Theoretical excessive excitation pressure.
The green algae Chlorella sp. DT (DT) and Chlorella pyrenoidosa 211-8b (8b) had similar cell growth rates and photochemical efficiency (Fv/Fm) when they were cultivated under a moderate irradiance of 120 μmol photons m−2 s−1 in combination with a series of temperatures that decreased from 32 to 7°C. Upon shifting the cultures to the relatively high irradiance of 240 μmol photons m−2 s−1, DT exhibited higher cell growth rates than 8b under the chilling temperatures of 20°C and 15°C and differences in the Fv/Fm and Chl a/b ratios from 8b. In particular, DT possessed more new differentially induced SOD isoforms than 8b.
- chilling temperature
- relatively high irradiance
- chlorophyll content
- Chl a/b
- superoxide dismutase
The unicellular green alga
Photosynthesis is the energy source for the growth and development of photosynthetic organisms. Photosynthetic efficiency is reliant on environmental conditions such as light and temperature. At low temperatures, algae experience reduced photosynthetic efficiency, whereas in high-light environments, they absorb more energy than they can consume in the photosynthetic processes . The absorption of too much energy can lead to an increase in the production of reactive oxygen species (ROS), which can damage the photosynthetic apparatus and further decrease photosynthetic efficiency . Therefore, in response to wide daily and seasonal fluctuations in temperature and light, algae must possess some protective and regulatory systems to avoid this “energy excess” [6, 7, 8, 9].
Upon initial exposure to low temperature or high irradiation, excessive excitation pressure may be induced between the rate of energy absorbed via the photosynthetic antenna and energy utilization [4, 5, 10, 11, 12]. One of the protection mechanisms that algae and higher plants employ to avoid receiving too much light energy is to adjust their chlorophyll (Chl) a/b ratios and the structure of the photosystem I and II (PSI and PSII) antenna complexes in response to different combinations of light intensity and temperature [2, 13, 14, 15]. Light-harvesting complexes (LHCs) with modified Chl composition have the ability to absorb different levels of light energy depending on the environmental conditions [16, 17, 18]. Another protective mechanism of algae and plants after receiving too much light energy is to adjust the antioxidant response of the scavenging system such that any excess excitation pressure is transferred to the superoxide radical (O2·−) pathway and other derived reactive oxygen species (ROS) . Superoxide dismutase (SOD, EC 126.96.36.199) is known as the first line of cellular defense against oxidative stress, and it catalyzes the dismutation of O2·− to H2O2 and O2. There are three distinct types of SOD classified on the basis of their metal cofactors: the copper/zinc (CuZnSOD), iron (FeSOD), and manganese (MnSOD) isoenzymes . SOD activity increases in cells in response to diverse environmental stresses including high light and chilling temperatures [21, 22, 23].
The primary objective of this present work was to explore combinations of light and temperature in algal cultures that may inform optimization of production system in manufacturing [24, 25]. The two warm-climate green algae,
2. Materials and methods
2.1 Chlorella culture and growing conditions
2.2 Chlorophyll (Chl) determination
The growth of algal cultures was monitored by measuring the total Chl (Chl
The cell specific growth rate (μ, day−1) was derived from the difference in cellular Chl content over time as follows:
2.3 Measurement of chlorophyll fluorescence parameters
Chlorophyll fluorescence was measured using a modulated chlorophyll fluorometer (Hansatech Instruments Ltd., Norfolk, UK). Algal samples were collected at the indicated times, adjusted to a concentration of 4 μg Chl mL−1, and dark adapted for a period of 10 min at room temperature before measurement. The fiber-optic probe of the fluorometer was then placed into the chamber with 2 mL of the algal samples. The minimum fluorescence (F0) with all open PSII reaction centers was determined by a weak non-actinic modulated light (<0.1 μmol m−2 s−1). The maximum fluorescence (Fm) with all closed PSII reaction centers was induced by a saturating pulse of white light (1 s, 13,000 μmol m−2 s−1) and measured after applying the actinic light (300 μmol m−2 s−1). The variable fluorescence (Fv) was calculated as Fm-Fo, and the ratio of the variable to maximum fluorescence (Fv/Fm) represented the maximal photochemical efficiency [29, 30].
2.4 Spectrophotometrical assay of SOD activity
Algal cells from the log phase were collected by centrifugation. The cell pellet was washed three times and resuspended with extraction buffer (100 mM K2PO4 and 5 mM EDTA, pH 7.0). Algal cells were then broken down by sonication (V500, SONIC, USA) and centrifuged at 10,000 × g (MK-201, Sigma, Germany) for 15 min. The supernatant obtained was the algal crude extract. Protein concentrations of algal crude extracts were determined by the method of Lowry et al.  using BSA as the standard. The algal crude extracts were then subjected to a SOD assay.
The activity of the SODs was determined by measuring the inhibited reduction of cytochrome c (cyt c) because the SOD competed with cyt c for superoxide radicals, thus inhibiting cyt c reduction . The reduction of cyt c was measured by monitoring the change in absorbance at 549 nm (cyt c absorption) when xanthine oxidase was added to a reaction mixture of K2PO4 (pH 7.8), xanthine, and cyt c (oxidized) at room temperature. After a few seconds, the algal crude extract was added to the reaction mixture. SOD activity was then calculated as 50% of the inhibited reduction rate of cyt c. An extinction coefficient of ε549nm = 21 mM−1 cm−1 for cyt c was used.
2.5 Native PAGE analysis of SOD
About 5–20 μg of protein from algal cell crude extracts suspended in sample buffer comprising 12.5 mM Tris–HCl (pH 6.8), 0.02% (w/v) bromophenol blue, and 4% (v/v) glycerol was loaded into each well. SODs in the algal extract were separated by native polyacrylamide gel electrophoresis (native PAGE) (10%). After electrophoresis the gels were washed with 100 mM K2PO4 buffer (pH 7.8) for 10 min and incubated in staining buffer composed of 20 mM K2PO4 buffer (pH 7.0), 0.05 mM riboflavin, 0.1 mM nitroblue tetrazolium (NBT), and 0.2% (w/v) TEMED in the dark at room temperature for 30 min . Then gels were washed twice with 100 mM K2PO4 butter (pH 7.8) and exposed to light until the development of colorless bands. The colorless bands on the purple-stained gel indicated the existence of SOD because the free radicals produced by riboflavin are removed by the SOD, and as a consequence the colorless oxidized NBT in the SOD band is not converted into its purple reduced form. The reaction was then stopped by immersing the gels in deionized water. The SOD isoenzymes were identified on the basis of their sensitivity to KCN (5 mM) or H2O2 (10 mM), which were added into the staining buffer when required. MnSODs are resistant to both inhibitors, Cu/ZnSODs are sensitive to both inhibitors, and FeSODs are resistant to KCN but sensitive to H2O2.
3.1 Enhanced inhibition of cell growth by a combined stress of doubled irradiance and chilling
The Chl content was monitored during acclimation because it represented not only the level of cell growth but also the capacity for light absorption . Under a moderate irradiance of 120 μmol photons m−2 s−1 at 32°C (Figure 1A), the Chl content of DT algal culture increased with acclimation time. The DT culture exhibited a maximum specific growth rate of 2.2 μg Chl mL−1 day−1 and reached a stationary phase on Day 3 at a content of 92 μg Chl mL−1. At 20°C, DT growth was inhibited on Day 1, but growth resumed at a slower rate than the control on Day 2. When the temperature was lowered to 15, 10, or 7°C, DT stopped growing and even showed negative growth rates; 15°C seemed to be a critical temperature at which no net growth was observed. Under a doubled irradiance of 240 μmol photons m−2 s−1 at 32°C (Figure 1B), DT cells exhibited a maximum specific growth rate of 2.7 μg Chl mL−1 day−1 and reached the stationary phase on Day 1 at a content of 70 μg Chl mL−1. At 20°C, DT cells initially ceased growth under this doubled irradiance but resumed growth on Day 3. Transferring cultures to temperatures below 20°C promoted cell death, while the critical temperature for avoiding the negative growth rate now rose to 17°C, two degrees higher than for moderate irradiation.
A similar response was observed in 8b cells during acclimation at the various temperatures. The 8b exhibited maximum specific growth rates of 2.1 and 2.7 μg Chl mL−1 day−1 at 32°C on Day 1 under irradiance of 120 and 240 μmol photons m−2 s−1, respectively, and reached the stationary phase at about 98 and 69 μg Chl mL−1 on Day 3 (Figure 2A
Therefore, although a slightly higher maximum specific growth rate was observed initially at 32°C, lower temperatures induced enhanced inhibition of cell growth in both DT and 8b, and this was further inhibited under doubled irradiance.
3.2 The readjustment of Chl a/b ratios
In order to understand the influence of Chl composition on excitation energy transfer, the Chl
Under 240 μmol photon m−2 s−1 irradiance, the Chl
3.3 Change in PSII photochemical efficiency
To assess the photochemical efficiency of PSII, the ratio of the variable to maximum fluorescence (Fv/Fm) was measured [28, 29]. In both algal cultures, the Fv/Fm ratios of the DT and 8b controls stayed initially in the 0.83–0.85 range at 32°C with an irradiance of 120 μmol photons m−2 s−1, but decreased slightly to 0.74–0.75 by the end of the acclimation period (Figures 1E
Under 240 μmol photon m−2 s−1 irradiance, at 32°C the Fv/Fm ratios of the DT and 8b strains also remained in 0.79–0.80 range (Figures 1F
3.4 Shielding effects of high algal cell concentrations on light absorption
In order to understand whether the initial concentration of algal cells affected light absorption during chilling acclimation, cell growth was measured at different initial concentrations of 2, 4, and 6 μg Chl mL−1 under the doubled irradiance of 240 μmol photons m−2 s−1.
By Day 3 following the initial cessation of growth at 20°C (Figure 3A
At 15°C, the cell growth of DT and 8b gradually declined with time regardless of the initial Chl concentrations. Nevertheless, at initial concentrations of 4 and 6 μg Chl mL−1, both strains were slower to die than cultures starting out at 2 μg Chl mL−1 (Figure 4A
The results suggested that the initial concentration (2, 4, and 6 μg Chl mL−1) of algal cells did affect light absorption, but temperature was the major factor determining cell growth (Figures 3, 4). DT had a slightly greater tolerance at 20°C than 8b because its Chl a/b ratios attained levels higher than the control (Day 0), even though the Chl a/b ratios of 8b also returned to slightly above the control level. However, neither DT nor 8b could overcome the stress of low temperatures of 15°C and below combined with the doubled irradiance of 240 μmol photons m−2 s−1.
3.5 Differential induction of multiple SOD isoforms
For the duration of the 15°C acclimation with 120 μmol photon m−2 s−1 irradiation, the specific growth rate of algal cells remained zero, implying that the energy input and output seemed to reach a critical point. To understand the contribution of antioxidants in scavenging ROS produced during chilling acclimation, the SOD activities were assayed with a spectrophotometrical method. It was found that DT had an approximately twofold higher rate of SOD activity (0.46 μmol mg−1protein sec−1) than 8b (0.21 μmol mg−1 protein sec−1). Moreover, when the expression of SOD isoforms was examined after activity staining on native PAGE, three distinct colorless bands were observed in DT (Figure 5) while only two bands were observed in 8b (Figure 6). The SOD activities of both strains were generally amplified with time and decreasing temperature. At the same time, some new SODs were induced, and some were diminished.
As shown in Figure 5, the DT control contained two DTMnSODs and three DTFeSODs, which were verified with inhibitors of H2O2 and KCN. Once the culture was moved to 15°C, the SOD activities of the DT increased greatly on Day-1 and reached a maximum on Day 2. By Day 4, at least 10 SOD isoforms were observed in DT including two new DTMnSODs and three new DTFeSODs. However, by Day 8, SOD activities declined, and some isoforms disappeared, leaving only three DTFeSODs and two DTMnSODs present. Similarly, as shown in Figure 6, the 8b control contained two 8bMnSODs and two 8bFeSODs. The SOD activities of 8b were amplified on Day 2 and reached a maximum on Day 4, while one new 8bMnSOD and two new 8bFeSODs were induced. At the end of acclimation, the SOD activity declined, and only two 8bFeSODs and two 8bMnSODs were present. These results suggest that DT and 8b utilize different strategies for scavenging O2·−. We found that MnSOD1 and FeSOD1 were the most abundant isoforms in both
For further identification of which SOD isoforms responded to light stress and to temperature stress, the SODs were analyzed under lower temperature or doubled irradiance. In the DT culture, the original SOD isoforms of DTFeSOD1, DTFeSOD2, DTMnSOD1, and DTMnSOD2 were amplified in response to both the lower temperature of 10°C (Figure 7A) and to a doubled irradiance of 240 μmol photons m−2 s−1 (Figure 7B). DTFeSOD3 disappeared on Day 1, probably because it was sensitive to both higher light and lower temperature. A newly induced DTFeSOD4 appeared on Day 1 in response to doubled irradiance, but it was not detected until Day 1 under moderate irradiance, implying that DTFeSOD4 was probably more sensitive to light than to low temperatures. In 8b culture, in addition to the original SOD isoforms of 8bFeSOD1, 8bFeSOD2, 8bMnSOD1, and 8bMnSOD2, some new isoforms were induced. They were amplified in response to the lower temperature of 10°C (Figure 7A) and the doubled irradiance of 240 μmol photons m−2 s−1 (Figure 7B) on Day 1. However, 8bMnSOD1 declined on Day 2. In spite of new SOD isoforms being amplified and in spite of the expectation that the SODs would prevent cell death, under the two combined stresses, the algal cells were still dying.
4.1 Imbalance in excitation pressure
The specific growth rates on Day 1 from DT and 8b were plotted as a function of the cultivation temperatures (Figure 8). This showed that the specific growth rates decreased exponentially with decreasing temperatures from 32 to 10°C. Our results did not follow the previous observation of Sandnes et al.  where the specific growth rate of the green alga
Furthermore, the relationship of specific growth rates versus cultivation temperatures was theoretically simulated in accordance with the excessive excitation pressure. The temperature coefficient (Q10) represents the factor by which the speed of a biochemical reaction approximately doubles for every 10°C rise. Although some evidence indicated that Q10 in plants is temperature dependent , a Q10 of 2 was used here to theoretically estimate excessive excitation pressure. Therefore, the excessive excitation pressure due to the reduction in biochemical processes was calculated as (T, temperatures below 32°C) so that the theoretical excessive excitation pressure of 2.3-fold at 20°C, of 3.3-fold at 15°C, and so on were calculated relative to the control (onefold at 32°C) (Table 1). The diminished activities caused by theoretical excessive excitation pressure were plotted as a function of acclimation temperature (Figure 8). Subsequently, another plot was obtained for the doubled irradiance of 240 μmol photons m−2 s−1, assuming that the excessive excitation pressure was twice the value under 120 μmol photon m−2 s−1 irradiance. However, the experimental curves did not follow the theoretical ones, implying that regulation of the response to the combined light and temperature stresses was more complicated than expected.
|Irradiation at 120 μmol photons m−2 s−1||Irradiation at 240 μmol photons m−2 s−1|
|Temperature||Theoretical excessive excitation pressure (fold)||DT cell specific growth rate (μ) on Day-1 (μg Chl day−1)||8b cell specific growth rate (μ) on Day-1 (μg Chl day−1)||Temperature||Theoretical excessive excitation pressure (fold)||DT cell specific growth rate (μ) on Day-1 (μg Chl day−1)||8b cell specific growth rate (μ) on Day-1 (μg Chl day−1)|
In our experiments, under a moderate irradiance of 120 μmol photons m−2 s−1, DT and 8b showed no significant differences in growth rates and photochemical efficiency when subjected to various low temperatures. However, under a doubled irradiance of 240 μmol photons m−2 s−1, DT had a slightly higher growth rate than 8b at temperatures below 20°C. This suggests that DT might possess a more efficient energy dissipation system against the combined stress of low temperatures and high irradiation than 8b. These results are in agreement with reports that the impact from photoinhibition due to low temperature and high light varies greatly across different green algal species [41, 43, 44, 45]. Although a greater specific growth rate was obtained under 240 μmol photon m−2 s−1 irradiance compared to 120 μmol photon m−2 s−1 irradiance, neither DT nor 8b favored high irradiance because a smaller Chl content was found during the stationary phase, that is, less biomass was generated.
In order to control light energy absorption and transfer, the LHC must modify the pigment composition of the Chl
Despite the apparent decrease in the Fv/Fm ratios in our 10 and 7°C acclimation experiments, an initial increase and then a quenching of Fo was observed (data not shown). This phenomenon has been found in
4.2 Differential SOD response
Since SOD is the first line of cellular defense against oxidative stress to remove O2·−, monitoring how SOD responds to photoinhibition during acclimation may provide more information about photoprotection . It is known that SOD activity increases in cells in response to diverse environmental stresses including high light intensities and low temperatures and that SOD isoforms are expressed differently to protect against a subset of oxidative stresses under various environmental conditions [46, 47]. In particular, each of the SOD isoforms is independently regulated according to the degree of oxidative stress experienced in the respective subcellular compartments .
At 15°C acclimation and 120 μmol photon m−2 s−1 irradiation, which was the point where the specific growth rate of the algal cells was zero, DT possessed higher SOD activities and more isoforms than 8b. To clarify further which SOD isoform responded to light or temperature, SOD activities were measured under the lower temperature conditions of 10°C and 120 μmol photon m−2 s−1 irradiance (Figure 7A) and at 15°C under the doubled irradiance of 240 μmol photon m−2 s−1 (Figure 7B). The results showed that the original SOD isoforms, which are likely sensitive to low temperature, were amplified by at 10°C and the newly induced SOD isoforms, which are likely sensitive to light, appeared under the doubled irradiance treatment.
Our data also suggested that the regulation of the antioxidant response to chilling was different from the response to irradiation. This raises the interesting question of why the regulation of antioxidant defenses is so highly complex and varied under a range of oxidative stresses even though they are targeting the same O2·− substrate [20, 21, 22, 23].
The green algae
The authors acknowledge that this work was partly supported by grants to Lee-Feng Chien from the National Science Council (now Ministry of Science and Technology) of Taiwan (NSC89-2312-B-005-007 and NSC93-2311-B-005-017). This article is dedicated in memory of Professor Pao-Chung Chen.
|ROS||reactive oxygen species|
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