Induced beta-carotene synthesis driven by triacylglycerol deposition in the unicellular alga dunaliella bardawil

Abstract

Under stress conditions such as high light intensity or nutrient starvation, cells of the unicellular alga Dunaliella bardawil overproduce beta-carotene, which is accumulated in the plastids in newly formed triacylglycerol droplets. We report here that the formation of these sequestering structures and beta-carotene are interdependent. When the synthesis of triacylglycerol is blocked, the overproduction of beta-carotene is also inhibited. During overproduction of beta-carotene no up-regulation of phytoene synthase or phytoene desaturase is observed on the transcriptional or translational level, whereas at the same time acetyl-CoA carboxylase, the key regulatory enzyme of acyl lipid biosynthesis, is increased, at least in its enzymatic activity. We conclude that under normal conditions the carotenogenic pathway is not maximally active and may be appreciably stimulated in the presence of sequestering structures, creating a plastid-localized sink for the end product of the carotenoid biosynthetic pathway.

Figure 1

Light induction of D. bardawil cells. A, N, noninduced cells; I, cells induced by high-light treatment; I + S, induced cells in the presence of 50 μm sethoxydim; I + C, induced cells in the presence of 8 μm cerulenin. All samples shown represent a concentrated cell culture harvested 48 h after the onset of the induction. B, Time course of β-carotene formation. ○, Control cells; •, induced cells. The data are the mean of two replicates. Vertical bars indicate ± se. prot, Protein. C, Time course of lipid formation (measured as total fatty acids upon saponification). □, Control cells; ▪, induced cells. The data are the means of two replicates. Vertical bars indicate ± se. prot, Protein. D, Electron micrographs of noninduced cells (1), induced cells after 72 h (2), and induced cells in the presence of 8 μm cerulenin after 72 h (3). N, Nucleus; P, pyrenoid; S, starch; L, lipid globules.

Figure 2

Analysis of lipid classes formed after 48 h of high-light treatment. A, TLC results. Std, Standard (tripalmitoylglycerol); N.I, noninduced cells; I, induced cells. Car, β-carotene; TAG, triacylglycerol; PL + Chl, phospholipids + galactolipids + chlorophyll. B, Distribution of radioactivity from NaH¹⁴CO₃ (incubation time, 48 h) into different lipid classes in control cells and in induced cells. Car, β-Carotene; GL, galactolipid; SL, sulfolipid; PL, phospholipid; and Chl, chlorophyll.

Figure 3

Cerulenin (A) and sethoxydim (B) inhibit high-light-induced β-carotene accumulation. Cells were induced by high light and cultured in the absence (control cells) or presence of different concentrations of inhibitors for 48 h and then analyzed. ▪, β-Carotene; •, total fatty acids. The data represent the means of three values ± se. prot, Protein.

Figure 4

Western analysis showing the abundance of phytoene synthase and phytoene desaturase during high-light treatment. Equal amounts of protein (30 μg per lane) were separated and electro-transferred as described in the experimental procedures. Primary antibodies were anti-phytoene synthase antibodies (A) and anti-phytoene desaturase antibodies (B). Secondary antibodies were linked to horseradish peroxidase. Detection was performed using the enhanced chemiluminescence system. C, The biotin-containing bands showing an increase during high-light treatment represent an internal control and were detected using alkaline phosphatase-conjugated streptavidin. Ct, Control (N. pseudonarcissus chromoplast extract).

Figure 5

Time course of the in vitro synthesis of acyl lipids and prenyl lipids in D. bardawil cell lysates at various times after onset of high-light induction of cells. A, The time course of the incorporation of radioactivity into acyl lipids (○; right scale) and prenyl lipids (•; left scale) from [1-¹⁴C]acetate and [1-¹⁴C]IPP, respectively. At the times indicated, cells were lysed and incubated in the presence of 37 kBq [1-¹⁴C]acetate or 28 kBq [1-¹⁴C]IPP for 2 and 6 h, respectively. Data are the means of three values. Vertical bars indicate ± se. B, ACCase activity (•) and lipid accumulation (○; measured as total fatty acids) during high-light treatment of the cells. Values are means ± se for three repetitions. prot, Protein.

Figure 6

RT-PCR analysis of phytoene desaturase (pds) and CBR (cbr) mRNA expression in D. bardawil during high-light induction. Total RNA was isolated at different times after the onset of high-light treatment. Left, RT-PCR reactions were performed with 100 (lanes 1), 33 (lanes 2), and 11 ng (lanes 3) of total RNA. The 487-bp band is derived from phytoene desaturase mRNA; 256-bp bands represent the CBR mRNA. Right, The two transcripts were co-amplified using all four specific primers. M, Marker (λ-DNA digested using EcoRI/HindIII).