Plant Physiol. (1994) 105: 535-543

Crowth at Low Temperature Mimics High-Light Acclimation in Chlorella vulgaris’

Denis P. Maxwell, Stefan Falk, Charles C. Trick, and Norman P. A. Huner*

Department of Plant Sciences, University of Western Ontario, London, Ontario, Canada N6A 567

Structural and functional alterations to the photosynthetic ap- paratus after growth at low temperature (5°C) were investigated in the green alga Chlorella vulgaris Beijer. Cells grown at 5’C had a 2-fold higher ratio of chlorophyll a/b, 5-fold lower chlorophyll content, and an increased xanthophyll content compared to cells grown at 27°C even though growth irradiance was kept constant at 150 pmol m-’s-l. Concomitant with the increase in the chloro- phyll a/b ratio was a lower abundance of light-harvesting polypep- tides in 5’C-grown cells as observed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and confirmed by western blotting. The differences in pigment composition were found to be alleviated within 12 h of transferring 5°C-grown cells to 27’C. Furthermore, exposure of 5°C-grown cells to a 30-fold lower growth irradiance (5 pmol m-’s-’) resulted in pigment content and composition similar to that in cells grown at 27°C and 150 pmol

temperature effects on COz-saturated Oz evolution, 5’C-grown cells exhibited light-saturated rates of O2 evolution that were 2.8- and 3.9-fold higher than 27’C-grown cells measured at 27°C and 5’C, respectively. Steady-state chlorophyll a fluorescence indicated that the yield of photosystem 11 electron transport of 5°C-grown cells was less temperature sensitive than that of 27°C-grown cells. This appears to be dueto an increased capacity to keep the primary, stable quinone electron acceptor of photosystem li (OA) oxidized at low temperature in 5°C- compared with 27°C-grown cells re- gardless of irradiance. We conclude that Chlorella acclimated to low temperature adjusts i ts photosynthetic apparatus in response to the excitation pressure on photosystem 11 and not to the absolute externa1 irradiance. We suggest that the redox state of Q,, may act as a signal for this photosynthetic acclimation to low temperature in Chlorella.

m-2 s -1 . Although both cell types exhibited similar measuring-

Under a given set of environmental conditions, photosyn- thetic organisms attempt to maintain a balance between energy supply through electron transport and energy con- sumption through carbon fixation. This balance is required to protect the organism from the detrimental effects of excess light while maintaining sufficient pools of ATP and NADPH for cellular metabolism. Sudden imbalances in the energy budget are countered by altering the efficiency of PSII pho- tochemistry via alterations in the trans-thylakoid pH gradient (Foyer et al., 1990). In addition, environmental changes may

This research was supported by Natural Sciences and Engineer- ing Research Council of Canada (NSERCC) Research Grants to N.P.A.H and C.G.T. D.P.M. was supported, in part, by an Ontario Graduate Scholarship. S.F. was supported by an NSERCC Intema- tional Postdoctoral Fellowship.

* Corresponding author; fax 1-519-661-3935. 535

induce structural and functional alterations to the photosyn- thetic apparatus, such as changes in photosynthetic unit size (Ley, 1986) or alterations in Rubisco activity (Mortain-Ber- trand et al., 1988), to maintain the energy balance.

A major environmental variable that can perturb the equi- librium between energy input and energy consumption and induce photosynthetic alterations is low temperature. Any phenotypic adjustment of functional or structural properties of the photosynthetic apparatus that can be modulated by environmental temperature is termed photosynthetic temper- ature acclimation (Oquist, 1983). Whereas photochemical reactions proceed at rates independent of temperature (Qlo of l), the rates of enzyme-mediated reactions decrease at lower temperatures (Qlo approximately 2) (Raven and Geider, 1988). Because of this, exposure of higher plants and algae to low temperature may result in the photosynthetic appa- ratus absorbing more light than can be readily dissipated through carbon fixation. From this it may be hypothesized that organisms that grow and develop at suboptimum tem- peratures would possess one or more mechanisms to maintain the energy balance. Possible mechanisms may include: (a) alterations in light harvesting and primary photochemistry to decrease the amount of light energy absorbed; (b) increased rates of cyclic or pseudocyclic electron transport (Mehler reaction); (c) increased rates of photorespiration or chloro- respiration; and (d) increased enzymic activity of the Calvin cycle, resulting in higher CO, fixation rates at low temperature.

In higher plants, a decreased measuring temperature leads to a reduction in PHM as well as a reduction in the irradiance required to achieve PMM (Stitt and Grosse, 1988). In addition, it has been reported that abrupt shifts of CJ plants from high

Abbreviations: F M and FM’, fluorescence when a11 PSII reaction centers are closed in dark- and light-acclimated cells, respectively; Fo and Fo’, fluorescence when a11 PSII reaction centers are open in dark- and light-acclimated cells, respectively; Fv and Fv’, variable fluorescence after dark acclimation (FM - Fo) and under light-accli- mated conditions ( F M ’ - Fo’), respectively; Fv/FM and Fv‘/FM’, the ratio of variable to maximum fluorescence as an expression of the maximum photochemdal yield of PSII in dark- and light-acclimated cells, respectively; LHCII, major Chl a / b light-harvesting complex of PSII; Qto, ratio of rates at temperatures differing by 10°C; Q& the primary, stable quinone electron acceptor of PSII; qp, photochemical quenching; @, apparent quantum yield of oxygen evolution; @ps,,,

quantum yield of PSII electron transport (@psn = qP X Fv’/FM’); P-, rate of photosynthesis at saturating irradiance; 8, convexity or rate of bending of a light-response curve; V-, the maximum rate of a substrate-saturated enzyme-catalyzed reaction.

536 Maxwell et al. Plant Physiol. Vol. 105, 1994

(20OC-30°C) to low (2°C-100C) measuring temperature re- sult in 0, insensitivity to CO, fixation, and that this reflects Pi limitation of photosynthesis (Sharkey, 1985). In vivo Chl a fluorescence has provided evidence that low measuring temperature causes an increase in the level of reduced Q A

under light-saturated conditions (Stitt and Grosse, 1988). However, after growth and development at 5OC, winter

wheat and rye exhibited saturating rates of O, evolution and CO, fixation that were greater than the rates from the same cultivars grown at 2OoC, regardless of measuring temperature between 5OC and 25OC (Huner et al., 1993). Similar trends have been reported for 3OOC- and 13OC-grown Brassica napus (Paul et al., 1990). The greater photosynthetic capacity found in winter wheat and rye is accompanied by a significantly greater V,,, for purified Rubisco at a11 measuring tempera- tures (Huner et al., 1993). qp analyses according to Genty et ai. (1989) and measured as a function of irradiance (50-2000 pmol m-’ s-’) and temperature (5OC-2SoC) indicate that 5OC- grown rye maintains a higher level of oxidized QA regardless of irradiance and measuring temperature (Oquist and Huner, 1993a). This is related to the finding of Mitchell and Barber (1986) that cold-grown pea plants had greater light-saturated uncoupled rates of full-chain electron transport than warm- grown plants. However, growth temperature does not affect CP for photosynthesis measured as either CPCOz or @O2 (Huner et al., 1993).

The short-term response of algae to changes in measuring temperature has been documented (Davison, 1991). As ex- pected, PMAx is directly related to measuring temperature up to a maximum in both temperate and isolated polar algae (Descolas-Gros and de Billy, 1987). In Laminaria sacckarina and Antarctic sea-ice diatoms, the initial slope of the light- response curve was shown to decline with increasing incu- bation temperature. Dark respiration generally increased with increasing assay temperature (Davison, 1991).

As observed for higher plants, long-term growth of Pkaeo- dactylum tricornutum (Li and Monis, 1982) and 1. sacckarina (Davison, 1987) at low temperature resulted in higher maxi- mum rates of CO, fixation when compared to cells grown at higher temperatures, regardless of measuring temperature. In both of these cases, and in Skeletonema costatum (Mortain- Bertrand et al., 1988), the higher photosynthetic rates at low growth temperature were correlated with increased Rubisco activity. Levasseur et al. (1990) showed that a temperature- induced decrease in carbon fixation rate in Dunaliella terti- olecta was correlated with a decrease in energy-transfer effi- ciency between the antenna and reaction center of PSII. Furthermore, growth at low temperature did not significantly change the PSII or Chl a content per cell. The latter is contrary to previous findings using the same Dunaliella species (Moms and Glover, 1974) and other algae (Thompson et al., 1992), where it was reported that Chl content decreases with a decrease in the growth temperature. However, in some algae such as S. costatum (Mortain-Bertrand et al., 1988) and many higher plants (Huner et al., 1993) Chl content is inversely related to growth temperature.

In this paper we address the following questions. (a) Since growth at low temperature can cause an imbalance between light capture and its utilization, does photosynthetic adjust- ment to low temperature in Chlorella reflect a temperature

response or an irradiance response? (b) How does photosyn- thetic acclimation to low-growth temperature in a green alga compare to photosynthetic acclimation to low-growth tem- perature in higher plants (Huner et al., 1993)T’ We have employed the green alga Chlorella vulgaris in this research for two reasons. First, it has a chloroplast struchire compa- rable to that of higher plants. Second, C. vulgaris grows well at both low and high temperatures, unlike Cklamydomonas reinkardtii (Falk et al., 1990) and species of Dunaliclla (Moms and Glover, 1974; Levasseur et al., 1990), which have low- temperature limits for growth of about 10°C-120C.

MATERIALS AND METHODS

Culture Conditions

The unicellular green alga Chlorella vulgaris Beijer (Univer- sity of Texas Culture Collection strain UTEX 265) was grown axe@cally in Bolds basal medium (Nichols and 1301d, 1965) supplemented with additional NaN03 (final concentration 500 mg/L), thiamine (0.1 ng/L), biotin (0.5 ng/L), and cya- nocobahmin (0.5 ng/L) and buffered with 5 m~ Hepes-KOH (pH 7.2).

A11 experiments were conducted using cells grcwn in 150- mL Pyrex glass tubes immersed in thermostated aquaria set at either 27OC f l0C (27OC cells) or 5OC & l0C (5OC cells). Each culture tube was aerated with sterile air (150 mL/min) and exposed to an irradiance of 150 pmol m-’ s-’ supplied by fluorescent tubes (Sylvania CW-40). The irradiance was measured from the middle of each culture tube using a quantum sensor (model 185A, Li-Cor, Inc., Lincoln, NE). Cultures were maintained in the exponential growth phase by daily dilution with fresh medium. Inocula for the cultures were maintained on agar slants for a minimum of 2 months at either 5OC or 27OC prior to use.

Determination of Crowth Rates

Cultures for growth experiments were inoadated from exponen tially growing cells exhibiting high PSII photochem- ical efficiency (Fv/FM = 0.700-0.750). Growth wa s measured spectrophotometrically as an increase in A750 (Shimadzu UV 160, Shimadzu Corp., Kyoto, Japan). Cell number was found to be dkectly correlated with A750 up to an A of 0.400. Cell samples with a higher A were diluted. Growth rates were determined using a logarithmic transformation B ccording to Guillard (1973). Qlo values were calculated using the Van’t Hoff equation.

Cell Size

Cell diameter was estimated by direct observa tion using a light microscope (Leitz Laborlux K) at lOOOX magnification. Cell volume was calculated for cells grown at both 5OC and 27OC asisuming they were spherical.

Chl Anallysis and Difference Spectra

Aliquiots of cells were centrifuged at 1500g for !j min in 15- mL Corex tubes. Each pellet was resuspended in ?O% acetone and trarisferred to a 2-mL screw-capped tube containing 0.1-

Low Temperature Acclimation in Chlorella 537

mm zirconium oxide beads (30%, v/v). Cells were broken using a Mini-BeadBeater (Biospec Products, Bartlesville, OK) for 90 s and subsequently centrifuged at 12,OOOg for 5 min. Chl in the supernatant was determined using the equations of Jeffrey and Humphrey (1975).

Difference spectra of total pigments were collected using the pigment extract from 27OC cells as the reference and compared to 5OC cells grown at either 150 or 5 pmol m-’ s-’. The concentration of the pigment extracts was adjusted such that both reference and sample gave equal A663, which cor- responds to the peak A of Chl a in 90% acetone.

Thylakoid Membrane lsolation

Cells were harvested during early exponential growth (1- 2 pg and 2-5 pg Chl/mL for 5OC and 27OC cells, respectively) and centrifuged at 30008 for 5 min. The pellet was resus- pended in wash buffer containing 10 m Tricine-NaOH (pH 7.2), 10 mM NaCl, 10 m~ KCl, 5 m MgQ, 200”M sorbitol, and 1 m PMSF. Samples were quick frozen in liquid nitro- gen before being stored at -8OOC. The thylakoid membrane fraction was isolated by passing thawed cell samples twice through a chilled French press at 55 MPa. The collected homogenate was centrifuged at 3000g for 5 min to pellet unbroken cells and cell debris. The supematant was centri- fuged at 106,OOOg for 30 min at 5OC. The pellet was washed once using the previous buffer and resuspended in 100 m Tris-HC1 (pH 7.8) containing 100 m DTT to a final Chl concentration of 1.67 mg Chl/mL before being stored at -8OOC.

Protein Determination

The protein concentration of the thylakoid membrane frac- tion was determined using the modified Bradford assay of Fanger (1987). Membrane samples (5-10 pL) were solubilized using 20 pL of a 50% (w/v) solution of octyl-b-D-glucopyr- anoside (Sigma) and incubated at 6OoC for 10 min. Solubi- lized samples were added to 5 mL of diluted Bio-Rad dye, and Asss was measured.

SDS-PAGE

Thawed membrane samples were solubilized in 5% (w/v) SDS:20% (w/v) Glc to give an SDS:protein ratio of 4:l and heated at 65OC for 20 min. Each lane was loaded with 20 pg of protein. SDS-PAGE was performed using a mini-Protein I1 apparatus (Bio-Rad), a 12% (w/v) polyacrylamide gel, and the buffer system of Laemmli (1970). Gels were run at 5OC and at a constant current of 15 mA for approximately 3 h. Gels were stained with Coomassie brilliant blue.

Western Blotting

Membrane polypeptides separated as above were electro- phoretically transferred to Immobilon (Millipore) according to Tsang et al. (1983) and probed with monoclonal antibodies raised against maize LHCII (MLH 9) (Darr et al., 1986). Blots were developed using horseradish peroxidase coupled to goat anti-mouse IgG (Sigma) with 4-chloro-1-naphthol as a color- genic substrate.

Measurements of O2 Evolution

O2 evolution and consumption were measured polaro- graphically using a thermostated, aqueous-phase Clark-type oxygen electrode (Hansatech Ltd., King’s Lynn, UK). Meas- urements were made using a 2-mL aliquot of cells with a Chl concentration of 1 to 2 r g and 3 to 5 pg Chl/mL for 5OC and 27OC cells, respectively. Prior to the measurements NaHC03 was added to a final concentration of 4 m. Each light- response curve was collected according to Falk and Samuels- soK(1992) using a single sample of cells exposed to 12 different light levels from O to 1600 pmol m-’ s-’. Actinic light was supplied by a 150-W halogen bulb using a fiber optic (Fiber-Lite, Dolan-Jenner Industries, Rochester, NH). PPFD was measured from the middle of the electrode cuvette using a photodetector (Hamamatsu G1125-02, Hamamatsu Photonic Systems Corp., Bridgewater, NJ) connected to a calibrated Li-Cor 185A quantum sensor. Ali data are pre- sented as pmol net oxygen evolution mg-’ Chl h-’.

The light-response curves were modeled using a nonrec- tangular hyperbola (Falk and Samuelsson, 1992) that has the form

ep2 - (91 + PM,)P +CPIPMM = 0.0

where P is the rate of photosynthesis ( y variable), I is the irradiance (x variable), CP is the maximum quantum yield, 8 is the convexity, and PM, is the light-saturated rate of 0 2

evolution.

Fluorescence Measurements

Daily measurements of the fluorescence parameter Fv/FM, which reflects the maximum photochemical yield of PSII, were collected during the growth experiments using a PSM Chl fluorometer (Biomonitor S.C.I. AB, Umei, Sweden) (Falk et al., 1990). Alga1 samples taken directly from the culture tubes were placed in 1.5-mL microcentrifuge tubes and dark adapted for 10 min at the growth temperature. Measurements were made by inserting the end of the instrument’s optical fiber into a tube.

Steady-state fluorescence measurements were collected using a PAM Chl fluorometer (PAM-101,103, H. Walz, Ef- feltrich, Germany) equipped with the manufacturer’s sup- plied KSlOl suspension cuvette and MKS 101 stirrer. Two Schott lamps (KL 1500, Schott Glaswerke, Mainz, Germany) provided saturating flashes and actinic illumination for pho- tosynthesis. Samples were dark adapted for 5 min in the presence of 4 m NaHC03 prior to a11 measurements. Fluo- rescence signals were recorded on a x-y recorder (Houston, Inc., Houston, TX). The Fo level was sensitized at an instru- ment frequency setting of 1.6 kHz, with the measuring beam set at the greatest amplification possible without causing photosynthetic induction. The parameters FM (determined after 5 min of dark adaptation) and F M ’ (determined in the presence of actinic light) were measured at an instrument frequency of 100 kHz by imposing 1-s flashes by the Schott flash lamp at an irradiance of 7400 pmol m-’ s-’. This was sufficient to close a11 reaction centers, since addition of 20 ~ L M

DCMU caused no further increase in the FM level. Specific fluorescence parameters were detennined using

538 Maxwell et al. Plant Physiol. Vol. 105, 1994

the PAM fluorometer in conjunction with the saturating- pulse technique described by Schreiber et al. (1986). Super- imposed upon actinic illumination were saturating pulses of 1 s duration occumng at 60-s intervals. At each of a series of light levels, once the steady-state leve1 was reached (5-10 min) the following parameters were calculated; Fv‘/FM‘ (Ha- vaux et al., 1991), qp (van Kooten and Snel, 1990), and aPsII = q p X Fv’/FM’ (Genty et al., 1989). Because of problems that this and other laboratories (Ting and Owens, 1992) have found using concentrated alga1 samples, a11 measurements were conducted using nonconcentrated samples of between 1 and 5 mg Chl/mL.

RESULTS

Effects of Temperature on Crowth Kinetics

During the exponential phase of growth Chlorella grown at 27OC had a doubling time of 8.6 -+ 0.6 h compared to 48.5 f 2.6 h for cells grown at 5OC (Fig. 1A; Table I). The Qlo for growth over a range of temperatures from 5OC to 27OC was found to be 2.26 (data not shown). At both 5OC and 2 7 T , growth of the cultures resulted in a gradual decrease in the Chl a/b ratio over time (Fig. 1B). This trend was also observed for experiments conducted at 12OC and 2OoC (data not shown). In addition, Fv/FM attained a maximum value early during growth and remained high throughout the exponen-

4

z 3 a!! 4 2 c

1 1

O

0.3t I , , , , , 4 O 48 96 144 192 240 264

Time ( h )

Figure 1. Changes in the exponential growth rate (A), Chl alb ratio (B), and Fv/FM (C) as a function of time after inoculation in C. vulgaris grown at 5°C (O) and 27°C (O). Bars represent SE; n = 3.

Table 1. Characteristics of C. vulgaris grown at 5°C and 27°C

acteristics were determined using exponentially growing cultures. Values represent means f SD of at least four experiments. Char-

Characteristic 5°C 27’C

Doubling time (h) 48.5 f 2.6 8.6 f 0.6 Chl alb 7.6 -C 0.54 3.7 f 0.41 Chl per cell (fg) 123 Ifr 28 623 f 43 Membrane protein/Chl (mg/mg) 9.1 f 1.1 4.8 f 0.3 Cell volume (pm’) 93.4 f 2.5 103.2 f 1.5

tia1 growth phase (Fig. 1C). However, at the onseí: of station- ary phase, Fv/FM decreased regardless of growth kmperature.

Although the PSM fluorescence system underestimates the absolute value of Fv/FM by approximately 25% compared with the PAM fluorescence system, the trend of decreasing Fv/FM during growth was observed using either the PSM or the PAM (data not shown).

Exponientially growing 5OC cells used for a11 subsequent experimlents had on average a 2-fold greater Chl a/b ratio, and five times less Chl per cell when compared to exponen- tially growing 27OC cells (Table I). The decrease in Chl per cell at low growth temperature could not be accounted for by a reduction in cell volume.

SDS-PACE and Western Blotting

In adldition to the changes observed in Chl per cell and Chl a/b, 5OC cells exhibited a membrane protein Chl ratio of 9.1 f 1 1, whereas 27OC cells had a membrane protein:Chl ratio of approximately 4.8 f 0.3 (Table I). Thus. membrane proteins, were solubilized on the basis of the SDS:protein ratio rafher than on an SDS:Chl ratio. Figure 2 A illustrates the meimbrane protein profiles of 5OC- and :27°C-grown Chlorella. It is apparent from Figure 2A that :17°C-grown cells have a greater abundance of the major polypeptides in the 28-kD region. These polypeptides were shown to be LHCII by cross-reactivity with monoclonal antibodies raised against maize LHCII using western blot analysis (Fig. 2B). This is consistent with the differences in the Chl a/b ratio. However, Figure 2A also shows that polypeptides in the range of 25 to 27 kD and 66 kD appear to be more abundant in 2 7 T than in 5OC cells. Furthermore, a 23-kD polypeptide that is present in 27OC cells appears to be absent in low- temperature-grown Chlorella. We have not yet identified these pdypeptides.

Effects of Measuríng Temperature on Photosynthesis

The culturing conditions used for Chlorella resulted in high Fv/FM values that were comparable to values reported for higher plants (Bjorkman and Demmig, 1987) and Chlamydo- monas reinhardtii (Falk and Samuelsson, 1992). In addition, it can be seen from Table I1 that growth and measuring temperature had minimal effects on Fv/FM.

A11 values derived from measurements of O2 evolution in Table [I are the modeled values calculated by using the quadratic equation presented earlier. By its implementation, values for both PMvIAx at infinite irradiance and 4, at infinitely

Low Temperature Acclimation in Chlorella 539

14.4*MN»

27°C 5°C

27°C 5°C

B

Figure 2. A, SDS-PACE of membrane polypeptides from C. vulgarisgrown at 27°C and 5°C. Each lane was loaded with 20 \i% of protein.Numbers at left indicate molecular masses (kD) of the standards inthe first lane. Arrowheads on the right indicate regions mentionedin the text. B, Western blot analysis of a gel similar to the one aboveusing monoclonal antibodies of maize LHCII.

low irradiance could be determined. When measured at 27°C,5°C cells had a PMAX that was 5.7 times greater than whenmeasured at 5°C (Fig. 3; Table II), but measuring temperaturehad no significant effect on $. Cells grown at 27°C had aPMAX that was 8.0 times greater and a 4> that was 20% greaterwhen measurements were made at 27°C compared withmeasurements made at 5°C.

0 as well as the initial slope ($) affect photosyntheticefficiency (Falk and Samuelsson, 1992). For example, when0 is low the light-response curve deviates from linearity atvery low irradiance, with the quantum yield of photosyn-thesis decreasing rapidly and continuously as the irradianceincreases. As shown in Figure 3 and Table II, there is a strongmeasuring-temperature effect upon G. When 5°C cells aremeasured at 27°C, 0 is reduced almost to zero compared to0.605 at 5°C. A lower 0 was also observed at the higher

O

500

400

300

200

O 10Q"5

-100 -<500 1000 1500 2000

—2 —1PPFD (/imol m s )

Figure 3. Light-response curves of Oz evolution of C. vulgaris grownat 27°C (squares) and 5°C (circles), measured at both 27°C (closedsymbols) and 5°C (open symbols). Bars represent SE (not shown ifsmaller than symbol), n = 3-5.

measuring temperature for 27°C cells, although the decreasewas not as great.

Effects of Growth Temperature on Photosynthesis

To assess fully the effects of different growth temperatureson photosynthesis, comparisons must be made at the samemeasuring temperatures. The results presented in Table IIand Figure 3 indicate that 5°C cells had a PMAX that was 2.8and 3.9 times greater than that of 27°C cells when measuredat 27°C and 5°C, respectively. When measured at 27°C, bothlow- and high-temperature-grown cells exhibited similar 4>,although 5°C cells had a much lower 0 (Table II). In contrast,when measured at 5°C both types of cells had similar 0, but27°C cells had a slightly lower *.

Effects of Growth and Measuring Temperature onRespiration

The rate of respiration under specific conditions is a keyparameter in evaluating the overall physiological state of anorganism. As expected, there was a strong measuring tern-

Table II. Photochemical efficiency of PSII (FV/FM) and modeled O2 evolution parameters for C. vulgarisgrown at 5°C and 27°C

Measurements were made at both temperatures for each growth regime. Values represent themean ± so. For FV/FM, n > 10; for all other parameters, n = 5.

Measured at 5°C Measured at 27°CParameter

5"C grown 27°C grown 5°C grown 27°C grown

0.720 ±0.01 3 0.735 ±0.011 0.745 ±0.021 0.751 ±0.022

e'max

Respiration1

0.726 ±0.1060.605

136.1 ± 5.227.8 ± 10.2

0.643 ± 0.0480.674

34.6 ± 1.26.4 ± 2.2

0.756 ± 0.0420.014

781. 9 ±59.598.4 ±21. 5

0.804 ± 0.0720.483

277.5 ± 13.574.7 ± 15.5

' Measured using the RAM. b /imol O2 (mg Chl)"' (mol PFD)~' m2. c /imol O2 (mg Chl)"' h

540 Maxwell et al. Plant Physiol. Vol. 105, 1994

perature effect on respiration in both 5OC- and 27OC-grown Chlorella (Table 11). The Qlo for respiration of 5OC cells was 1.77 compared to 3.06 for 27OC cells. However, Table I1 also shows that regardless of measuring temperature 5OC cells had a greater rate of respiration than 27OC cells. This is consistent with data published for higher plants (Huny et al., 1992).

Effeds of Crowth and Measuring Temperature on Steady-State Chl Fluorescence

To investigate steady-state photochemical processes of PSII in vivo, we employed modulated fluorescence (Schreiber et al., 1986). Figure 4C indicates that both 5OC and 27OC cells exhibited a reduced @psII when measured at 5OC. However, the @psII of 27OC cells appeared to be more temperature sensitive than that of 5OC cells. Since the yield of electron transport through PSII is given by @psII = qF X Fv’/FM’ (Genty et al., 1989), one can determine which of the parameters contributing to @’psII is affected by changes in growth and measuring temperature. Figure 4B shows that unlike 27OC cells, qp of 5OC cells was insensitive to changes in measuring

0.8

2 LL 0.6 Y LL’

0.4

1 .o

0.8

mn 0.6

0.4

0.2

0.0 1 O 500 1000 1500

-2 -1 PPFD (pmol m s )

Figure 4. Steady-state fluorescence parameters Fv‘/FM‘ (A), qp (B), and @psII (C) as a function of irradiance for C. vulgaris. Circles represent 27°C-grown cells measured at 27°C (O) and 5°C (O). Squares represent 5°C-grown cells measured at 27°C (O) and 5°C (a). Bars represent SE (not shown if smaller than symbol). n = 4-8.

W o c o

O cn n Q

e

0.3

0.2

o. 1

0.0

400 500 600 700

Wavelength (nm)

Figure 5. Difference spectra of pigments of C. vulgaris in 90% acetone. The pigment extract from cells grown at 27°C and 150 pmol m-’s-’ was used as the reference against extracts from cells grown at 5°C and 150 pmol m-* s-’ (-) or cells growri at 5°C and 5 pmol m-’ s-l (- - -). Extracts were diluted to give zero difference at the Chl a peak at 663 nm.

temperaíure. Thus, the sensitivity of @psll to meamring tem- perature in 5OC cells can be accounted for solely by changes in FV’/FlLI’ (Fig. 4A). In contrast, the sensitivity of @psll in 27OC cells to measuring temperature appears to be due to a combination of both FV’/FM’ and qF. Thus, it appears that 5OC cells have a greater capacity to keep QA oxidized at low temperaíture than 27OC cells, regardless of irradiance.

Effect of Crowth lrradiance on Pigment Compo!iition

Low temperature may reduce rates of photosyní hesis, lead- ing to the absorption of light energy in excess of demand. Thus, we hypothesized that the changes in pigment and polypepítide composition observed between 5OC- and 27OC- grown cells were due to excessive excitation pressure experi- enced by the cells grown at 5OC, even though the growth irradiance was kept constant. If this is correct, then growth of 5OC cells at a lower irradiance should eliminate these differences in pigment composition. Figure 5 presents differ- ence spectra of alga1 pigments extracted in 90% acetone and normalized at 663 nm (Chl a maximum). When on e compares 5OC and 27OC cells grown at the same irradiance (Fig. 5, solid line), low-temperature-grown cells have less Chl! b, as indi- cated by the negative minimum at 645 nm, but appear to have a higher content of xanthophylls, as indiated by the positive absorption maxima at 423, 445, and 481 nm (Diaz et al., 19190). This has been confirmed by preliminary HPLC analysis (our unpublished data). To assess further the effect of irradimce at low growth temperature, cells were grown at 5OC and 5 @mo1 m-’s-’. When comparing 27OC cells grown at 150 pmol m-’s-’ with 5OC cells grown at lov~ irradiance (Fig. 5, dashed line), no difference in Chl b content was found, and the higher levels of xanthophylls in the 5OC cells grown at 150 pmol m-* s-I were eliminated. The ratio of Chl a/b in 5OC Chlorella grown at 5 pmol m-2 s-’ was 3.5 k 0.2, which i!< similar to the ratio in 27OC-grown control cells (Table I).

To separate the effects of growth temperature f rom growth

Low Temperature Acclimation in Chlorella 54 1

irradiance on pigment composition, we followed the kinetics of changes in Chl per cell and the ratio of Chl a/b as a function of time after shifting an exponentially growing culture of 5OC cells to 27OC while maintaining a constant irradiance of 150 pmol m-’ s-’. Figure 6 illustrates that a decrease in Chl a/b and an increase in Chl per cell was detected within 1 to 2 h after the shift and that the growth temperature effect on the ratio of Chl alb was complete after 12 h. By 12 h the Chl a/b ratio had been reduced by 50%, concomitant with a 6-fold increase in Chl per cell, such that both pigment content and composition were similar to 27OC- grown control cells. We conclude that the alterations in pigment content and composition of 5OC-grown Chlorella reflects an irradiance response.

DISCUSSION

This investigation has shown that growth irradiance can play a key role in low-temperature acclimation in algae. This is supported by the steady-state fluorescence measurements (Fig. 4). qp reflects the redox state of QA (Bradbury and Baker, 1981) and thus is an estimate of the ratio of (QA)ox/[(Qn)red +

At constant irradiance, low temperature should cause a decrease in qp as a result of the low-temperature-induced reduction in the rates of photosynthesis. This effect is illus- trated in Figure 48 when one compares the qp of 27OC-grown Chlorella measured at a constant irradiance of 150 pmol m-’ s-’ but at either 5OC (Fig. 4B, O) or 27OC (Fig. 4B, O). Thus, exposure to a combination of low temperature and an irra- diance of 150 pmol m-’ s-’ potentially increases the excitation pressure on PSII, as reflected by a significant decrease in qp. One may expect that cells grown at 5OC and 150 pmol m-2 s-’ to exhibit a qP similar to that observed for 27OC cells measured at 5OC and 150 pmol m-’s-*. However, Figure 4B (W) shows that this is not the case. In fact, Chlorella grown at 5OC and 150 pmol m-’s-’ exhibits a qF similar to 27OC cells measured at 27OC and 150 pmol m-’s-’. Clearly, growth at low temperature has allowed Chlorella to adjust photosyn- thetically to reduce the excitation pressure on PSII and keep a greater proportion of Q A in the oxidized state.

600

500

a, 400 0

L 300 O

0-l

200

1 O0

- -

1

L

I O 4 8 12 16 20 24

Time after shift (h)

Figure 6. Change in Chl content (fg Chl/cell, O) and the ratio of Chl a/b (O) as a function of time after shifting an exponentially growing culture of C. vulgaris from 5°C to 27°C. The irradiance remained constant at 150 pmol m-’ s-’.

We suggest that the photosynthetic adjustment is reflected, in part, by severa1 phenomena. First, by a reduction in Chl per cell, by a higher ratio of Chl alb, and by a decreased content of LHCII polypeptides. These adjustments would act to reduce the probability of light absorption. Second, the greater xanthophyll content in 5°C-grown cells may decrease the excitation pressure on PSII at low temperature by increas- ing the capacity for the nonradiative dissipation of excess light energy as heat (Demmig-Adams, 1990). Third, the in- creased photosynthetic capacity of 5OC- compared to 27OC- grown cells (Fig. 3) will also decrease the excitation pressure on PSII through concomitant increases in the flux of electrons (@psiI x I ) through PSII (Fig. 4C). Furthermore, we have shown that 5OC-grown Chlorella is much more resistant to photoinhibition than cells grown at 27OC (Maxwell et al., 1993).

We believe that these data support the hypothesis that cells grown at 5OC, in addition to being acclimated to low temperature, are acclimated to growth under an excessive excitation pressure on PSII even though the extemal growth irradiance at both 5OC and 27OC was identical(l50 pmol m-’ s-I). Thus, Chlorella may be responding to modulation of the redox state of PSII by growth temperature. This may explain why Chlorella (Maxwell et al., 1993), winter rye (Huner et al., 1993; Oquist and Huner, 1993a), and winter wheat (Hurry et al., 1992; Huner et al., 1993) exhibit an increased resistance to high light (1500-2000 pmol m-’ s-I) after growth at low temperature and low to moderate irradiance (150-250 pmol m-’ s-I).

Additional support for the idea that excitation pressure plays a key role in low-temperature acclimation comes, first, from growth of Chlorella at constant low temperature but decreasing growth irradiance and, second, from shifting a 5OC-grown culture to 27OC while maintaining constant irra- diance. A 30-fold reduction in growth irradiance at a constant growth temperature of 5OC resulted in Chl a/b and Chl per cell similar to that observed for 27OC cells. Furthermore, the rapid alterations in pigment composition (Chl per cell, Chl a / b ) that occurred within hours of shifting a 5OC-grown culture to 27OC illustrates that low temperature presents a thermo- dynamic constraint to the metabolism of Chlorella cells that is quickly released upon shifting to a higher temperature.

Our hypothesis is further supported by the fact that growth of Chlorella at 5OC and 150 pmol m-‘ s-’ mimics many of the changes reported for irradiance stress in Chlorella (Ley, 1986; Senge and Senger, 1990) and related species such as Dunaliella (Smith et al., 1990; Hamson et al., 1992). Figure 6, which shows changes in Chl content and Chl a/b ratio after shifting a 5OC-grown culture to 27OC, is very similar to the results of the work by Hamson et al. (1992), in which changes in Chl content and Chl alb ratio were followed after restoration of irradiance-stressed Dunaliella to normal growth conditions. In addition, Ley (1986) showed that the ratio of Chl a/b in Chlorella increased 10-fold and that Chl per cell decreased by 80 times when grown at light levels ranging from 0.5 pmol m-’s-’ to over 4000 pmol m-’ s-’. It has also been shown in Chlorella pyrenoidosa (Fujita et al., 1989) and Dunaliella salina (Smith et al., 1990) that the increase in Chl alb during growth at high irradiance is correlated with a decrease in the abundance of LHCII polypeptides. Increased

542 Maxwell et al. Plant Physiol. Vol. 105, 1994

carotenoid content as well as an increase in the thylakoid protein:Chl ratio (Smith et al., 1990) are also characteristic of growth at high irradiance.

Certain changes observed in PSII in Chlorella are different from that reported for cold-tolerant higher plants. Growth of both winter rye (Oquist and Huner, 1993a) and winter wheat (Huny et al., 1992) at 5OC resulted in a 40 to 60% increase in Chl per unit leaf area and no change in Chl a / b or content of LHCII polypeptides when compared with plants grown at 20 to 25OC. In those experiments the irradiance was main- tained at 250 pmol m-'s-'. However, the greater photosyn- thetic capacity of Chlorella grown at 5OC compared to 27OC is similar to the findings for cold-tolerant higher plants, including winter rye and winter wheat (Huner et al., 1993). The greater photosynthetic capacity found in winter rye is reflected in a greater ability to keep Q A in the oxidized state (greater qp) regardless of measuring temperature (Oquist and Huner, 1993a). In Chlorella, when measurements were made at 5OC, qp was strongly inhibited in 27OC cells compared to cells grown at 5OC. This is consistent with a recent report conceming the temperature sensitivity of qp in 5OC- and 20°C-grown winter rye (Oquist and Huner, 1993b). This inhibition of primary photochemistry in 27OC cells measured at low temperature may indicate that growth of Chlorella at low temperature may result in alterations to the lipid com- ponents of the thylakoid membrane, allowing for higher photochemical activity at low 'temperature. Acclimation of Dunaliella to 12OC caused a pronounced increase in the level of fatty acid unsaturation in phosphatidylglycerol and phos- phatidylcholine, which was correlated with a decrease in the threshold temperature for thermal stability of the thylakoid membrane (Lynch and Thompson, 1984). This is currently under investigation for Chlorella.

As is shown in Figure 4, the higher capacity for photosyn- thesis in low-temperature-grown cells measured at 27OC, compared with 27OC cells, is not achieved through a higher yield of PSII electron transport. This suggests that the greater capacity for photosynthesis in 5OC cells at the high measuring temperature is due to alterations in carbon fixation. This notion is further supported by the low convexity of the light- response curves when 5OC cells are measured at 27OC. The low convexity indicates that the overall rate of photosynthesis is probably limited by the tumover of PSII and not by other processes such as the reoxidation of the plastoquinone pool or carbon fixation. The finding that the convexity of 27OC cells does not decrease to the extent of 5OC cells at the high measuring temperature suggests that the capacity for electron transport (reoxidation of plastoquinone) and/or the capacity of the Calvin cycle is greater in 5OC cells. However, this must by confirmed by further experimentation.

In summary, the results presented support the hypothesis that, due to thermodynamic constraints, C. vulgaris accli- mated to low temperature adjusts its photosynthetic appara- tus in response to the excitation pressure on PSII imposed by light in excess of that required for photosynthesis and not the absolute irradiance. This has important implications for a11 studies that address the effects of environmental temper- ature on plant physiology, biochemistry, or molecular biol- ogy, since any change in temperature will result in concomi- tant changes in excitation pressure. Thus, under normal

growth conditions, temperature effects per se can be assessed accurately only if they are examined in parallel with changes in excitation pressure. The photosynthetic adjujtments of Chlorella to growth at low temperature are similar to those described in published reports of photosynthetic acclimation to irradialnce stress in algae. We propose that the redox state of Q A miay act as a signal for photosynthetic acchation to low temperature in higher plants and algae. This signal may then trigger structural and/or functional adjustments at the level of the thylakoid membrane and photosynthetic carbon metabolism. This is currently under investigation.

Received October 18, 1993; accepted February 14, 1994. Copyright Clearance Center: 0032-0889/94/105/0535/09.

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