Carotenoid accumulation during Arabidopsis callus development.

<p>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 <i>CCD/NCED</i> mutants is reduced only in <i>ccd1</i>, <i>ccd4</i> and <i>ccd1 ccd4</i> 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.</p

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids

<div><p>The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through <i>PSY</i> overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g^-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.</p></div

Carotenoid accumulation during Arabidopsis callus development.

<p>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 <i>CCD/NCED</i> mutants is reduced only in <i>ccd1</i>, <i>ccd4</i> and <i>ccd1 ccd4</i> 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.</p

Carotenoid contents in carotenoid pathway mutants calli.

<p>A, Seed-derived calli were generated from Arabidopsis carotenoid pathway mutants. Only the <i>crtISO</i> mutant <i>ccr2</i> 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192158#pone.0192158.g001" target="_blank">Fig 1</a>). 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 <i>ccd1</i> and <i>ccd4</i> excludes phytoene as <i>in vivo</i> CCD substrate. Callus from homozygous <i>pds</i> mutant accumulate phytoene in absence of NFZ while expression of the bacterial desaturase CrtI (<i>35S</i>:<i>crtI</i>) bypasses NFZ-inhibition. Results are mean ± SD from three biological replicates. Significant difference (<i>P</i><0.05) *rel. to WT, 14d (A, B); *rel. to WT-NFZ, 14d (C). Carotenoid differences between WT and <i>35S</i>:<i>CrtI</i> calli were non-significant (<i>P</i><0.05).</p

Carotenoid pathway flux determines β-carotene accumulation in Arabidopsis callus.

<p>A, Seed-derived calli were generated from <i>AtPSY</i>-overexpressing Arabidopsis lines with low (<i>At13</i>, <i>AtU16</i>) and high (<i>At12</i>, <i>At22</i>) PSY protein levels. <i>At13</i> and <i>AtU16</i> 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 <i>At12</i> and <i>At22</i> 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, <i>P</i><0.05. B, <i>In vitro</i> 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, *<i>P</i><0.05.</p

Carotenoid crystal formation in Arabidopsis.

<p>A to H, one <i>AtPSY</i>-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 <i>AtPSY</i>-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.</p

Carotenoid synthesis and cleavage pathway in higher plants.

<p>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-<i>cis</i>-epoxycarotenoid dioxygenase; CCD, carotenoid cleavage dioxygenase; D27, β-carotene isomerase; CrtI, bacterial carotene desaturase; AGs, apocarotenoid glucosides; NFZ, norflurazon.</p

β-Apocarotenoids in Arabidopsis callus.

<p>A, β-Apocarotenals were determined in seed-derived calli from <i>AtPSY</i>-overexpressing (<i>At12</i>, <i>At22</i>) and <i>ZmPSY1</i>-expressing (<i>Zm18</i>) 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, *<i>P</i><0.05.</p

Apocarotene-dialdehydes in Arabidopsis callus.

<p>Apocarotene-dialdehydes were determined in seed-derived calli from <i>AtPSY</i>-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 (*<i>P</i><0.05). Numbers indicate dialdehyde hydrocarbon chain length, ranging from C10 to C5 (A); relative glyoxal and methylglyoxal levels were determined for WT and <i>At12</i> calli and are shown in B. For detailed compound names, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0192158#pone.0192158.s007" target="_blank">S2 Table</a>.</p

Image_4.pdf

<p>Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C₁₃ α-ionol/C₁₄ mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides “just in time”-delivery of precursors for apocarotenoid formation.</p