence: Tel.: +34-953-212791; Fax:+34-952-211873; E-mail: [email protected])

Received 14 December 2004; in revised form 29 September 2005; accepted 2 October 2005

Key words: carbonic anhydrase, fluorescence, nitrate reductase, photosynthesis, ultraviolet

Abstract

Thalli of the intertidal Phaeophyte Fucus spiralis L. and the subtidal Chlorophyte Ulva olivascens Dangeardwere exposed to artificial UV-A, UV-B and photosynthetically active radiation (PAR) by combination ofPAR + UV-A + UV-B (PAB), PAR + UV-A (PA) and PAR (P) treatments. UV-A enhanced photo-synthesis and stimulated carbonic anhydrase (CA) and nitrate reductase (NR) in F. spiralis whilst PAR onlyhad an inhibitory effect in this species. U. olivascens suffered chronic photoinhibition in all the treatments asevidenced by reduced maxima photosynthesis (Pmax) and photosynthetic efficiency (a). Non stimulatoryeffect was observed upon CA and NR in this species. Our results showed that artificial UV radiationtriggered opposite responses in both species. We suggest that differences shown by both species might berelated to their location in the rocky shore and their ability to sense UV. We propose that the ratioUV:PAR acts as an environmental signal involved in the control of photosynthesis as shown by pro-nounced inhibition in samples exposed to only PAR. We also suggest that UV-regulated photosynthesiswould be related to carbon (C) and nitrogen (N) cycles, regulating feedback processes that control C and Nassimilation.

Introduction

Macroalgae are sessile organisms subjected tostrong fluctuations in both light quantity andspectral light quality (Kirk, 1976; Hader &Figueroa, 1997). Light plays an essential rolecontrolling both plant physiology and morphologyin two ways: i) as a source of energy, and ii) as anenvironmental signal acting on specific photore-ceptors (Dring et al., 1996b; Segovia et al., 2001;Gordillo et al., 2004). Both terrestrial plants andmacroalgae have developed several mechanisms tosense light to overcome changes in their environ-ment in order to optimise their survival. They can

detect almost all facets of light such as direction,duration, spectral quality, quantity, and solarangle, through specialised photoreceptor mole-cules that allow them to sense light within thevisible spectrum (Sharma, 2001). This is of vitalimportance because canopies and water produce adrastic modification in the light spectrum due todifferential light absorbance and dispersion. Inaddition, plant density and morphology decreasethe overall irradiance that reaches the leaf or thallisurface (Smith, 1994). The enzymes from theCalvin cycle have been reported to be activated byblue light (Dring, 1984) by means of a blue-UVphotoreceptor.

Hydrobiologia (2006) 560:31–42 � Springer 2006DOI 10.1007/s10750-005-1097-1

The increase in solar UV-B radiation is a con-sequence of the stratospheric ozone layer depletion(Hofmann, 1996; Fraser & Prather, 1999). Underthe actual solar UV-B levels, marine ecosystemsare already subjected to an enormous stress thatwill increase during the first quarter of our cen-tury, when ozone will achieve its lowest, as pre-dicted by Madronich (1994). UV radiation hasbeen described to have many effects upon macro-algal physiology. UV alters DNA by formingthymine dimmers (Karentz, 1994); UV-B alsoalters photosynthetic activity causing photoinhi-bition (Flores-Moya et al., 1998; Figueroa et al.,2002). In terms of nutrient incorporation andassimilation UV has shown to exert both inhibi-tory (Dohler, 1996) and stimulating effects(Kumar et al., 1996) upon inorganic N incorpo-ration, altering enzymatic activities responsible forN metabolism (Dohler et al., 1995; Sinha et al.,1995; Rai et al., 1998). UV also has effects uponpigment content (Post & Larkum, 1993; Figueroaet al., 2002), and it is responsible for algal growthinhibition (Dring et al., 1996a).

Little is known about the cellular and molecu-lar mechanisms of UV perception in algae orabout the effect of UV upon the processes involvedin C and N incorporation and assimilation.Underwood et al. (1999) showed that photopro-tective mechanisms against UV radiation might beenhanced by visible radiation.

We have shown in this work the effect of arti-ficial UV radiation upon some aspects of thephysiology of F. spiralis and U. olivascens. Wedemonstrate that UV-A plays an important roleregulating photosynthesis and key enzymes of theC and N metabolism, such as carbonic anhydrase(CA) and nitrate reductase (NR), in F. spiralis andU. olivascens, regardless the presence of UV-B andvisible light.

Material and methods

Plant material and culture conditions

Whole thalli of the intertidal Phaeophyte Fucusspiralis L. and the subtidal Chlorophyte Ulvaolivascens Dangeard were collected in Tarifa(Cadiz, Southern Spain). Healthy thalli freefrom macroscopic epibiota were selected and

maintained in natural aerated seawater at 17 �Cfor not more than 2–3 days. Water was changedevery day. Light was provided by means of whitefluorescent lamps (W) (F20 W/CW Osram, Ger-many, Fig. 1) at 12 light: 12 dark photoperiod anda photon fluence density (PFD) of 100 lmolquanta m)2 s)1 (21.74 W m)2). The containers forthe incubations were 5L Plexiglas cylinders, nottransparent to UV. Filters for the light treatmentswere placed on top of the cylinders. Bubbling waslocated on the bottom part of the containers.Three independent thalli were taken from eachcontainer for every measuring period.

Light treatments

Fresh thalli were taken from culture and exposedto artificial UV radiation (Fig. 1) in filtered sea-water. PAR radiation was provided by a TrueLitePlus (Duro-Test, USA) lamp at an irradiance ofca. 340 lmol quanta m)2 s)1 (74.26 W m)2). UV-A was provided by a QP-340 (Q-panel, USA) at anirradiance of 4.29 W m)2. UV-B was provided bya TL 40 W/12 (Philips, The Netherlands) at anirradiance of 0.54 W m)2. Combination of threedifferent light quality treatments were used forexperimental set-up: (i) PAR + UV-A + UV-B(PAB); (ii) PAR + UV-A (PA); and (iii) PAR (P).Different UV selective cut-off filters were used inorder to achieve these light treatments accordingto Figueroa et al. (1997). Ultraphan 295 (DigefraGmbH, Munich, Germany) excludes UV-C, pro-viding PAB (PAR+UV-A+UV-B). Folex 320(Folex GmbH, Dreieich, Germany) excludes UV-B and UV-C providing PA (PAR + UV-A).Finally, Ultraphan 395 (Digefra GmbH, Munich,Germany) excludes UV-A, UV-B and UV-C,providing P (PAR). For the different light sourcesapplied (lamps + filters), spectral PFDs in therange 350–800 nm were measured each nm bymeans of a Licor-1800 UW spectroradiometer(Licor, Lincoln, Nebraska, USA) provided with acosene-corrected planar sensor (2p). Samples wereexposed to UV for 24 h, sampling at 0, 6 and 24 h.

In vivo fluorescence measurements

Parameters of in vivo induced chlorophyll a fluo-rescence of photosystem II were estimated bymeans of pulse amplitude modulated fluorescence

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Figure 1. Emission spectra (relative units) of the lamps used during the incubations under artificial PAR/UV radiation. (a) Solar

irradiance; (b) TrueLight Plus; (c) Q-Panel-340; (d) TL40 W/12. Solar irradiance spectrum is included for comparison.

33

(PAM2000; Waltz, Effeltrich, Germany), accord-ing to Schreiber et al. (1995). Optimal quantumyield of fluorescence of PSII (Fv/Fm) was calcu-lated according to Krause & Weis (1991), as:

Fv ¼ Fm � Fo;

Fv=Fm ¼ ðFm � FvÞ=Fm

being Fo the initial fluorescence in the dark-adapted state (when all PS II reaction centres arereduced) and Fm the maximal fluorescence in thedark-adapted state (when all PS II reaction centresare oxidized). In order to obtain this parameterthe thalli were pre-incubated for at least 15 minin darkness prior to the measurement. Non-photochemical quenching of fluorescence (NPQ)was also determined in order to have an insight onenergy dissipation processes during photosynthesisin the thalli. This parameter was calculated as:

NPQ ¼ 1� ð½Fm0 � Fo

0�=½Fm � Fo�Þ;where Fm and Fo are the maximal and the minimalfluorescence of a light-adapted plant, respectively.NPQ was determined in thalli directly under thelight treatments.

Photosynthetic oxygen evolution

Oxygen evolution rates for F. spiralis andU. olivascens were measured under increasingwhite light from 0 to 1200 lmol quanta m)2 s)1, ina custom-made transparent Plexiglas chamber of10 ml volume equipped with an oxygen Clark-typeO2 electrode (YSI 5331), a stirring device at con-stant temperature (17 �C) and two TrueLightCompact (Duro-Test, USA) lamps as the lightsources. For every sample in each treatment aphotosynthesis-irradiance (P–I) response curvewas carried out. For this purpose 0.15 g fresh-weight samples were placed for 5 min in darknessand then 1.5 min under each irradiance for aconstant O2 evolution rate. This process was con-stantly repeated in three replicates for each treat-ment. The P–I curve was fitted to the equation ofJassby and Platt (1976):

GP ¼ Pmax � tanh ðaI=PmaxÞ þ Ro

where GP is the gross photosynthesis, Pmax is themaximum net photosynthetic (NP) rate, tanh is the

hyperbolic tangent, a is the photosynthetic effi-ciency at low irradiance, I is the incident irradi-ance, and Ro is the dark respiration rate.

Total carbonic anhydrase (CA)

Total carbonic anhydrase activity was measuredpotentiometrically, according to Haglund et al.(1992). The assay was carried out at 0–2 �C deter-mining the time taken for a linear drop in pH in therange 8.5–7.5 in a 3 ml volume cuvette containing abuffer (50 mM TRIS, 25 mM ascorbic acid and5 mM EDTA). Small pieces of algae with a totalweight of 10–20 mg FW were washed with distilledwater and placed in the cuvette. The reaction star-ted by adding 1 ml of ice-cold CO2-saturated dis-tilled water. One unit of relative enzymatic activitywas defined as (to/tc))1, where to and tc are the timetaken for the pH change in the absence and thepresence of the alga, respectively. Enzymatic acti-vity was expressed by fresh weight of algae.

Nitrate reductase (NR) activity

Nitrate reductase activity of fresh material wasmeasured following the in situmethod according toCorzo & Niell (1991). The in situ method hasbecome a suitable method when the extractionprocedure for in vitro determination of NR is diffi-cult or even impossible without the inactivation ofthe enzyme (Gordillo et al., 2001). In the in situprocedure the enzyme is assayed in its original cel-lular location. The assaymedium contained a buffer(0.2 M H2PO4

) and 1 mM EDTA; pH 8), a com-pound able to permeabilise the membrane (0.1%propanol v/v), nitrate in excess (50 mM NaNO3),and a source of reducing power (10 lM glucose).The reaction was carried out by placing 15 mg FWof alga in a test tube containing 2 ml of the assaymedium. The incubation time was 30 min at atemperature of 30 �C. The assay medium wasbubbled with N2 gas for 2 min before and 2 minafter placing the algal sample in order to remove thedissolved O2, that competes with NO3

) in its reduc-tion to NO2

). The incubation was performed indarkness to prevent further reduction of NO2

) toNH4

+. The activity is measured as the rate of NO2)

produced. The observed activity is a potentialmeasure of the NR of the cell under the conditionsprior to the assay.

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Statistics

Data presented are the means of 3 measurementsfor CA, NR and P–I curves and 6 measurementsfor the fluorescence parameters. All the measure-ments were performed on independent thalli fromeach treatment. Data for the different light treat-ments were compared by means of one-wayANOVA analyses followed by Fisher’s LSD test(Sokal & Rohlf, 1987).

Results

Photosynthetic rates

Figure 2 shows the photosynthesis-irradiance(P–I) response curves for both species prior to theUV treatments. U. olivascens showed both higherPmax and photosynthetic efficiency (initial slope, a)than F. spiralis. Corresponding parameters aredescribed in Table 1. Under UV radiation photo-synthetic parameters showed significant differences(p<0.01). Pmax increased in F. spiralis in all thetreatments, being such increase notably higher in

the absence of UV-B and the presence of UV-A(Fig. 3a). Exposure to UV promoted an increase ina (Fig. 3b) in all the treatments. Non significantdifferences were observed between the initial timeof non-exposed thalli and 24 h after exposure(p<0.01).

The response found for U. olivascens wasdifferent. Pmax decreased 4-fold in thalli exposed toPAB as opposed to thalli under P or PA radiation(Fig. 3c). Both P and PA treatments showedsimilar patterns. The photosynthetic efficiency (a)dropped constantly in time in the P treatmentwhen compared to PA and PAB. The absence ofUV radiation caused the lack of recovery of a, butafter 24 h under PAB and PA a was 50% recov-ered (Fig. 3d).

Fluorescence measurements

There were no significant differences in Fv/Fm val-ues for F. spiralis (around 0.65) (Fig. 4a). Fv/Fm

for U. olivascens was lower in the PAB treatmentthan under PA and PAR (Fig. 4c), and it did notchange until after 2 h exposure to PAB. After 6 hexposure Fv/Fm dropped from 0.6 to 0.4. Fv/Fm did

Figure 2. Photosynthesis versus irradiance curves determined in F. spiralis (d) and U. olivascens (o) prior to incubation under the

different light treatments. Experimental data were fitted to the equation of Jassby & Platt (1976) in order to obtain the photosynthetic

parameters. Symbols are the mean of three replicates with error bars indicating standard deviation.

35

not vary after 24 h exposure under PA and Pirradiation.

Non-photochemical quenching (NPQ) reachedhigh values (0.6) in F. spiralis after 6 h in all thetreatments (Fig. 4b). NPQ for U. olivascens at

initial points was low (0.3) and remained soalmost until the end of the exposition period.Then, NPQ increased under the PAB and Ptreatments (Fig. 4d).

CA and NR activities

CA activity in F. spiralis reached the highest valueunder the PAB treatment (700 AER g)1 FW).Activity increased from 0 to 6 h, then it showed a2-fold decrease to recover initial levels after 24 h.UV-A had the same stimulating effect on theenzymatic activity but it occurred later on time(Fig. 5a). In U. olivascens the stimulating effectupon CA activity did not occur up to 24 h. Thisindicated that the UV dose (accumulating effect ofUV radiation) needed by this alga was muchhigher than for F. spiralis (Fig. 5c). However, inthe PA treatment the highest CA activity wentdown to initial levels after reaching the maximum(150 AER g)1 FW). PAB and P graduallyincreased the enzymatic activity with exposure

Table 1. Photosynthetic parameters before UV exposure of

photosynthesis-light quantity (P–I curves) in white light for

F. spiralis and U. olivascens, derived from Jassby and Platt

equation (Jassby & Platt, 1976). Results are the mean of trip-

licates. Standard deviations are given in brackets

U. olivascens F. spiralis

Pmax (lmol O2 g)1 FW h)1) 59.72 (0.01) 24.96 (5.16)

a (lmol O2 g)1 FW

h)1 lmol)1 quanta m)2 s)1)

1.206 (0.544) 0.165 (0.060)

Ro (lmol O2 g)1 FW h)1) 14.07 (4.74) 2.47 (0.54)

Ik (lmol quanta m)2 s)1) 61.19 (20.61) 165.92 (27.00)

Ic (lmol quanta m)2 s)1) 11.67 (3.93) 14.92 (2.25)

Pmax: maximum net photosynthetic rate; a: photosynthetic

efficiency; Ro: dark respiration; Ik: saturation constant; Ic: light

compensation point.

Figure 3. Light saturated net photosynthetic rate (Pmax) and photosynthetic efficiency at limiting irradiances (a) determined in thalli of

Fucus spiralis (a and b) and Ulva olivascens (c and d) incubated under three different artificial light treatments. P: photosynthetically

active radiation (PAR) (light bars); PA: PAR +UV-A (grey bars); PAB: PAR+UV-A+UV-B (dark bars). Symbols are the mean of

three replicates with error bars indicating standard deviation.

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time, but activity was never as high as in PA. NRactivity seemed to be stimulated equally by UV-Aand UV-B radiation (PAB and PA treatments)whilst P did not stimulate NR. Enzymatic activitystarted to increase from the beginning in F. spiralis(Fig. 5b) and after 6 h for U. olivascens (Fig. 5d).In both species the increase in activity dependedon the accumulated UV dose although the activitywas 4-fold higher in F. spiralis.

Discussion

The pattern observed in the P–I curves and CAand NR activities was the expected attending tothe ecological distribution of both species in therocky shore. F. spiralis was located at the higherintertidal of the rocky shore and subjected to tidalcycles suffering daily desiccation periods which arecoincident with the highest levels of irradiance.On the contrary, U. olivascens was collected at

subtidal locations of the rocky shore where lightintensity was attenuated. During the tidal cycleU. olivascens was completely submerged andsubsequently this species presented the samecharacteristics as those expected from shadowplants. Given that F. spiralis receives higher lightdoses than U. olivascens it would be expected thatincubation with artificial UV radiation would havea greater photoinhibitory effect on U. olivascenssince it may lack the acclimation mechanisms tocope with UV. However, although U. olivascensshowed a decrease in Pmax and a, which might beunderstood as a short-time response of dynamicphotoinhibition, it still remained a certain recoveryability. Indeed, similar levels of photosyntheticefficiency as those measured at the beginning of theexperiment were found in the PAB and PA treat-ments after 24 h of incubation, suggesting a med-ium to long-time acclimation mechanism to UVradiation that would not be expressed in its naturallocation under low light conditions. The same

Figure 4. Variation of PS II optimum quantum yield (Fv/Fm) and non-photochemical quenching (NPQ) determined in F. spiralis

(a and b) andU. olivascens (c and d) incubated for 24 h under three different artificial light treatments: P, only photosynthetically active

radiation (PAR) present (light bars); PA: PAR + UV-A (grey bars); PAB: PAR+ UV-A + UV-B (dark bars). Symbols are the mean

of three replicates with error bars indicating standard deviation.

37

effect has been observed in several Ulva species(Bischof et al., 2002). These authors have studiedthe photoinhibitory effects that individual Ulvathalli presented under natural solar radiationduring daily cycles. This inhibition was even morepronounced in samples only exposed to photo-synthetically active radiation. Heat dissipationstrongly increased in these samples indicating theactivity of regulatory mechanisms involved indynamic photoinhibition. Our results have shownthat both Fv/Fm and NPQ varied in U. olivascens.This was probably due to the lack of UV-acclimating mechanisms of the thalli when theywere transported from the rocky shore to the UVradiation conditions in the laboratory. Similarresults have been observed in three species ofLaminaria and three species of subtidal red algae(Delesseria sanguinea, Plocamium cartilagineumand Phyllophora pseudoceranoides). Fv/Fm valueswere reduced to minimal values after 4 h under P,PA, UV-A+UV-B or UV-A treatments (Dringet al., 2001). In the more resistant species

(Laminaria spp. and P. pseudoceranoides) Fv/Fm

was higher after shorter exposures to UV radiationalone than to PAR with or without UV. Therecovery of Fv/Fm in all species was also faster inthe two treatments that contained UV radiationalone than in those that included PAR. Theseresults suggest that it is the high irradiance of PARin natural sunlight which is responsible for thephotoinhibition of photosynthesis of subtidalseaweeds. The quantity of energy associated toeach wavelength is a limiting factor in terms ofdeveloping a rapid response because only in 2 hunder PAB the drop in the optimal quantum yieldwas the same as under PA and P. The decrease offluorescence yield accompanied by the increase inNPQ is a dynamic mechanism for dissipating theexcess of energy in order to avoid irreversiblepigment degradation as a result of energy flowfrom antennae complex toward reaction centers inPSII (Hader et al., 2002). The drop of fluorescenceyield due to UV-B was faster than under PA andP. Therefore, the mechanisms in charge of energy

Figure 5. Evolution of total carbonic anhydrase (CA) and in situ nitrate reductase (NR) activities in thalli of F. spiralis (a and b), and

U. olivascens (c and d), incubated for 24 h under artificial light (P: photosynthetically active radiation (PAR) (light bars); PA: PAR +

UV-A (grey bars); PAB: PAR + UV-A + UV-B (dark bars). Symbols are the mean of three replicates with error bars indicating

standard deviation.

38

dissipation would be activated by a signaldepending on energy quantity below the thresholdreached under the different UV treatments anddose. On the contrary, the lack of changes in Fv/Fm

and NPQ in F. spiralis suggests that the lightconditions and incubation time did not representany stress for this alga in terms of the quantity ofenergy required to produce any effect in the reac-tion centres. As adverse effects of UV-B radiationon photosynthesis were only observed in combi-nation with high levels of PAR, it might reflect thesynergistic effects of the two wavelength ranges. Insamples exposed either to PA or to UV-B+UV-Awithout PAR, no inhibition of photosyntheticquantum yield was found in the course of the day.Other authors have also pointed out the photoin-hibitory mechanisms as a response to cope withhigh levels of UV (Hader et al., 1996; Figueroaet al., 1997; Flores-Moya et al., 1998). In Rissoellaverruculosa PAR levels of 2000 lmol quantam)2 s)1 promoted a degree of photoinhibitionsimilar to that obtained under P + UV radiation.On the contrary, the red macroalga Porphyraleucosticta showed higher inhibition under naturalsolar radiation than when UV cutting filters wereused (Figueroa et al., 1997). Our results showedthat artificial UV radiation triggered oppositeresponses in both species according to Pmax and a(Table 1). It has been previously suggested thatUV-A exerts an activating effect on the photo-synthetic capability (Perez-Rodrıguez et al., 1998).The photosynthetic enhancing effect of UV-A isshown in F. spiralis (Figs. 3a, b). The light receivedby the alga might be activating uptake systems andstimulating enzymes from the Calvin cycle whichhas a direct consequence upon Pmax, that increasedafter 6 h of irradiance. The mechanism of activa-tion triggered by UV radiation required higherdoses of UV to be activated. This was probablybecause to achieve the critical UV doses able topromote activation in the rocky shore, a goodnumber of hours under irradiation are required.The ecological implication of the advantageousrole of UV-B in well-acclimated marine plants tohigh irradiances was previously proposed byFigueroa et al. (2002). Also, studies on dailyphotoinhibition and full recovery in intertidalMediterranean algae suggested that photoinhibi-tion is a photoprotective mechanism against highsolar radiation as in higher plants and that

patterns of photoinhibition and recovery areaffected by accumulative dose (Figueroa &Vinegla, 2001).

A correlation between Fv/Fm and the photo-synthetic efficiency should be expected. However,the photosynthetic efficiency showed a 2-foldincrease in all the treatments in F. spiralis(Fig. 3a), while no significant differences wherefound in the optimal quantum yield of fluores-cence (Fig. 4a). Fv/Fm is a variable that is reflectingthe efficiency of the photosynthetic apparatusconverting light energy in chemical energy, and itis expressed in terms of relative units. This meansthat the variable is restricted when the actual valueis near the limit. Thus, the sensitivity of bothvariables is completely different since great chan-ges in photosynthetic efficiency cannot be followedby changes in Fv/Fm or at least changes in Fv/Fm

cannot have the same range of variation when thisvariable is close to, for example, 0.8. Thus, thevariation which may be taking place in bothvariables cannot be detected in Fv/Fm due to itsrange of variation (from 0 to 1) and then thecorrelation that should appear is lost.

In U. olivascens the PAB and PA treatmentsshowed significantly higher values in photosyn-thetic efficiency than those found in the P treat-ment after 24 h of incubation (Fig. 3d). However,no differences where found among the threetreatments in Fv/Fm (Fig. 4c) indicating that theerror has a greater effect than the treatments in thisvariable and the differences found in the photo-synthetic efficiency are now minimized due to theparticular range of variation of Fv/Fm.

The inhibitory effect of UV-B radiation uponinorganic nitrogen incorporation in algae is wellknown (Dohler et al., 1995; Sinha et al., 1995;Dohler, 1996). However, there are other reportsthat did not found any effects of UV-B on N and/or NH4

+ accumulation (Braune & Dohler, 1996).Surprisingly, other authors showed that NRactivity was stimulated after UV-B exposure(Kumar et al., 1996).

We have shown in this work the differencesbetween CA and NR activities depending on thepresence or absence ofUV-A and/orUV-B. CAandNR activities are strongly regulated by environ-mental factors such as nutrient availability (Wheeler& Weidner, 1983; Davison & Stewart, 1984), andnitrogen (N) and carbon (C) assimilation (Turpin,

39

1991). There is not a clear correlation betweenchanges of energy dissipation and fluorescence ofPSII and changes inCAandNR, suggesting that theregulatory mechanisms under UV radiation forthese processes follow diverting pathways.

In situ NR activity was dependent on UVradiation in both species, as demonstrated by thelack of differences between PAB and PA treat-ments and the low activity obtained under P. After24 h exposure under these two light regimes in situNR increased 4-fold in U. olivascens and 3-fold inF. spiralis. On the contrary, under P radiation, theactivity remained very low during the wholeincubation period. Flores-Moya et al. (1998)observed that in thalli of red alga Rissoella verru-culosa exposed to full-spectrum solar radiation,NR activity tended to increase in the afternoon indaily cycles. This effect has not only been observedin marine macroalgae. Exposure of cultures in thecyanobacterium Nostoc calcicola to UV-B for aslittle as 30 min caused complete inactivation ofnitrogenase activity whereas NR activity wasstimulated 2-fold in comparison to one exposedto fluorescent white light (Kumar et al., 1996).Reports in the N2 fixing cyanobacterium Anaba-ena doliolum showed a significant increase in NO3

)

uptake and nitrate reductase activity, after expo-sure to UV-B. Kinetic studies of all the processesdemonstrated that the UV-B-induced structuralchanges in the enzymes/carriers could be respon-sible for uptake and assimilation of these nutri-ents (Rai et al., 1998). NR activity was enhancedby UV-A radiation in stem tissue in radish plants.These data suggest that promotion of plantgrowth by UV-A radiation involves the promo-tion of carbon and nitrogen metabolism in radishplants (Tezuka et al., 1994). Figueroa & Vinegla(2001) also suggested that UV radiation acts asan environmental signal involved in the control ofC and N cycles, regulating feedback processesthat control N assimilation as a function of Ccontent in Ulva rigida and Plocamium cartilagi-neum. Due to NR’s ability to absorb UVradiation, the enzyme has been proposed to be apossible blue and UV-A light photoreceptor(Quinones & Aparicio, 1990; Aparicio &Quinones, 1991; Stohr et al., 1995). We suggestthat UV-A is responsible for the higher photo-synthetic rate observed and as a consequence it isenhancing inorganic nitrogen uptake.

CA activity seemed to be clearly dependent onUV-A radiation in F. spiralis. Activity was rapidlystimulated between 0 and 6 h to fall down tothreshold levels. A possible explanation for thisbehaviour is that as Pmax increases, there is ademand on CO2 from Rubisco. P and PAB filledsuch demand after 24 h as shown by the highestlevels of CA activity. However, between 0 and 6 hthe highest CA activity was achieved under PA.The enzymatic activity due to PA would probablyfacilitate that Pmax at 24 h was higher. UV-Amight be stimulating C concentrating mechanismsin F. spiralis whereas UV-B promoted an earlyinhibition that was totally recovered after 24 h.The opposite was observed in U. olivascens. In thiscase UV-A had no effect on the enzymatic activity,since there were no significant differences for thelight treatments (p>0.01).

The different behaviour of CA activity inF. spiralis and U. olivascens can be understood interms of their different life strategies to deal withtheir different environments. F. spiralis is subjectedto tidal cycles, suffering daily desiccation periodsat the higher intertidal of the rocky shore. Thus, itcannot incorporate CO2 during its emersion perioddue to the desiccation process that it is coincidentwith the highest daily levels of irradiance (dynamicphotoinhibition) and has to increase dramaticallyits CA activity while submersed to compensate forthe period of the day when it is desiccated.

CA activity was dependent in light qualityduring incubation, whereas NR showed a commonvariation pattern in both species and for Nincorporation the mechanisms would be similar inboth species. The different response for CA sug-gests that might be different mechanisms of Cincorporation in both species. In U. olivascens Cincorporation would only depend on the amountof dissolved HCO3

) and for F. spiralis the source ofC under the desiccation periods at low tide wouldbe atmospheric CO2. The high levels of CAactivity found in the latter species were similar tothat found in other algae subject to the same dailyvariations (Flores-Moya et al., 1998) and seemedto be related to inorganic C during desiccation. Wehave shown in this work the effect of artificial UVradiation upon some aspects of the physiology ofF. spiralis and U. olivascens. We demonstratethat UV-A plays an important role regulatingphotosynthesis and key enzymes of the C and N

40

metabolism in F. spiralis and U. olivascens such asphotosynthesis, carbonic anhydrase (CA) andnitrate reductase (NR), regardless the presence ofUV-B and visible light.

Acknowledgements

This work was supported by the European Unionproject ENV4-CT96–0188.

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