Fig 1.
Carotenoid synthesis and cleavage pathway in higher plants.
Enzymes (in bold) and corresponding Arabidopsis mutant names (in italics) are given; carotenoids accumulating in WT tissues are underlined; boxes indicate colour code used for carotenoids in subsequent figures. GGPP, geranylgeranyl diphosphate; PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; eLCY, ε-cyclase; bLCY, β-cyclase; CYPA3, cytP450 hydroxylase 97A3; CYPC1, cytP450 hydroxylase 97C1; BCH1/2, β-carotene hydroxylase 1/2; NXS, neoxanthin synthase; NCED, 9-cis-epoxycarotenoid dioxygenase; CCD, carotenoid cleavage dioxygenase; D27, β-carotene isomerase; CrtI, bacterial carotene desaturase; AGs, apocarotenoid glucosides; NFZ, norflurazon.
Fig 2.
Carotenoid accumulation during Arabidopsis callus development.
A, Carotenoid content in Arabidopsis leaves (lvs) decreases strongly in callus developed for 28 d on callus-inducing medium in darkness (CIM; lvs callus-28d). Seedlings light-germinated for 5 days on CIM (sdl) contain carotenoid levels almost similar to leaves and progressively lose carotenoids during 7 and 14 days of callus development in darkness (callus-7d/14d). Pie charts show carotenoid patterns. B, Carotenoid breakdown in Arabidopsis CCD/NCED mutants is reduced only in ccd1, ccd4 and ccd1 ccd4 double mutant and in WT callus generated in presence of the water-soluble tocopherol analogue trolox. Results are mean ± SD from at least three biological replicates. Significant difference to the wt, *P<0.05.
Fig 3.
Carotenoid contents in carotenoid pathway mutants calli.
A, Seed-derived calli were generated from Arabidopsis carotenoid pathway mutants. Only the crtISO mutant ccr2 accumulated high levels of non-coloured pathway intermediates, while carotenoids in other mutants are almost similarly degraded. Genes affected by the mutation are given below mutant names (see Fig 1). B, Active carotenogenesis during etiolated callus development evident by phytoene accumulation in WT callus generated in presence of the PDS inhibitor norflurazon (NFZ) for 7 and 14 days. C, Similar phytoene levels in NFZ-treated calli from ccd1 and ccd4 excludes phytoene as in vivo CCD substrate. Callus from homozygous pds mutant accumulate phytoene in absence of NFZ while expression of the bacterial desaturase CrtI (35S:crtI) bypasses NFZ-inhibition. Results are mean ± SD from three biological replicates. Significant difference (P<0.05) *rel. to WT, 14d (A, B); *rel. to WT-NFZ, 14d (C). Carotenoid differences between WT and 35S:CrtI calli were non-significant (P<0.05).
Fig 4.
Carotenoid pathway flux determines β-carotene accumulation in Arabidopsis callus.
A, Seed-derived calli were generated from AtPSY-overexpressing Arabidopsis lines with low (At13, AtU16) and high (At12, At22) PSY protein levels. At13 and AtU16 accumulated carotenoid levels almost like WT despite two-fold increased pathway activity concluded from the phytoene content in presence of NFZ. Further increased pathway activity as in lines At12 and At22 results in β-carotene accumulation. Different PSY activities are reflected by different PSY protein levels shown by immunoblotting using 80 μg of callus protein below. Actin levels are included as loading control. Results are mean ± SD from at least three biological replicates. Significant difference to the WT (*) and WT-NFZ (**), respectively, P<0.05. B, In vitro PSY activities were determined in isolated callus plastid membrane fractions by incubation with DMAPP, [14C]-IPP and a recombinant GGPP synthase and quantification of [14C]-phytoene levels. Results are means ± SD from three biological replicates. Significant difference to WT, *P<0.05.
Fig 5.
β-Apocarotenoids in Arabidopsis callus.
A, β-Apocarotenals were determined in seed-derived calli from AtPSY-overexpressing (At12, At22) and ZmPSY1-expressing (Zm18) Arabidopsis lines by LC-MS analyses. Apocarotenoid amounts were expressed in ng g-1 DM. β-IAc, β-ionylidene acetaldehyd. The amounts of β-carotene (in μg g-1 DM) are given for comparison. B, β-ionone, C, β-cyclocitral and D, 5,6-epoxy-β-ionone are expressed as peak areas normalized to internal standards and dry mass. Results are means ± SD from three biological replicates. Significant difference to the WT, *P<0.05.
Fig 6.
Carotenoid kinetics during prolonged callus development.
WT and ZmPSY1-expressing seeds (Zm) were germinated for 5 days on callus-inducing medium (CIM) under long day, followed by 2 weeks etiolation. Thereafter, calli were transferred on CIM (-) or CIM supplemented with 1 μM NFZ (+) and developed further in the dark. Samples were taken after 0, 5, 10, 15 and 20 days and carotenoids were quantified by HPLC (A) while β-apocarotenals were quantified at 0 days and after 10 and 20 continued etiolation on NFZ by LC-MS (B). Note that carotenoids are given in μg g-1 DM, while β-apocarotenals are in ng g-1 DM. All data are mean +/- SD from three biological replicates. Significant difference to preceding time point of same sample type, *P<0.05).
Fig 7.
β-Carotene oxidation products.
β-Carotene oxidation primarily yields β-apocarotenals of various chain lengths. Capital letters indicate the reacting double bond position; the corresponding cleavage product pairs are depicted below from A to E. Secondary oxidation results in the release of the β-ionone ring moiety, e.g. as β-cyclocitral, and linear apocarotene-dialdehydes. Methylglyoxal and glyoxal represent end products after continued oxidation of carotene dialdehydes.
Fig 8.
Apocarotene-dialdehydes in Arabidopsis callus.
Apocarotene-dialdehydes were determined in seed-derived calli from AtPSY-overexpressing Arabidopsis lines by LC-MS analyses. Peak areas were normalized to internal standards and dry mass and are expressed relative to one WT sample. Data are mean ± SD from three biological replicates, significant difference rel. to WT (*P<0.05). Numbers indicate dialdehyde hydrocarbon chain length, ranging from C10 to C5 (A); relative glyoxal and methylglyoxal levels were determined for WT and At12 calli and are shown in B. For detailed compound names, see S2 Table.
Fig 9.
Carotenoid crystal formation in Arabidopsis.
A to H, one AtPSY-overexpressing line was crossed with an Arabidopsis line expressing a plastidic marker protein fused to cyan fluorescent protein (CFP). Two different callus protoplasts are shown (A to D and E to H). CFP fluorescence (A, E) largely overlaps with carotenoid crystals visualized using a (polarized) laser beam at 543 nm (C, G), confirming intraplastid localization of carotenoid crystals. B and F, bright field images; D and H, overlay of all images. Slight mismatches between CFP and crystal birefringence are due to plastid movements between two image acquisitions. Bar = 5 μm. I to N, TEM of roots sections of an AtPSY-overexpressing line (I to K), and roots of Arabidopsis wild type (L to N). Cw, cell wall; Pt, plastid; Mt, mitochondrium; Nc, nucleus; pg, plastoglobuli; G, Golgi; C, membrane remnants of carotenoid crystal. Bar = 0.1 μm.