Phytoene desaturase is localized exclusively in the chloroplast and up-regulated at the mRNA level during accumulation of secondary carotenoids in Haematococcus pluvialis (Volvocales, chlorophyceae)

Abstract

The unicellular green alga Haematococcus pluvialis Flotow is known for its massive accumulation of ketocarotenoids under various stress conditions. Therefore, this microalga is one of the favored organisms for biotechnological production of these antioxidative compounds. Astaxanthin makes up the main part of the secondary carotenoids and is accumulated mostly in an esterified form in extraplastidic lipid vesicles. We have studied phytoene desaturase, an early enzyme of the carotenoid biosynthetic pathway. The increase in the phytoene desaturase protein levels that occurs following induction is accompanied by a corresponding increase of its mRNA during the accumulation period, indicating that phytoene desaturase is regulated at the mRNA level. We also investigated the localization of the enzyme by western-blot analysis of cell fractions and by immunogold labeling of ultrathin sections for electron microscopy. In spite of the fact that secondary carotenoids accumulate outside the chloroplast, no extra pathway specific for secondary carotenoid biosynthesis in H. pluvialis was found, at least at this early stage in the biosynthesis. A transport process of carotenoids from the site of biosynthesis (chloroplast) to the site of accumulation (cytoplasmatic located lipid vesicles) is implicated.

Figure 1

Pathway of secondary carotenoid synthesis in H. pluvialis. Data are presented in this paper for the enzymes shown in black boxes. Enzyme designation is according to the corresponding gene: CRTL-B, lycopene β-cyclase; CRTO, β-carotene oxygenase; CRTR-B, β-ring hydroxylase; GGPS, geranylgeranyl diphosphate synthase; IPI, isopentenyl diphosphate isomerase; PDS, phytoene desaturase; PSY, phytoene synthase; ZDS, ζ-carotene desaturase.

Figure 2

Induction of PDS during the synthesis of secondary carotenoids in H. pluvialis. A, Typical nitrocellulose blot probed with PDS antiserum (1:6,000). From left: 0.05 μg of the antigen (A); marker (M); total protein extracts from 10⁵ flagellates, before (0), 2, 4, or 7 d after the onset of inductive conditions for secondary carotenoid biosynthesis. B, Densitometric results for the induction of PDS on a cell base (▪) and the drop of total protein content per cell (□). Bars indicate se of 17, 15, 2, 17, and 17 experiments for 0, 2, 3, 4, and 7 d after onset of induction, respectively, and of four experiments for total protein content.

Figure 3

Changes in the mRNA content of H. pluvialis PDS and CRTO during secondary carotenoid biosynthesis measured by RT-PCR. A, Typical autoradiograph of the RT-PCR products. The relative amount of RNA indicated as rRNA on an ethidium bromide-stained agarose gel is shown below. Lanes from left: M, PCR from pds in pBluescript (used as a marker); lanes 2 to 4, RT-PCR from 16.7, 50, and 150 ng total RNA of the extract 2 d after onset of inductive conditions; lanes 5 to 13, RT-PCR products from 50 ng total RNA for the different days after induction start; M, PCR from crtO in pBluescript (used as marker); lane 15, PCR from 50 ng total RNA as used in lane 2 (RT reaction omitted). B, Results from the phosphor imager quantification analyses of the RTR-PCR products for crtO (▪) and pds (□). Bars indicate the se of four parallels.

Figure 4

Immunogold labeling of PDS in a flagellate of H. pluvialis exposed for 2 d to SC-inducing conditions. A, Typical part of a flagellate showing all major cell compartments. Bar represents 1 μm. B, Magnification of the chloroplast. Bar represents 0.2 μm. Ch, Chloroplast; Ex, extracellular matrix; Li, lipid vesicles; Nc, nucleolus; Nu, nucleus; St, starch; Va, vacuole.

Figure 5

Distribution of PDS in cellular fractions of H. pluvialis flagellates exposed for 4 d to N deprivation and high light. A, Coomassie-stained SDS-PAGE gel of chloroplastic (1), supernatant (2), microsomal (3), and lipid vesicle fraction proteins (4). Each lane was loaded with 15 μg of protein. B, Western-blot analysis with antibodies raised against recombinant PDS (1:4,000). Lanes 1 to 4 are as indicated in A, but were loaded with cell fraction aliquots of 10⁵ cells.