Photosynthesis and growth response of the oceanic picoplankter Pycnococcus provasolii Guillard (clone Ω48-23) (Chlorophyta) to variations in irradiance, photoperiod and temperature

J. Exp. Mar. Biol. Ecol., 168 (1993) 239-251 0 1993 Elsevier Science Publishers B.V. All rights reserved

JEMBE

239 0022-0981/93/$06.00

01951

Photosynthesis and growth response of the oceanic picoplankter Pycnococcus provasolii Guillard (clone a48-23) (Chlorophyta) to variations in irradiance, photoperiod and temperature Arantza

Iriarte and Duncan

A. Purdie

Department of Oceanography, The University, Southampton, UK (Received

8 June

1992, revision received

12 January

1993, accepted

19 January

1993)

Abstract: The growth, photosynthesis and respiration rates of the green picoplanktonic algae Pycnococcus provasolii Guillard were measured as a function of irradiance, temperature and photoperiod. The algae showed positive photoadaptation to low irradiance and from an analysis of the photosynthesis versus irradiance curves, it is suggested that this is achieved mainly by increasing the size of the photosynthetic units. In accordance with this conclusion, chlorophyll b to a ratios increased with decreasing photon flux density. The algae further compensated for low light energy supply by reducing the rates of respiration. Values of the initial slope of the growth versus irradiance curve were higher than average (0.0016-0.0022 h-’ (nEm-z~s-‘)-’ at 20 “C). It is thus concluded that P. provasolii Guillard is a well suited organism to grow at sites of low irradiance and this may explain its success in colonizing the pycnocline area of stratified oceanic waters. This capacity, however, was not accompanied by a reduced ability to photosynthesize at high irradiances. A 24 h light regime did not seem harmful to P. provasolii Guillard, however, light energy was utilized less efficiently under 24 h than under 12: 12 h photoperiod. Values of Q,, for P,,,,, and p,,,_ were in the region of 2. Key words: Growth;

Irradiance;

Photosynthesis;

Picoplankton;

Pycnococcus provasolii; Temperature

INTRODUCTION

Until comparatively recently, phytoplankton in the picoplankton size range (i.e. < 3 pm, Sense Stockner & Antia, 1986) have been largely overlooked in the marine microbial flora (Johnson & Sieburth, 1979; Waterbury et al., 1979; Murphy & Haugen, 1985). It is now well established, however, that these minute phytoplankters are major contributors (> 50%) to the water column primary production in oceanic oligotrophic waters (Li et al., 1983; Platt et al., 1983; Glover et al., 1985; Iturriaga & Mitchell, 1986). A most interesting feature of picophytoplankton populations is that they seem to show defined spatio-temporal distribution and productivity patterns (Li et al., 1983; Platt Correspondence address: ton, SO9 5NH, UK.

A. Iriarte, Department

of Oceanography,

The University,

Highfield,

Southamp-

230

A. IRL4R-I-E AND D.A. PURDII:

et al., 1983; Murphy & Haugen, 198.5; El Hag & Fogg, 1986; Odate, 1989). In order to provide a reliable physiological basis for interpreting these biomass and productivity patterns observed in natural assemblages, a need has been identified to study the growth response of picophytoplankton isolates to environmental parameters under laboratory controlled conditions. To date, culture work on picophytoplankton has been almost exclusively devoted to studying aspects of the photosynthetic and growth response of various strains of cyanobacteria of the genus $vnechococcus (e.g. Morris & Glover, 1981; Barlow & Alberte, 1985, 1987; Kana & Glibert, 1987a,b). A few investigations have studied eukaryotic members of the picophytoplankton in culture (e.g. Glover et al.. 1987). A major factor contributing to this imbalance between the prokaryotic and the eukaryotic research component is probably the scarcity of information on how representative the particular eukaryotic isolates that have recently been brought into culture are of the marine picophytoplankton. This, in turn, must be mainly attributed to the difficulties associated with the fixation and taxonomic identification of many of these eukaryotic algae. It is worth noting, however, that molecular taxonomy has revealed a great diversity of prokaryotic picophytoplankton in the marine environment (Giovanonni et al., 1990). The high chlorophyll b:u ratios systematically found at the chlorophyll maximum layer in open ocean environments (Jeffrey, 1976; Gieskes et al., 1978; Vernct & Lorenzen, 1987) are suggestive of a relative abundance of phytoplankton containing chlorophyll b, i.e. chlorophytes and/or euglenophytes. More recently, detailed pigment analysis has revealed prasinophyte (micromonadophyte)-like organisms as a potentially important group of the oceanic picoplankton (Hooks et al., 1988). A comparatively recently isolated green algal strain, i.e. C!48-23. now positioned taxonomically in the class Micromonadophyceae and the species Py~nococcus provcwlii Guillard (Guillard et al., 1991) has been claimed to be a very common picoplanktonic form (1.5-4 pm in diameter) in oceanic waters of the northwestern Atlantic and the Gulf of Mexico (Campbell et al., 1989; Guillard et al.. 1991). The vertical distribution and photosynthetic pattern of picophytoplankton in stratified oligotrophic waters of the open ocean is suggestive of a capacity to grow efficiently deep in the euphotic zone (Li et al., 1983; Platt et al., 1983; Murphy & Haugen, 1985; Prezclin et al., 1986). In recent years much effort has been placed on investigating the photophysiology of Synechococcus spp., results suggesting that the strains investigated display positive photoadaptation to low irradiance (Barlow & Alberte. 1985; Kana & Glibert, 1987a,b), but also show a capacity to adjust to a wide array of light levels (Kana & Glibert, 1987a,b). Some evidence has been put forward to suggest that in the open ocean eukaryotic picoplankton have their maxima below the peak of the picocyanobacteria (Murphy & Haugen, 1985), a pattern that has been ascribed to differences in their response to light quality; cukaryotic algae having greater photosynthetic efficiency under blue-violet and blue wavelengths than cyanobacteria (Wood, 1985; Glover et al., 1987).

PHOTOSYNTHESISANDGROWTHOFPYCNOCOCCUS

Picocyanobacteria

abundance

PROVASOLII

has also been shown to be positively

correlated

241

with

temperature, both on a latitudinal basis (Murphy & Haugen, 1985; Joint, 1986) and on a temporal basis in temperate regions (El Hag & Fogg, 1986; Shapiro & Haugen, 1988; Odate, 1989). Few studies have attempted to relate temperature to eukaryotic picophytoplankton abundance. Similarly to picocyanobacteria, they have been shown to peak in late summer in Funka Bay, Japan (Odate, 1989) and Southampton Water, UK (Iriarte, 1991). However, the effect of temperature on the growth physiology of picophytoplankton has not been addressed in any detail. We present here data on the growth and photosynthetic response of laboratory grown cultures of P. provasolii Guillard (strain R48-23) to acclimation at various conditions of h-radiance, photoperiod and temperature.

MATERIALSAND

METHODS

Pycnococcus provasolii was grown in natural seawater (salinity 3 33 ppt) enriched with Guillard’s f/2 nutrient recipe without added silicate (Guillard, 1980). Growth rates, determined as the rate of increase in various biomass indicators: cell number, chlorophyll a and particulate organic carbon, were measured over a range of photon flux densities (3 to 540 pEm-2*s-1 PAR), at each of three temperatures (10, 15,20 o C), and under both 12:12 h Light:Dark cycle and 24 h light regime (20 “C only). Non-axenic cultures for growth rate experiments were incubated in 2 1 conical Pyrex flasks containing 1.5 1 of medium and incubated in temperature controlled water tank incubators illuminated from below with 30 W cool white fluorescent tubes (for further details of incubation tanks and illumination conditions see Garcia & Purdie, 1992). A range of irradiances were obtained using various combinations of layers of neutral density screens. Irradiance was measured with a quantum meter Model Q.101 connected to a cosine collector sensor (Macam Photometrics Ltd.). All flasks were wrapped in aluminium foil to remove the influence of ambient light and cells were preconditioned to each light level for 2 weeks. Cells were maintained in suspension by shaking the flasks periodically (i.e. twice daily). Aliquots for the determination of cell counts (1 to 10 ml), chlorophyll a and b concentration (5-25 ml) and particulate organic carbon (lo-75 ml) were removed from the flasks once daily. The use of preservatives was avoided (Murphy & Haugen, 1985) and cells were counted within 30 min of sampling, by filtering them onto 0.45 pm cellulose nitrate Whatman filters and counting autofluorescent cells under a Zeiss epifluorescence microscope. 30 to 100 non-overlapping fields of an eye piece counting grid were counted per filter, enumerating not less than 100 cells. For cultures grown under 12:12 h L:D cycle, chlorophyll a and chlorophyll b were determined fluorometrically using a Perkin Elmer model LS5 luminescence spectrometer, following a procedure similar to that described in Boto & Bunt (1978). The instrument was calibrated using purified commercial standard chlorophyll a (from Ana-

‘42

A. fRIARTE

AND D.A. PURDIE

c~~rrisnidulans) and chlorophyll b (from spinach) (both obtained from Sigma Ltd.), as described in Iriarte (1991). Chlorophylls were extracted from samples filtered onto Whatmann GF/F filters and homogenized in 90% acetone. For cultures grown under continuous light chlorophyll b values were not measured and chlorophyll a concentration was determined fluorometrically using an. AMINCO fluoro-calorimeter, without correcting for phaeopigments, as described in Parsons et al. (1984). The difference between estimates of chlorophyll a concentration obtained using this procedure (i.e. broad band excitation and emission filters, with no correction for phaeopigments) and using the monochromatic wavelength technique was shown to be < 10% (Iriarte, 1991). Particulate organic carbon (POC) was determined by combustion in oxygen of samples retained on precombusted GF/F filters and measurement of the CO, produced in an infra-red gas analyzer. Samples from growth rate experiments at 10 and 15 “C were analyzed for POC using an elemental anaiyzer (Carlo Erba Model 1108). Acetanilide was used as standard. Growth rate (p, in units of h- ‘) was calculated as the slope of the regression line of the semilogarithmic plot of the corresponding biomass index against time in hours. Following Langdon (1987), the growth rate versus irradiance relationship was fitted, using the nonlinear regression analysis routine contained in the Statgraphics package, to a hyperbolic tangent expression of the kind: 11 = (/l,,Xnn+ K> tanh (Us JI(~,,,;,, + cl& - pcl where p = growth rate (h ‘); p,max= light saturated growth rate (11. ‘): p, = intercept of the g vs. I curve, i.e. maintenance respiration rate (h -I>; X~= initial slope of the p vs. 1 curve, i.e. growth efficiency (h -’ (~Em-~‘~s-~‘)--‘); /= irradiance {PEm .2*s-‘). The irradiance for saturation of growth (I,,,) was derived as pmax/rg (in units of p Em -* *s -’ ). Rates of photosynthesis and respiration were determined for cultures preconditioned at various irradiances, and at both 12:12 h photoperiod (at both 10 and 20 “C) and 24 h light regime (at 20 ’ C), using the oxygen light and dark bottle technique. Measurements were made in mid-exponential growth phase and at algal cell concentrations greater than 2 x 10s cells*mll’. Preliminary size fractionated respiration rate measurements (> 1 pm and < 1 pm size fractions) showed that at these algal concentrations the < 1 pm size fraction accounted for less than ZOO-; of total respiration rate (Iriarte, 1991). Samples for photosynthesis measurements were incubated for 3 h over a range of irradiances (lo-2500 yEm_‘.s -‘I) in a light gradient incubator as described in Garcia & Purdie (1992). Illumination was provided by a 500 W tungsten halogen lamp (Thorn EMI, ODW 500). Dissolved oxygen concentration was measured using a precise, automated and microprocessor-controlled Winkler titration technique (Williams & Jenkinson, 1982). The gross photosynthetic rate versus irradiance relationship was fitted to the equation of Platt et al. (1980) (including the photoinbition parameter), and photosynthetic parameters estimated as previously described in Garcia & Purdie ( 1992).

PHOTOSYNTHESIS AND GROWTH OF PYCNOCOCCUSPROVASOLII

243

RESULTS Cellular chlorophyll a, chlorophyll b, and POC content

For algae grown under 12: 12 h photoperiod cellular concentrations of chlorophylls exponentially with increasing growth irradiance at all temperatures and photoperiods tested (Fig. 1). The relative change was greater for chlorophyll b (ca. 6-fold) than for chlorophyll a (ca. 4-fold) at all three temperatures. As a consequence, chlorophyll b to chlorophyll a ratios increased with decreasing growth photon flux density (Fig. 1). Growth irradiance, however, did not significantly influence the cellular carbon content at any of the temperatures (Fig. 2). The C:chl a (0, in units of g.g-‘) versus irradiance (1, in units of pEmd2* s-l) relationship from cultures grown at 15 “C can be formulated as 8 = 47.5 + 0.21 x Ig (2 = 0.95, p< 0.001) and for cultures grown at 20°C as 8=42.3 +0.16 xIg (?=0.98, p
Photosynthesis versus irradiance curves

Figure 3 shows typical photosynthesis versus it-radiance (P vs. I) relationships fitted to the equation of Platt et al. (1980). The parameters of the P vs. Z curves from cells of P. provasolii grown under various conditions of temperature, irradiance and photoperiod are shown in Table II. At 20°C under both photoperiods tested, the initial slope of the P vs. Z curve, i.e. the efficiency of light utilization at subsaturating h-radiances (cI), normalized to cell number exhibited a tendency to be highest at low to intermediate light levels 25110 pEm-2.s-1 and decreased clearly at growth photon flux densities above llO140 pEm-2.s-1. At 20 “C and continuous it-radiance tl is also expressed normalized to cell carbon content and this parameter decreased with increasing light level. In contrast, when normalized to chlorophyll a concentration, CIshowed a clear increase with growth photon flux density at all temperatures and photoperiods tested. A comparison between continuous light and 12:12 h 1ight:dark cycle showed c( values normalized to cell number to be 1.4 to 1.7-fold a values under 24-h light dose. There was no clear effect of temperature on a. Light saturated rates of photosynthesis (Pm,,) showed generally similar patterns of variation to those of a. At 20 “C, under both photoperiods, expressed on a cell number basis, highest values were determined at low to intermediate irradiances (25110 pEm-2.s-1), decreasing at higher irradiances. At 10 “C the trend of variation of P max per cell with growth photon flux density was not clear. When normalized to

A. IRIARTE

244

AND

D..A.

PURDIE

0.14

0.12 0.10 0.08 0.06 0.04 0.02 0.00 o.,40

0

1

J 50 I”“““”

’

100

’ ’ ’ 1 ’ 150200250300350400450500550

’

’

’

’

I

0.06

, ,. 0 I

50

100

’

”

150

200 ”

250

300 ”

350

400 ’

450 ”

500

550

0.8

0.6

0.51 0

’

50

100

’ ’ ’ ’ 150200250300350400450

’

’

-.I!

500550

Fig. 1. Effect of growth irradiance and temperature on the (A) cellular chlorophyll u and (B) cellular chlorophyll b concentrations and (C) the chlorophyll h to (I ratio in P. prowwlii grown under 12: 12 h L:D cycle at (0) 10 “C, at (0) 15 “C and at (A) 20 “C. Standard error bars are included when greater than symbol.

PHOTOSYNTHESIS

I

0 0

50

0

50

AND GROWTH OF PYCNOCOCCUS PROVASOtfi

I

I

I

1

I

I

I

I

I

I

I

245

100150200250300350400450500550

150

7

0,

9

100

d

YE 0 . .

50

0

1

0

I

,

1

,

I

I

I

100150200250300350400450500550 I4 (/JE m-‘s-l)

Fig. 2. Effect of growth irradiance and temperature on the (A) cellular carbon content and (B) carbon to chlorophyll a ratio in P. provasoliigrown under 12:12 L:D cycle, at (a) 15 “C and (A) 20 “C. Standard error bars are included when greater than symbol.

chlorophyil a concentration, Pm&xincreased with growth irradiance at both temperatures and photope~ods tested. Estimates of P,,,, both expressed on a cell number and chlorophyll a content, were

TABLE I

Effect of irradiance (I) on the cellular chlorophyll a (chl a) and carbon (C) content, and C:chl a ratio (B) of cells of P. provasolii grown at 20 “C under continuous light. Values in parenthesis are standard deviations as a percentage of the mean. I

chlx low2 a

C

e

25 65 140 260 370

7.82 (4.5) 8.03 (2.5) 7.47 (12.1) 2.69 (4.7) 2.32 (1.8)

2.91 (0.6) 3.73 (1.8) 3.61 (3.6) 3.02 (0.0) 3.30 (3.0)

37 47 49 113 142

I(‘JIE~-~,s-‘);

chla, C (pgceh’);

B (g-g-‘).

246

A. IRIARTE 1.25

_

1

AND

I

D.A.

PURDIE

I

I

1 /

t.OO

~

?,:----

i

,

I 0.75

0.50

0.25

0.00 0

500

1000 lrradiance

1500

(pE

2000

2500

3000

m-*.s-‘)

Fig. 3. Typical P vs. I curves for cells of P. ~ro~u.~of~j~~wn at 20 “C and 12: 12 h L:D cycle at irradiances: (Of 360. (0) 110 and (V) 30 PEm -‘.s I

various growth

greater (Q,, greater than 2) at 20 ‘C than at 10 ‘C under 12: 12 L:D cycle. Also, at 20 o C growth under 12: 12 h 1ight:dark cycle resulted in an enhancement of P,,,, in relation to growth under 24 h light dose. 1, exhibited a general pattern of increase with increasing irradiance, i.c. at low growth photon flux density celis required lower it-radiance to saturate photosynthesis. At IO “C 1, values were lower (66-llOgEm_‘.s-‘) than at 20°C (151-222gEm-‘.s-I). No substantial difference could be observed between photoperiods. The P vs. I curves generally showed little photoinhibition (as indicated by the parameter I,,). The exception was for cells grown at low irradiances at 20°C and continuous light, but these low values of It, were possibly not related to photodestructive processes, but were rather the consequence of increased rates of oxygen consumption due to increased rates of photorespiration at these high irradiances. The observed decrease in photosynthetic quotient with increasing irradiance at irradiances over the saturation level for cells of P. provasolii grown at low photon flux densities provides some evidence to support this hypothesis (Iriarte, 1991).

Respiration

rates

Table III shows values of respiration rate determined at 10 and 20 “C and under continuous light and L:D cycle regime. Rates of respiration increased markedly with

PHOTOSYNTHESIS

AND GROWTH

OF PYCNOCOCCUS

PROVASOLII

247

TABLE II The effect of photon

flux density on the photosynthetic characteristics of P. provusoliigrown under continuous light (20 “C) and 12:12 h photoperiod (10 and 20 “C).

P

a

nlax

Cdl

C

chl

25 65 140 260 370

28.5 32.8 29.4 20.6 21.5

9.74 8.66 8.11 6.78 6.55

0.36 0.41 0.39 0.77 0.93

30 110 360

50.6 56.9 43.0

nd nd nd

0.49 0.83 1.20

20 110 360

17.1 15.5 20.3

nd nd nd

0.24 0.39 0.58

cell

A: 2O”C, 24 h 0.21 73.7 0.25 66.3 0.18 49.6 0.11 35.3 0.14 41.6 B: 2O”C, 12:12 h 0.33 nd 0.35 nd 0.19 nd C: lO”C, 12:12 h 0.26 nd 0.17 nd 0.18 nd

P,,,,x: cell (fmolO,.cell‘.h- ‘); C (nmolO,.pgCcc cell (fmolOzxell~‘~h-’ @Em-‘.s-‘)-I); -z.sml (PEm 1. nd: not determined.

temperature and riod. Q,, values (Qt,, = 2.2) and with respiration

C

4

Ib

4

2.75 3.12 2.39 4.00 5.90

132 130 163 191 157

837 973 751 1514 4552

8 15 27 30 30

3.26 5.19 5.14

151 159 222

2925 6504 13047

8 5 3

3.59 3.66 5.27

66 108 110

4729 9778 5613

3 7 27

chl

‘.h- ‘); chl (~molO,.~gchl.a‘.h- ‘). C (pmolO,.pgC-‘.h-’ @Em-‘.s-‘)-I);

Ig, Ikr I,, I,

it-radiance, but did not appear to be greatly affected by the photopefor respiration could be derived at two irradiances, at 55 ~Ern-‘~s-’ 360 pErn-*.s-t (Qr, = 2.1). Growth rates were positively correlated rates (? = 0.661, p
Growth rates

Figure 5 shows the growth rate (in terms of increases in cell number) versus irradiante relationship for cells of P. provasolii cultured at the three temperatures and two photoperiods tested. The best fit values for the parameters of the growth versus irradiance curves are presented in Table IV. Estimates of rxedenoted steep slopes, however, curves showed signs of saturation at low irradiances (lo-25 PErn-*es-‘), alvalues were achieved at ii-radiances higher than 100 ~Em-*.s-‘. In the though Max majority of cases xs was calculated from the first two points of the curve and the availability of and/or differences in growth rates at the very low photon flux densities (lower than 10 pErn-*. s-‘) appeared critical for the estimation of txe. Given the standard errors involved in the estimation of the growth efficiencies, ae was not significantly different between 10 and 20 “C under L:D cycle, nor between 10 and 15 o C under L:D cycle, nor between L:D and continuous light at 20 “C. as was significantly higher at

A. IRIARTE

248

AND D.A. PURDIE TABLE III

‘.h- ‘) at various growth irradiances (1. in WErn ‘.s I). temperaRates of respiration (R, in fmolOz.cell tures and photoperiods for cells of P. provasofii. Values of growth rate (p, in h I) are also given. R

I’

A: 10 ‘C, 12:12 I1 I). 14 0.51 0.78 0.79 I.61 1.93 B: 2O’C. 12:12 h 2.20 I.62 1.78 3.93 3.10 c: 20 ;c, 24 h I.83 3.71 4.X6 3.21 4.23

3 I0 20 55 110 360 22

30 55

I10 360 25 65 140 260 370

nd

0.005 0.009 0.0 I 1

0.013 nd 0.037 nd 0.020 0.026

0.03 1 0.016

0.027 0.029 0.033 0.031

nd: not determined.

20 “C than at 15 “C under 12: 12 h photoperiod, but this may have been influenced the unavailability of growth rate data at 3 PEm ~‘.s-’ at 15 ‘C.

/

0.05

7-

,

/

/ *

0.04 -

0.00

/ 0

by

1

Respiration

1

1

2

3 (fmo102

/ 4

5

cell-‘h-l)

Fig. 4. Relationship between growth rate and respiration rate in P. provasolii. Data have been combined for various growth conditions: (0) 12: 12 h L:D cycle and 10 “C; (A) 12: 12 h L:D cycle and 20°C and (A) continuous light and 20 “C.

PHOTOSYNTHESIS

AND GROWTH

OF PYCNOCOCCUS

PROVASOLII

249

- 0.030 7 5 0.025 0 5 0.020 $ 0.015 2 0 0.010 0.005 0.000 0

50

100

150200250300350400450500550

lrradiance

(/IE mW2s-‘)

0.035 -i f m 2 r 5 F 0

0.030

P

-

n

0.025 0.020 0.015 0.010

-

0.005 0.000 0

I, I, I i I I 50 100 150200250300350400450500550 It-radiance

(pE

m

-2

s

I

/

_

-1

)

Fig. 5. (A) A comparison of p vs. I curves for cells of P.provusolii grown under 12: 12 h L:D cycle at various temperatures: (0) 10 “C, (0) 15 “C and (A) 20 “C, determined from increases in cell number. (B) A comparison of p vs. I curves for cells of P.provasolii grown at 20 “C under two photoperiods: (A) continuous light and (A) 12:12 h L:D cycle, determined from increases in cell number.

Pmax was markedly

influenced by temperature (Q,, values of ca. 2). Algae grown at 20 “C under continuous light showed enhanced (1.3-fold) pL,,, in relation to algae grown at the same temperature and 12:12 h photoperiod. However, the enhancement was very moderate considering the 2-fold increase in the total daily irradiance. Irradiances for saturation of growth (I,,) were remarkably low, generally below 25 ~Em-2.s-1. Estimates of the intercept on the y axis (,uJ yielded positive values at 20 “C, but the standard errors involved in the estimation were large enough to make values of p0 statistically not different from very small negative values. Compensation

A. IRIARTE

250

AND D.A. TABLE

Parameters photoperiods,

,

I

10

15

20

20*

PURDlr;

IV

of the p vs. I curves derived for cells of P. pmvusolii grown at various temperatures (7‘) and using different biomass indicators: cell number (cell), chlorophyll CI(chl(0 and carbon (C). Values in parenthesis are standard errors. Biomass Cdl chl a cell chl CI C cell chl n c CCH

0.0125 0.0129 0.0281 0.0284 0.0164 0.0260 0.0246 0.0196 0.0361

0.0003 (0.0030) 0.0002 (0.0026) 0.0060 (0.0019) - 0.0040 (0.0019) - 0.0010 (0.0028) 0.00 18 (0.0043) 0.0005 (0.0014) 0.0022 (0.0026) 0.0034 (0.007 1)

(0.0005) (0.0012) (0.0012) (0.0010) (0.0017) (0.001s) (0.0006) (0.0009) (0.0017)

pm, and n_ (h-r): xp (h-’ (PEm -‘.s-‘) * 24 h continuous light; the rest of values obtained

T (“3

irradiances derived as ~,;a, showed generally lower than 5 pErn-‘.s‘.

0.0011 t1.0010 0.0007 0.0009 0.0009 0.0019 0.0020 0.0022 0.0017

(0.0006) (0.0010) (0.0002) (0.0002) (0.0005) (0.0007) (0.0003) (0.0007) (0.0007)

13 13 42 31 I8 I? 12 9 21

‘): I,,, (nEm ‘.s ‘). under 12: 12 h L:D cycle.

variability

upon

biomass

indicators,

but were

DISCUSSION Pigment and carbon content

A very generalized response in plants to variations in irradiance involves changes in the concentration and relative composition of photosynthetic pigments. The 4-fold exponential decrease in cellular chlorophyll a concentration measured for P. provasolii between 3 and 400 pEme2.s -’ is in good agreement, both in magnitude and pattern, with the typical variations in chlorophyll a concentration observed in phytoplankton (Kirk, 1983). The elevated chlorophyll b to chlorophyll a ratios with decreasing levels of irradiance are a clear indication that P. provasolii responds to a reduction in irradiante enhancing primarily its light harvesting capacity in relation to reaction centres. This strategy of increasing the ratios of accessory pigments to chlorophyll a at low light has been shown for phytoplankton from a variety of taxa (Falkowski & Owens, 1980; Kana & Glibert, 1987a; Coats & Harding, 1988). Some phytoplankters do not show appreciable increases in chlorophyll a per cell; instead, they reduce their cell size, i.e. reduce the cell components other than the photosynthetic machinery (Perry et al., 1981; Langdon, 1987; Garcia & Purdie, 1992) mainly reserve compounds (Morris et al., 1974), so that with the same photosynthetic potential there is less carbon to be turned over. This is not the case for P. provasolii, since the cellular carbon content remained similar at all growth irradiances. A similar

PHOTOSYNTHESIS

AND GROWTH

OF PYCNOCOCCUS

PROVASOLII

251

pattern has been observed for the picoplanktonic cyanobacterium Synechococcu.~ spp. (Kana & Glibert, 1987a). It may not be feasible for small phytoplankton to undergo significant reductions in cell size or carbon content because of minimum requirements of incompletely scalable cell components. Photosynthesis versus k-radiance curves

Despite early claims of insufficient evidence to suggest that phytoplankton grown at low n-radiances show enhanced ability to utilize low light levels (Beardall & Morris, 1974), higher values for a normalized to cell number for cells grown at lower light have been found to be a common feature (Richardson et al., 1983; Langdon, 1988). If results obtained at 20 “C (both, 12:12 h and 24 h photoperiods), i.e. non-temperature limiting conditions, are considered, P. provasolii also exhibited reduced CInormalized to cell number, when grown at high photon flux density. Furthermore, growth at lower ii-radiances also enhanced, although to a much lesser extent, the light saturated rates of photosynthesis (Pm,,) on a per cell basis. When P,,,,, and CIare expressed per unit of chlorophyll a biomass, however, clearly high light grown cells (i.e. cells with lower chlorophyll content) displayed elevated rates at all, subsaturating to saturating, ii-radiances. The loss of efficiency of the chlorophyll a in producing photosynthetic output, as the cells accumulate more chlorophyll a per unit volume, has been ascribed to a reduction in the absorption cross section normalized to chlorophyll a, i.e. increased package effect (Geider & Osborne, 1986). Changes in the photosynthesis versus irradiance (P vs. I) curves, and hence in the photosynthetic parameters, with the growth n-radiance have also been interpreted in terms of changes in the number or size of the photosynthetic unit (PSU) (Prezelin and Sweeney, 1979; Prezelin, 1981; Ramus, 1981; Richardson et al., 1983). The photosynthetic unit represents the organized assemblage of pigment molecules on photosynthetic membranes which carry out the complete set of light reactions of photosynthesis (see Prezelin, 1976). Lower Pm,, and CInormalized to chlorophyll a for low light grown cells have been suggested to be indicative of increases in the size of PSUs, i.e. larger increases in the light harvesting pigments in relation to reaction centre chlorophylls, and the subsequent loss of energy in the transference of energy from the former to the latter. For P. provaso2ii this latter interpretation is to some extent supported by the increased chlorophyll b to chlorophyll a ratios at low irradiance. The increased values of P,,,,, per cell at low growth light, on the other hand, although a less pronounced effect, could be interpreted as a slight increase in PSU numbers as well. Even though possibly one of the two strategies, i.e. increases in the size or in the number of PSUs, appears generally dominant, the possibility of species combining to some extent both strategies has not been contemplated when interpreting the various models of theoretical responses in the P vs. Z curves of algae expressing different strategies of photoadaptation (Prezelin & Sweeney, 1979: Prezelin, 1981; Ramus, 1981; Richardson et al., 1983). However, this has been suggested to be the case for several phytoplankters, as deter-

251

A. IRIARTE AND D.A. PURDIE

mined from methods other than the analysis of the P vs. I parameters (Kana & Glibert, 1987a; Coats and Harding, 1988). Differences in the strategy of light-shade adaptation have been shown to be closely related to differences in ecological niches (FaIkowski and Owens, 1980). The strategy of increasing primarily the size of the PSUs is more effective at subsaturating irradiantes and it seems appropriate for an organism like P. provasolii, which is generally found under conditions of almost permanent low light i.e. the pycnocline area of stratified oceanic waters. A photoadaptive strategy involving mainly increases in the number of PSUs, although energetically more costly (Prtzelin, 1987) has the advantage of an increased photosynthetic output under conditions of high irradiance. This strategy has been described for phytoplankton typical of shallow waters (Falkowski & Owens, 1980) and for species that experience a dynamic light field, such as some dinoflagellates during vertical migrations in the water column (Garcia & Purdie, 1992). Despite P. provasolii showing characteristics of positive photoadaptation to low light, there was hardly any indication of photoinhibition of photosynthesis at high irradiance. A similar feature has been described for the picoplanktonic cyanobacterium Synechococcus strain WH7803 (Kana & Glibert, 1987b). Cells of P. provasolii showed enhanced photosynthetic rates when grown under 12:12 h photoperiod than under continuous light, Both, the photosynthetic efficiency and the photosynthetic capacity, were higher under the short photoperiod, not only when normalized to cell number, but also on a chlorophyll a basis. This suggests an increased number of PSUs under 12: 12 h 1ight:dark cycle in comparison to growth under 24 h photoperiod. Increasing the number of PSUs may be more advantageous than increasing the size of PSUs when changing to growth conditions of shorter photoperiod, since the instantaneous light dose does not become more limiting. The most pronounced effect of growth at 10 “C, in comparison to growth at 20 “C, was a reduction in P,,,,, (by more than 2-fold), both when expressed per unit cell number and per unit chlorophyll n concentration. This is probably due to the lower thermal energy supply for the catalysts of the dark reactions (Davison, 1991). The similarity of c(normalized to chlorophyll a concentration at different temperatures, for cells grown at similar growth photon flux densities, suggests the specific reaction rates for the light reactions of photosynthesis are independent of temperature, and thus have little or no enzymatic control. This contrasts with other studies in which a positive relationship of a with temperature was found, and for which an enzymatic control was suggested (Verity, 1981; Palmisano et al., 1987). As a consequence of the higher Q,, for P,,,;,, than for J, photosynthesis saturated at a lower irradiance at low temperature for P. provasolii. This pattern was also observed with the cryptophycean Cryptomonus erosu (Morgan & Kalff, 1979). This lowering of I, was clearly not indicative of an enhanced performance at low light when grown at the lowest temperature, and shows the need for knowledge of both parameters, P,,,,, and s(, when interpreting the parameter I,.

PHOTOSYNTHESIS

AND GROWTH

OF PYCNOCOCCUS

PROVASOLZZ

253

Growth rates The minimum generation times (i.e. In 2/p,,,) recorded of 19.2 h in continuous light and 24.4 h in 12:12 h photoperiod at 20 “C compare well the 19 h estimated for this isolate by Glover et al. (1987) in 14/10 h L:D cycle at saturating irradiance and at the same temperature. A comparison of the two different light regimes: 24 h and 12: 12 h photoperiod, shows that P. provasolii is not harmed by continuous light. However, the algae attain similar growth rates even with half the amount of light on a daily basis at low irradiances, and the increased pL,,, under 24 h irradiance is less than proportional to the increase in daily light dose. This type of response is not uncommon for phytoplankton (Foy & Gibson, 1976; Yoder, 1979; Brand & Guillard, 1981). Brand & Guillard (1981) observed no phylogenetical trend in the response of phytoplankton to continuous light and L:D cycle. The authors could only suggest a slight tendency for coastal species to reproduce at the same rate or even more rapidly under continuous light, whilst many oceanic species seemed to be harmed by continuous light. P. provasolii does not seem to conform with this pattern. A comparison between temperatures (10 and 20 ‘C) showed a clear decrease in P,,,=~ at low temperature, a feature well documented in the literature for phytoplankton (Cloern, 1977; Yoder, 1979; Meeson & Sweeney, 1982; Verity, 1982; Post et al., 1985). Since a similar pattern of decrease with decreasing temperature was observed for Pm,,, it is likely that the decrease in pmax is also related to some extent to a reduced activity of the enzymes of the dark reactions of photosynthesis. For cultures of P. provasolii grown at 15 and 20 “C under 12: 12 h L:D cycle, growth rates were shown to be maintained at near maximal rates only in a certain range of C:chl a ratios. C:chl a ratios below this range were accompanied by decreased growth rates (Fig. 6). This pattern has been observed for other phytoplankton species (Geider, 1987) and can be interpreted as decreases in C:chl a ratio not being able to fully compensate for the light limitation below a certain level of luminous energy supply. Despite the difficulties encountered in determining reliable values of OLD, given the onset of deviation from linearity with increasing light supply at low photon flux densities observed for the isolate in this study, it appears to be higher than average, following Langdon’s (1988) review. This can be interpreted as an efficient management of resources at the very low photon flux densities, these algae being better adapted to low light than the majority of phytoplankton species. Saturation of growth occurred owing to both the steep initial at very low photon flux densities, 13-42 pEm-*.s-’ slopes of ap and low pmaX. Ik for photosynthesis, however, was around 66239 ~Em-*~s-‘. Saturation of growth rate at lower irradiance than photosynthesis as derived from short term incubation measurements is a common feature in phytoplankton (Beardall & Morris, 1976; Prezelin & Sweeney, 1978; Morris & Glover, 1981; Verity, 1982) although the reasons for this are as yet poorly understood. It was also difficult to obtain reliable values of compensation irradiance (i.e. large standard errors

A. IRIAR’TE AND

D.A. PURDIE

0.03

0 6 _c 5 L? m

4

l

7 z

h A

a

l

0.02 h

0

0

0.01 a

I

0.00 0

25

/

I

50

C:chi Fig. 6. Relationship

I

75

100 a

I

125

150

1(g g-‘)

between growth rate and carbon to chlo~o~hyi~ u ratio in cells of P. /~r(~~~,~ulij grown under ~~:~2hL:Dcycleat(~)lS°Cand(~)2~~C.

of estimates). This can be accounted for by the difficulties associated with accurately measuring low irradiances, and those related with obtaining a range of irradiances in the very low end of the irradiance spectrum, with the technique used for varying irradiance in this study (i.e. combination of layers of neutral density filters) and also with the difficulties associated with the measurement of slow growth rates. These pitfalls have also been experienced in other studies (e.g. Verity, 1982). P~~.~iolo~ic~iresponses and ~cologic~i niche 2 are consistent Values of Q,e for the parameters P,,, and c(,,,~+of approximately with the typical values found for ph~~topI~kton growth rate (Raven & Geider, 1988) and indicate that the physiological effect of temperature on P. prowsok is of similar magnitude as for the majority of phytoplankton. If temperature is a limiting factor for phytoplankton with increasing latitude, we should not expect this species to be more severely limited with increasing latitude than the majority of phytoplankton. fycnococcus provasolii appears to be an organism well suited to growth at low irradiance, a capacity achieved mainly by increasing the light harvesting potential, aided to some extent by reducing carbon losses through reduced rates of respiration. This capacity to grow with low fluxes of light, together with previous findings of efficient growth under blue-violet and blue light (i.e. the wavelength bands with deepest penetration in the water column) (Giover et al., 1987), are a good reflection of the general habitat of this species, the deep region of the euphotic zone in open ocean waters (Guillard et al., 1991).

PROVASOLII

PHOTOSYNTHESISANDGROWTHOFPYCNOCUCCUS

255

ACKNOWLEDGEMENTS

We would like to thank Dr. G. Dixon for providing a culture of Pycnococcusprovusolii Guillard (clone R48-23). Financial support for this research was provided by grant from the Departamento de Educacibn, Universidades e Investigacidn de1 Gobierno Vasco to A. Carte.

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