Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains

Abstract

The accumulation of bioenergy carriers was assessed in two starchless mutants of Chlamydomonas reinhardtii (the sta6 [ADP-glucose pyrophosphorylase] and sta7-10 [isoamylase] mutants), a control strain (CC124), and two complemented strains of the sta7-10 mutant. The results indicate that the genetic blockage of starch synthesis in the sta6 and sta7-10 mutants increases the accumulation of lipids on a cellular basis during nitrogen deprivation relative to that in the CC124 control as determined by conversion to fatty acid methyl esters. However, this increased level of lipid accumulation is energetically insufficient to completely offset the loss of cellular starch that is synthesized by CC124 during nitrogen deprivation. We therefore investigated acetate utilization and O(2) evolution to obtain further insights into the physiological adjustments utilized by the two starchless mutants in the absence of starch synthesis. The results demonstrate that both starchless mutants metabolize less acetate and have more severely attenuated levels of photosynthetic O(2) evolution than CC124, indicating that a decrease in overall anabolic processes is a significant physiological response in the starchless mutants during nitrogen deprivation. Interestingly, two independent sta7-10:STA7 complemented strains exhibited significantly greater quantities of cellular starch and lipid than CC124 during acclimation to nitrogen deprivation. Moreover, the complemented strains synthesized significant quantities of starch even when cultured in nutrient-replete medium.

Fig. 1.

Cell counts for CC124, the sta6 and sta7-10 mutants, and the sta7-10:STA7 complemented sta7-10[c5] and sta7-10[c19] strains. Growth curves from a representative experiment are shown and were constructed from cell counts at resuspension (0 h) until 96 h, as indicated, in TAP (A) or nitrogen-depleted TAP-N (B) medium. Each data point represents three replicates, except for sta7-10[c5], which represents two biological replicates. Error bars indicate standard deviations.

Fig. 2.

Starch analyses. Cells were analyzed for glucose derived from starch after amyloglucosidase digestion at resuspension (0 h) and after 96 h in nitrogen-replete TAP or nitrogen-depleted TAP-N medium. (A) Starch-derived glucose per million cells. (B) Starch-derived glucose per ml of culture. Error bars indicate standard deviations. (C) Verification of starch phenotype in the CC124, sta6, and sta7-10 strains. Cells were spotted on TAP and TAP-N agar plates, as indicated. After 7 days the cells were imaged on TAP plates, and the TAP-N plate was stained with iodine vapors and imaged (far right) to indicate the presence of starch (deep purple color).

Fig. 3.

GC-FID quantification of fatty acid methyl esters derived from CC124, sta6, sta7-10, sta7-10[c5], and sta7-10[c19] lipids at the indicated times of culturing in nitrogen-depleted TAP-N medium. Values are representative of triplicate biological samples. Lipid was quantified per million cells (A) or by ml of culture (B). Error bars indicate standard deviations.

Fig. 4.

Representative GC-FID chromatogram. Fatty acid methyl esters derived from the sta7-10 mutant after 96 h of nitrogen deprivation are shown. 18:1/C18:3(9,12,15) indicates a peak containing both 18:1 and 18:3(9,12,15). 13:0/IS indicates a tridecanoate internal standard.

Fig. 5.

Laser scanning confocal fluorescence microscopy images merged with transmitted light images. CC124, sta6, sta7-10, sta7-10[c5], and sta7-10[c5] cells are shown. The two leftmost columns show cells stained with the nonpolar lipid fluorophore Bodipy 493/503. Nonpolar lipid bodies are visualized by green Bodipy fluorescence after 96 h (left, TAP; right, TAP-N). The two rightmost columns show differential interference contrast images after 96 h (left, TAP; right, TAP-N). Oil bodies are visible inside the cells as blue-green-tinted vesicles. Chlorophyll autofluorescence is red. All scale bars represent 10 μm.

Fig. 6.

(A) Concentrations of acetate remaining in CC124, the sta6 and sta7-10 mutants, and the sta7-10:STA7 complemented sta7-10[c5] and sta7-10[c19] strains in TAP-N medium at the indicated culturing times. Cultures were inoculated at approximately 2.0 × 10⁶ to 2.5 × 10⁶ cells/ml. The initial acetate concentration in TAP and TAP-N media was 17.5 mM. Values are representative of triplicate biological samples. (B) Oxygen evolution from 0.8-ml aliquots of the indicated strains after 24 h in nitrogen-depleted TAP-N medium. Oxygen evolution is shown on the basis of cell number and chlorophyll (Chl). Cultures were inoculated at approximately 1.0 × 10⁷ cells/ml to produce O₂ at levels sufficient for detection. Error bars indicate standard deviations.

Fig. 7.

Chlorophyll content per ml of culture in a representative experiment at the specified time points for the CC124, sta6, sta7-10, sta7-10[c5], and sta7-10[c19] strains in nitrogen-replete (TAP) (A) or nitrogen-depleted (TAP-N) (B) medium. Values are representative of triplicate biological samples. Cells were precultured to late log phase and then resuspended at 2.0 × 10⁶ to 2.5 × 10⁶ cells/ml in either TAP or TAP-N medium. Error bars indicate standard deviations.

Fig. 8.

(A) Variable chlorophyll a fluorescence (F_v/F_m) of the CC124, sta6, and sta7-10 strains plated on TAP-N agar (five replicate colonies for each strain) as measured by the PSI FluorCam imager indicated. (B) Nonvariable chlorophyll a emission (F_o) of the CC124, sta6, and sta7-10 strains plated on TAP-N agar (five replicate colonies for each strain) as measured by the PSI FluorCam imager. Error bars indicate standard deviations.