Conserved carotenoid pigmentation in reproductive organs of Charophyceae

Abstract

Sexual reproduction in Charophyceae abounds in complex traits. Their gametangia develop as intricate structures, with oogonia spirally surrounded by envelope cells and richly pigmented antheridia. The red-probably protectant-pigmentation of antheridia is conserved across Charophyceae. Chara tomentosa is, however, unique in exhibiting this pigmentation and also in vegetative tissue. Here, we investigated the two sympatric species, C. tomentosa and Chara baltica, and compared their molecular chassis for pigmentation. Using reversed phase C₃₀ high performance liquid chromatography (RP-C₃₀-HPLC), we uncover that the major pigments are β-carotene, δ-carotene and γ-carotene; using headspace solid-phase microextraction coupled to gas chromatography equipped with a mass spectrometer (HS-SPME-GC-MS), we pinpoint that the unusually large carotenoid pool in C. tomentosa gives rise to diverse volatile apocarotenoids, including abundant 6-methyl-5-hepten-2-one. Based on transcriptome analyses, we uncover signatures of the unique biology of Charophycaee and genes for pigment production, including monocyclized carotenoids. The rich carotenoid pool probably serves as a substrate for diverse carotenoid-derived metabolites, signified not only by (i) the volatile apocarotenoids we detected but (ii) the high expression of a gene coding for a cytochrome P450 enzyme related to land plant proteins involved in the biosynthesis of carotenoid-derived hormones. Overall, our data shed light on a key protection strategy of sexual reproduction in the widespread group of macroalgae. The genetic underpinnings of this are shared across hundreds of millions of years of plant and algal evolution. This article is part of the theme issue 'The evolution of plant metabolism'.

Keywords: Charophyceae; apocarotenoids; carotenoids; sexual reproductive structures; streptophyte algae.

Figure 1.

Investigation of two sympatric Chara species. (a) Location of sampling near Michaelsdorf in the brackish ‘Bodstetter Bodden’ of the Darss Zingster Bodden chain. Map from www.openstreetmap.org. (d) Male C. tomentosa, dashed line indicates cutting to obtain the two respective sample groups for this organism. On the right, two pictures show a zoom-in (of a different individual) on the morphological details of the whorls; note the spotted pigmentation (the two pictures were reproduced with permission from Holzhausen [42]). (b) Chara baltica. In (b) and (d), white arrows indicate antheridia, in male C. tomentosa only in top parts, and in C. baltica distributed over the branches of the whole plant (WP). (c) Principal component analysis (PCA) of global differential gene expression of the three sample groups indicated by colour and shape as displayed on the right side. (e,f) Maximum-likelihood phylogenies of genes salient to salt tolerance. Black circles are scaled and represent the ultrafast bootstrap (UFBoot) support (see legend). Teal hues label highlight sequences from Charophyceae; C. baltica sequences are labelled with a dark turquoise circle, and C. tomentosa sequences are labelled with an orange triangle. Blue boxes indicate significant downregulation, and purple boxes indicate significant upregulation of a minimum of twofold in the comparison C. tomentosa sexual reproductive tissue (SRT) versus C. tomentosa VT.

Figure 2.

(Apo)carotenogensis in the investigated Charophyceae. (a) Non-normalized chromatograms of replicate 1 of the three sample groups. Purple, C. tomentosa SRT; orange, C. tomentosa VT; teal, C. baltica WP; i, violaxanthin ; ii, 9-cis-neoxanthin; iii, antheraxanthin; iv, chlorophyll b; v, lutein; vi, zeaxanthin; vii, chlorophyll a; viii, 15-cis-β-carotene; ix, α-carotene; x, β-carotene; xi, 9-cis-β-carotene; xii, putative torulene or some cis-δ-carotene or cis-γ-carotene; xiii, δ-carotene; xiv, γ-carotene; xv, lycopene. (b) Schematic of the apocarotenoid and carotenoid pathway in the investigated species. (c) Pigment pools of the three sample groups with area relative to pool size, legend on the left. (d) Headspace solid-phase microextraction coupled to gas chromatography equipped with a mass spectrometer (HS-SPME-GC-MS) chromatogram of C. tomentosa. Data shown were derived from VT replicate 1.

Figure 3.

Pigment levels and ratios in C. tomentosa and C. baltica. (a) Individual pigment levels or pigment ratios: purple, C. tomentosa SRT; orange, C. tomentosa VT; teal, C. baltica WP. p-values indicate results from Kruskal–Wallis test to test for significant differences between the three sample sets. For significant results, a post hoc Conover–Iman test was performed for pairwise comparisons with p1, C. tomentosa SRT versus C. tomentosa VT; p2, C. tomentosa SRT versus C. baltica; p3, C. tomentosa VT versus C. baltica WP. (b–e) Maximum-likelihood phylogenies of genes salient to carotenoid metabolism and photoprotection. Black circles are scaled and represent the ultrafast bootstrap (UFBoot) support (see legend). Teal hues label highlight sequences from Charophyceae; C. baltica sequences are labelled with a dark turquoise circle, C. tomentosa sequences are labelled with an orange triangle. Blue boxes indicate significant downregulation, and purple boxes indicate significant upregulation of a minimum of twofold in the comparison C. tomentosa SRT versus C. tomentosa VT.

Figure 4.

Ecophysiological model for C. baltica and C. tomentosa (male). Small arrows indicate elevation or depletion.