Subfunctionalization of D27 Isomerase Genes in Saffron

Abstract

Chromoplasts and chloroplasts contain carotenoid pigments as all-trans- and cis-isomers, which function as accessory light-harvesting pigments, antioxidant and photoprotective agents, and precursors of signaling molecules and plant hormones. The carotenoid pathway involves the participation of different carotenoid isomerases. Among them, D27 is a β-carotene isomerase showing high specificity for the C9-C10 double bond catalyzing the interconversion of all-trans- into 9-cis-β-carotene, the precursor of strigolactones. We have identified one D27 (CsD27-1) and two D27-like (CsD27-2 and CsD27-3) genes in saffron, with CsD27-1 and CsD27-3, clearly differing in their expression patterns; specifically, CsD27-1 was mainly expressed in the undeveloped stigma and roots, where it is induced by Rhizobium colonization. On the contrary, CsD27-2 and CsD27-3 were mainly expressed in leaves, with a preferential expression of CsD27-3 in this tissue. In vivo assays show that CsD27-1 catalyzes the isomerization of all-trans- to 9-cis-β-carotene, and could be involved in the isomerization of zeaxanthin, while CsD27-3 catalyzes the isomerization of all-trans- to cis-ζ-carotene and all-trans- to cis-neurosporene. Our data show that CsD27-1 and CsD27-3 enzymes are both involved in carotenoid isomerization, with CsD27-1 being specific to chromoplast/amyloplast-containing tissue, and CsD27-3 more specific to chloroplast-containing tissues. Additionally, we show that CsD27-1 is co-expressed with CCD7 and CCD8 mycorrhized roots, whereas CsD27-3 is expressed at higher levels than CRTISO and Z-ISO and showed circadian regulation in leaves. Overall, our data extend the knowledge about carotenoid isomerization and their implications in several physiological and ecological processes.

Keywords: apocarotenoids; carotenoids; expression; isomerase activity; leaves; mycorrhiza; root; stigmas.

Figure 1

Carotenoid biosynthetic pathway from geranylgeranyl diphosphate to β-carotene. The steps in which carotenoid isomerases are known to be involved are indicated by the name of the corresponding enzymes. The described isomerase activity of D27 over β-carotene is indicated in red.

Figure 2

Sequence characteristics of CsD27 enzymes. (a) Amino acid sequence alignments among CsD27 enzymes. The amino acid sequence alignments were made by CLUSTAL omega. Conserved cysteines (C) are highlighted in grey, to indicate structural conservation. Amino acids that constitute the conserved domain of D27 isomerase in higher plants are framed in black. Asterisks denote fully conserved residues, while colons and dots denote partially conserved residues. (b) Percent identity matrix among the CsD27 amino acid sequences.

Figure 3

Phylogenetic tree of the CsD27 amino acid sequences with other plant members of the D27 family. The unrooted phylogenetic tree was constructed using MEGA7 from the D27 sequences retrieved from the GenBank database. Evolutionary relationships were inferred using the Neighbor-joining method with 2500 bootstrap re-sampling strategy. The D27 sequences from saffron are highlighted by black dots and red arrows.

Figure 4

Relative expression levels of CsD27-1, -2 and -3 in vegetative and reproductive tissues investigated by qRT-PCR. (a) Transcripts levels in corm, leaf, root, stamen and tepal. (b) Expression levels in six developmental stages of the stigma. Bars represent mean ± SD (n = 3 biological replicates).

Figure 5

HPLC analysis of the reaction catalyzed by CsD27-1 in vivo on β-carotene produced by E. coli cells. (a) pThio+CsD27-1, converts all-trans-β-carotene (III) to 9-cis-β-carotene (IV), 13-cis-β-carotene (II) and to15-cis-β-carotene (I). (b) Percentages of all the cis-β-carotene isomers were significantly increased in the cells expressing the CsD27-1 enzyme. Statistical analysis was performed using student-t test. A designation of ** = p < 0.01 (t-test). n = 3 independent biological replicate experiments. Error bars represent SD.

Figure 6

In planta activity of CsD27-1. (a) HPLC analysis of in vivo CsD27-1 activity in leaves of N. benthamiana plants grown during 48 h in dark conditions. (b) HPLC analysis of in vivo CsD27-1 activity in leaves of N. benthamiana plants grown under standard light conditions. (c) Percentage of cis-9-β-carotene isomer was significantly increased in N. benthamiana leaves agroinfiltrated with 35S::CsD27-1 in both dark and under standard light conditions. Ratio was calculated based on peak area of 9-cis-β-carotene to all-trans-β-carotene in the chromatograms in (a,b). (d) Increase in apocarotenoid levels in N. benthamiana leaves agroinfiltrated with 35S::CsD27-1 in both dark and under standard light conditions. Statistical analysis was performed using the Student t-test. Asterisks indicate differences from wild type (** = p < 0.01, *** = p < 0.001 t-test). n = 3 independent biological replicate experiments. Error bars represent SD.

Figure 7

Analysis of CsD27-3 activity in E. coli cells accumulating the linear carotenoids ζ-carotene and neurosporene. (a) HPLC analyses of carotenes extracted in the absence (pThio-θ) and presence (pThio+CsD27-3) of CsD27-3. MaxPlot chromatograms showing each peak at its spectra are presented. Peak I: 9,15,9′-tri-cis-ζ-carotene; peak II: 9,9′-di-cis-ζ-carotene; peak III: all-trans-ζ-carotene. Absorption spectra of specific peaks are presented in inset boxes. 9,15,9′-tri-cis-ζ-carotene is distinguished from the 9,9′-cis-isomer by the typical absorbance at 296 nm. (b) Percentage of 9,15,9′-tri-cis-ζ-carotene isomer was significantly increased in the cells expressing the CsD27-3 enzyme. A designation of ** = p < 0.01 (t-test). n = 3 independent biological replicate experiments. Error bars represent SD. (c) HPLC analyses of carotenes extracted in the absence (pThio-θ) and presence (pThio+CsD27-3) of CsD27-3. MaxPlot chromatograms showing each peak at its spectra are presented. Peak I: 15-cis-neurosporene; peak II: 13-cis-neurosporene; peak III: 9-cis-neurosporene, and peak IV: all-trans-neurosporene. Absorption spectra of specific peaks are presented in boxes. (d) Percentage of all cis-isomers was significantly increased in the cells expressing the CsD27-3 enzyme. Statistical analysis was performed using the Student t-test. A designation of *** = p < 0.001 (t-test). n = 3 independent biological replicate experiments. Error bars represent SD.