COORDINATED CAROTENOID AND LIPID SYNTHESES INDUCED IN PARIETOCHLORIS INCISA (CHLOROPHYTA, TREBOUXIOPHYCEAE) MUTANT DEFICIENT IN Δ5 DESATURASE BY NITROGEN STARVATION AND HIGH LIGHT¹

Cultivation conditions. The Δ5-desaturase mutant of P. incisa, P127, was obtained in the Microalgal Biotechnology Laboratory, J. Blaustein Institutes for Desert Research by nitrosoguanidine mutagenesis (NNG). The mutant was cultivated on complete, [+N], and nitrogen-free, [−N], BG-11 medium (Stanier et al. 1971) in 1 L glass columns (6 cm ID) under constant illumination (by daylight fluorescent lamps) of three different intensities (35, 130, and 270 μE · m⁻² · s⁻¹ PAR as measured in the center of the empty column) and with constant bubbling of CO₂: air mixture (1: 99, v/v) at 25°C. Prior to the experiment, cultures were daily diluted to maintain logarithmic growth. In all cases, initial chl content and DW were maintained at 30 mg · L⁻¹ and 1 g · L⁻¹, respectively, to prevent photodamage of the cultures at high irradiance (Solovchenko et al. 2008a). The nitrogen content in the medium was checked during the experiment using the nitrate assay kit (Merckoquant 1.10020.001, Merck, Darmstadt, Germany). For nitrogen-starvation, cells were washed three times with sterilized distillated water and resuspended in [−N] BG-11. The DW, TFA, and pigment contents and composition (see below) were determined at d 0 and following 3, 7, 10, and 14 d of growth.

Fatty acid analysis. Capillary gas-chromatography was used for fatty acid quantification; the analysis was performed according to Cohen et al. (1993). The data shown represent mean values with a range of <5% for major peaks (>10% of fatty acids) and 10% for minor peaks, of at least two independent samples, each analyzed in duplicate.

Isolation thylakoids and OB. Thylakoid- and OB-enriched fractions were isolated from the cells of 14 d cultures following the rupture of the biomass in liquid nitrogen as described in (Solovchenko et al. 2008a). The OB fractions were immediately extracted with 4 mL of diethyl ether and the thylakoids—with 5 mL of chloroform-methanol mixture (2:1, v/v) per 2 mL of the thylakoid fraction. After centrifugation (Beckman L5-75B, Beckman Instruments Inc., Palo Alto, CA, USA) of the extracts for 10 min at 1,500g for phase separation, the chloroform phase was collected, evaporated in a stream of nitrogen to dryness, and redissolved in acetone for further analysis. All manipulations were carried out under dim illumination at 4°C.

Spectral measurements. The spectra of P127 cell, thylakoid, and OB extracts were recorded with the Cary 50 Bio spectrophotometer. Due to rapid cell sedimentation, the optical properties of the P. incisa P127 suspensions were determined using glass-fiber filter technique (Mitchell 1990). The spectra were taken upon cell deposition on 25 mm GF/F glass-fiber filters (Schleicher & Schuell Inc., Keene, NH, USA) against an empty filter soaked in BG-11 medium. The wet filters were mounted as overheads (deposited cells facing the detector) on the output windows of cuvette compartment of the spectrophotometer. Optical density (D) of the filters was expressed as −log₁₀T, where T is transmission.

Statistical treatment. The experiments were carried out in two biological replications with three analytical replications for each of them. In the figures, average values together with standard deviations are presented. The significance of differences was tested using analysis of variance (ANOVA) via Microsoft Excel (Microsoft Corp., Redmond, WA, USA).

Accumulation of biomass. The time-course of the changes in biomass of the P. incisa P127 cultures under different conditions is plotted in Figure 1. Under low PAR (LL, 35 μE · m⁻² · s⁻¹) intensity, the cultures showed relatively slow increase in biomass (Fig. 1, curves numbered 1) regardless of the medium composition (average increase rates of ca. 0.16 mg DW · d⁻¹ for LL–N and LL+N) and attained DW of ca. 2.2 g · L⁻¹ by the end of cultivation period (14 d). Under irradiances of 130 μE · m⁻² · s⁻¹ (medium light, ML), the [ML+N] cultures possessed higher final biomass (ca. 6.1 g · L⁻¹ by 14 d at the average rate of 0.47 mg DW · d⁻¹) than [ML−N] cultures; the latter reached DW of ca. 4.1 g · L⁻¹ at the average rate of 0.29 mg DW · d⁻¹ (Fig. 1, curves numbered 2). Under high PAR irradiance (HL, 270 μE · m⁻² · s⁻¹), the [HL+N] cultures reached the highest biomass (ca. 7 g · L⁻¹; curve 3 in Fig. 1a) at the average rate of 0.47 mg DW · d⁻¹, whereas the biomass and its accumulation rate in the [HL−N] culture did not differ significantly from [ML−N] (cf. curves 2 and 3 in Fig. 1b).

Fatty acid accumulation. In all cultures except for [LL+N], the volumetric content of TFA built up with time along with an increase in DW (Fig. 2). In cultures grown on complete medium, the extent of TFA accumulation depended on the irradiance. Specifically, in the [LL+N] culture, TFA did not show significant changes (Fig. 2, a and c, curves numbered 1). In [ML+N], the TFA percentage increased, after a 5% dip for the first 3 d, reaching 20% by day 14 (Fig. 2a, curve 2); volumetric content in this case also increased (Fig. 2c, curve 2). The highest volumetric contents of TFA were detected in the [HL+N] cultures (Fig. 2c, curve 3).

In all cultures grown on N-free medium, TFA content continuously increased (Fig. 2, b and d). The [LL−N] cultures accumulated more TFA than the corresponding [+N] cultures (cf. Fig. 2, b and d, and Fig. 2, a and c, curve 1). The [ML−N] and [HL−N] cultures demonstrated a similar increase in TFA percentage (Fig. 2b, curves 2, 3) close to that of the [HL+N] cultures (Fig. 2a, curve 3). Due to slow down of an increase in biomass (Fig. 1), cultivation under [HL−N] and [ML−N] conditions resulted in a lower volumetric contents of TFA (ca. 1.5 g · L⁻¹; Fig. 2d, curves 2, 3), which was only a little higher than in the case of [ML+N] (Fig. 2c, curve 2). Notably, the [HL+N] cultures accumulated ca. 40% more TFA per unit volume than the corresponding nitrogen-starved cultures (cf. curves numbered 3 in Fig. 2, c and d). However, under ML and LL, the TFA volumetric contents were higher in the [−N] cultures.

The DGLA percentage on a DW basis demonstrated a linear relationship (r²∼ 0.80) with the percentage of TFA (Fig. 3). Volumetric accumulation of DGLA followed approximately the same trends as TFA (not shown). Accordingly, the highest percentages of DGLA were seen in the [HL+N] and [HL−N] cultures, whereas the highest volumetric DGLA content was observed in the case of [HL+N] (Fig. 3).

Changes in the contents of chl and car and their ratio. The dynamics of chl and car contents as well as their ratios are shown in 4, 5. Relatively slow and nearly linear increase in chl was observed in the [LL+N] cultures (Fig. 4a, curve 1). Chl content in the [HL+N] cultures peaked on day 7 of cultivation, reaching 75 mg · L⁻¹, and underwent a gradual decrease thereafter, apparently manifesting the depletion of the nitrogen from the medium (Fig. 4a, curve 3). The [ML+N] cultures possessed the highest chl content, reaching ca. 130 mg · L⁻¹ by day 10 (Fig. 4a, curve 2). The chl content in cultures grown on N-free medium (Fig. 4b) declined, after a moderate increase during the first 3 d of cultivation, as in the case of [LL−N] and [ML−N] (Fig. 4b, curves 1 and 2, respectively), or immediately after the start of the experiment ([HL−N]; Fig. 4b, curve 3). Regardless of the experimental conditions, the chl a/b ratio did not change significantly and remained in the range of 2.3–2.5 (not shown).

On complete medium, a distinct irradiance-dependent increase in car content took place (Fig. 4c). A moderate increase in car following an S-shaped trend was recorded in the [LL+N] culture (Fig. 4c, curve 1); cultures grown at medium and high irradiances showed steeper increase in car content, which was the highest in the [ML+N] culture (Fig. 4c, curve 2) and less pronounced, tending to decline after day 10 in the [HL+N] culture (Fig. 4c, curve 3). Under N-deprivation, car content was considerably lower relative to that observed during cultivation on complete medium and did not exceed 11 mg · L⁻¹ in all these cases (Fig. 4d).

According to the dynamics of the changes in chl and car contents described above, the ratio of those pigments depended on cultivation conditions (Fig. 4, e and f). Thus, in the [LL+N] and [ML+N] cultures, the car/chl ratio did not change significantly (Fig. 4e, curves 2, 1) and remained almost the same on the background of a considerable increase in chl. The [HL+N] culture displayed ca. 50% increase in the car/chl ratio (Fig. 4e, curve 3), which rose more rapidly with an increase in chl. The most pronounced (up to 4-fold in [HL−N]) irradiance-dependent rise in the car/chl ratio was recorded in the absence of N: the higher the PAR irradiance, the higher the extent of a linear increase in car/chl with cultivation time (Fig. 4f). As could be seen from Figure 5, the car/chl ratio rapidly increased along with the decline in chl following similar nonlinear trend regardless of the irradiance level.

Car composition of the P127 cells, thylakoids, and OB. The proportions of the individual carotenes and xanthophylls were monitored at the beginning and at the end of the experiment (day 14). Regardless of cultivation conditions, car of the P127 was composed of neoxanthin, violaxanthin, anteraxanthin, lutein, zeaxanthin, and β-carotene (Fig. 5). The car profile of the P127 underwent similar irradiance-dependent changes in the [+N] and [−N] cultures. Among these changes, the most conspicuous was a decrease in lutein and an increase in β-carotene at higher irradiances (see, e.g., Fig. 5, inset); these trends were more pronounced in the [−N] cultures.

To reveal the subcellular localization of pigments, their composition was determined in isolated thylakoid- and OB-enriched fractions (see inset in Fig. 5). On the whole, the car composition of the thylakoid fraction resembled that of whole cells with significantly lower proportion of β-carotene (cf. curves a and b, the inset of Fig. 5). On the contrary, β-carotene was the dominant car in OB (inset in Fig. 5, curve c) regardless of cultivation conditions. Only traces of xanthophylls and chl (probably due to contamination of the OB preparations with chloroplast membranes in the course of isolation) were detected in OB. The changes in absolute β-carotene content in OB and thylakoids followed the same trends as total car content determined for the whole cells (Fig. 4, c and d). The highest and lowest β-carotene contents were found in the OB isolated from [HL+N] and [LL+N] cells, respectively. By 14 d of cultivation under high-light conditions, ca. 70% of the total β-carotene was localized in the OB, whereas that of total β-carotene and that of thylakoid-localized β-carotene amounted to ca. 30% (inset in Fig. 5).

Relationships between the changes in fatty acid and pigment contents and ratio. The car/chl ratio exhibited a tight, nonlinear relationship with TFA contents per DW or suspension volume unit (Fig. 6; r² = 0.86–0.99) regardless of the cultivation conditions, whereas a weak (r² < 0.3) correlation was found between individual car or chl content and volumetric content of TFA (not shown). The relationships “TFA_%DW versus [car]/[chl]” (Fig. 6a) and “TFA_vol versus [car]/[chl]” for all cultures were successfully fitted with exponentials (Fig. 6b).

Relationships between lipid and pigment content and algal cell spectral absorption. Figure 7 displays the spectra of P127 cells recorded on the glass-fiber filters, normalized at 678 nm, D(λ) · D₆₇₈⁻¹. The difference, ΔD(λ) · D₆₇₈⁻¹, spectra (see insets in Fig. 7) contained features in the 600–720 nm range evident of progressive narrowing of the red chl absorption peak and a shift of its maximum toward shorter wavelengths. As could be seen, an increase in car/chl ratio taking place in P127 under higher irradiances (Fig. 4) was accompanied by a considerable increase in absorption in the blue region of the spectrum. This effect was weaker in the [+N] cultures (Fig. 7a), whereas in the [−N] cultures, it was two to three times higher (Fig. 7b). The difference spectra (insets in Fig. 7) had maxima near 480, 460, and 420 nm, attributable to car.

The relationship of D(λ) with absolute car or chl content was weak (r² < 0.5; not shown); by contrast, that of “car/chl versus D(λ) · D₆₇₈⁻¹” was characterized by a tight correlation, which varied from strong negative (–0.98) in the 600–700 nm band governed by chl absorption to strong positive (>0.99) in the region of combined absorption by car and chl (Fig. 8). As a result of a tight interrelationship between the car/chl ratio, TFA and DGLA contents (3, 6), a strong correlation was found between the volumetric content as well as percentages of the FA contents and D(λ) · D₆₇₈⁻¹ (Fig. 8). For both [+N] and [−N] cultures, the highest correlation (r² > 0.95) was found in the blue-green range of the spectrum, whereas negative peaks were observed in the red region of the spectrum dominated by chl absorption. In the NIR, the region where pigments do not absorb, a weak correlation (r < 0.5) was observed.

The correlation spectra, r{D(λ) · D₆₇₈⁻¹ vs. [DGLA]}, were essentially the same as those, r{D(λ) · D₆₇₈⁻¹ vs. [TFA]}, presented in Figure 8. However, the parameters of approximation of these relationships for the [+N] and [−N] were different. In the 400–500 nm band containing high values of correlation coefficient (see, e.g., 8, 9), exponential models provided the best fit of the “D₄₈₀ · D₆₇₈⁻¹ versus [TFA_%DW]” (Fig. 9a). Essentially, the same models related the percentage and volumetric content of TFA with D₄₈₀ · D₆₇₈⁻¹ (Fig. 9, a and b, curves numbered 1); at the same time, the relationship “D₄₈₀ · D₆₇₈⁻¹ versus [TFA_vol]” was linear (Fig. 9b, curve 2). Based on these findings, different algorithms for estimation of the contents of TFA and DGLA in the P127 cell suspensions grown on complete and N-free media were suggested (Table 1). These algorithms allowed the determination of the TFA percentage in the full range studied (8%–40% DW) with an RMSE of 2.20%. A similar algorithm provided the accuracy of the volumetric TFA assay of 0.13 g · L⁻¹ in the TFA range of 0.1–2.8 g · L⁻¹ (Table 1).