Synthesis of hydroxylated sterols in transgenic Arabidopsis plants alters growth and steroid metabolism

Abstract

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels.

Figure 1.

Chemical structure of end product desmethylsterols and of different forms of hydroxycholesterol. A, Chemical structure of the plant sterol precursor cycloartenol, and the sterol end products cholesterol, campesterol, and sitosterol, representing the C8, C9, and C10 side chain sterols, respectively. B, Cholesterol hydroxylated at the C-7, C-24, C-25, and C-27 positions.

Figure 2.

General appearance of wild-type Arabidopsis (Col.) plants and representative CH transformants. A, Wild-type plants (left) and homozygous CH7, CH24, CH25, and CH27 transformants grown 12 d in soil. B, Wild-type (left) and CH25 transformants in heterozygous (middle) and homozygous (right) form, grown 12 d in soil. C and D, The same lines and order as in B but grown in soil for 3 weeks (C), or 12 weeks (D). The enlargement in D illustrates aberrant flower structures formed in CH25 homozygotes. A more detailed illustration of the CH25 floral morphology is shown in Supplemental Figure S2. [See online article for color version of this figure.]

Figure 3.

GC-MS analysis of the hydroxysterol profile in wild-type Arabidopsis (Col.) plants. Hydroxysterols were extracted from rosette leaves, enriched using the SPE technique, and TMS-ether derivatized prior to GC-MS analysis using 19-hydroxycholesterol as an added internal standard. A, GC-MS chromatogram of the hydroxysterol fraction. Peaks were identified as 7α-hydroxycholesterol (I), 7α-hydroxycampesterol (II), 7β-hydroxycholesterol (III), 7α-hydroxysitosterol (IV), 7β-hydroxycampesterol (V), and 7β-hydroxysitosterol (VI). Additional peaks could not be identified with certainty as hydroxysterols from the GC-MS analysis. B and C, Mass spectrum of the peaks identified as 7α-hydroxycholesterol (B), and 7β-hydroxycholesterol (C) from comparisons of the retention time and fragmentation pattern of authentic standards, or identified as 7α-hydroxysitosterol (D), based on published spectra.

Figure 4.

GC-MS analysis of the hydroxysterol profile in CH Arabidopsis transformants. Hydroxysterols were extracted from rosette leaves, enriched using the SPE technique, and TMS-ether derivatized prior to GC-MS analysis using 19-hydroxycholesterol as an added internal standard. Gas chromatograms of hydroxysterols isolated from CH7 (A), CH24 (C), and CH25 (E) transgenic lines, and mass spectra for compounds determined as 7α-hydroxycholesterol (B), 24-hydroxycholesterol (D), and 25-hydroxycholesterol (F). Different types of GC columns were used in E as compared to A and C.

Figure 5.

Sterol profiles in wild-type Arabidopsis (Col.), and CH24 and CH25 transformants. Total sterols were isolated from rosette leaves, separated by TLC into desmethyl-, 4-monomethyl-, and 4,4′-dimethylsterol fractions, and analyzed by GC-MS as TMS-ether derivatives. A, Desmethylsterols in wild-type plants (white), CH24 homozygous line (gray), and CH25 homozygous line (black). X and Y denote two unidentified sterols with m/z 470, indicative of a C9 sterol side chain. B, 4-Monomethylsterols and 4,4′-dimethylsterols (C) in the same lines as above. Mean value ± sd of at least three biological replicates.

Figure 6.

Hypocotyl elongation in response to BRs for control Arabidopsis and CH25 transformants. Seeds were germinated for 14 d on half-strength Murashige and Skoog medium containing kanamycin, and resistant seedlings were transferred to half-strength Murashige and Skoog medium containing no, or increasing concentrations of BRs and grown for 6 d in vertical position. A, Seedling hypocotyl length in BL-supplemented medium for Arabidopsis empty-vector transformants (white squares), and a CH25 line in heterozygous form (dark triangles) and homozygous form (dark circles). B, Seedling hypocotyl length in CS-supplemented medium. Same materials and symbols as in A. Mean value of 10 seedlings ± se.

Figure 7.

CH25 transformants display increased drought tolerance. Top row shows empty-vector control Arabidopsis transformants, homozygous CH24 and heterozygous CH25 transformants, and the BR-biosynthesis mutant det2-1. Plants received equal watering with equal volumes at each watering occasion. Bottom row shows the same genotypes grown in parallel but subjected to a drought stress with no watering for 14 d. [See online article for color version of this figure.]