The Specialized Roles in Carotenogenesis and Apocarotenogenesis of the Phytoene Synthase Gene Family in Saffron

Abstract

Crocus sativus stigmas are the main source of crocins, which are glucosylated apocarotenoids derived from zeaxanthin cleavage that give saffron its red color. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis in plants. Four PSY genes encoding functional enzymes were isolated from saffron. All the proteins were localized in plastids, but the expression patterns of each gene, CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3, in different saffron tissues and during the development of the stigma showed different tissue specialization. The CsPSY2 transcript was primarily detected in the stigmas where it activates and stimulates the accumulation of crocins, while its expression was very low in other tissues. In contrast, CsPSY1a and CsPSY1b were mainly expressed in the leaves, but only CsPSY1b showed stress-light regulation. Interestingly, CsPSY1b showed differential expression of two alternative splice variants, which differ in the intron retention at their 5' UTRs, resulting in a reduction in their expression levels. In addition, the CsPSY1a and CsPSY1b transcripts, together with the CsPSY3 transcript, were induced in roots under different stress conditions. The CsPSY3 expression was high in the root tip, and its expression was associated with mycorrhizal colonization and strigolactone production. CsPSY3 formed a separate branch to the stress-specific Poaceae homologs but was closely related to the dicot PSY3 enzymes.

Keywords: activity; apocarotenoids; carotenoids; expression; mycorrhiza; phytoene synthase; root; stigmas.

FIGURE 1

PSY amino acid sequence similarities. (A) Pairwise percentage similarity of the Crocus PSY mature amino acid sequences without the putative signal peptide. (B) Alignment of the amino acid sequences of the putative CsPSYs. With red background: aspartate rich regions and substrate-Mg2+-binding sites (DXXXD); black underline: active site lid residues. Green background: SQS-PSY domain 1; and blue background: SQS-PSY domain 2.

FIGURE 2

Three dimensional models and location of the saffron phytoene synthase enzymes. (A) Three-dimensional models of the CsPSY enzymes. The models for CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 were created using the PPM Server (http://opm.phar.umich.edu/server.php). The α-helices, and loops are depicted as pink and blue, respectively. Blue dots indicate the membrane surface. (B) Subcellular localization of GFP fusion proteins of CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 in agro-infiltrated tobacco leaves after 5 days as detected with confocal laser scanning microscopy and enhanced green fluorescent protein (eGFP) expression. Chlorophyll auto-fluorescence in red (left panel), eGFP fluorescence is shown in green (middle panel) and a merged overlay of the eGFP/chlorophyll fluorescence (right panel) is shown in yellow.

FIGURE 3

Dendrogram of the CsPSYs amino acid sequences with other plant members of the PSY family. The unrooted phylogenetic tree was constructed using MEGA7 from the PSY sequences retrieved from the GenBank database. Evolutionary relationships were inferred using the Neighbor-joining method with 2500 bootstrap re-sampling strategy. The PSYs sequences from Crocus species are indicated by different colors.

FIGURE 4

Relative expression levels of the CsPSYs in vegetative and reproductive tissues investigated by qRT-PCR. (A) Transcripts levels in the tepals at different developmental stages, and in the stigma and stamens at anthesis. (B) Expression levels in seven developmental stages of the stigma. (C) Transcripts levels in the cormlet, corm, leaf and roots. Scale bars indicate five biological replicates ± SD.

FIGURE 5

Stress regulation of the CsPSYs transcripts in leaf and root tissue. (A) qRT-PCR analysis of the CsPSY gene expression in leaves at different developmental stages and along the mature leaf (L, low part of the leaf; M, medium part of the leaf; Up, upper part of the leaf). (B) Transcript levels in leaves dark-adapted for 2 h and upon illumination with white light for 4 h. (C) Transcript levels of the CsPSYs genes in the roots of dry plants, re-watering plants and in plants treated with 200 mM NaCl. Error bars indicate five biological replicates ± SD. Asterisks mark statistically significant differences relative to reference (no NaCl addition) samples (P < 0.05).

FIGURE 6

Expression analysis of the CsPSY genes in the root tissue. (A) The root system of saffron plants. i, the complete root system; ii, the root tip (RT); iii, the mature root (MR); iv, the corm showing the radicular bottom (RB), the initiation of the roots (IR), and the dissection of the IR, showing the root vascular cylinder (IRI). Bar = 50 μm. (B) qRT-PCR-based expression analysis of CsPSY genes in different parts of saffron roots. Expression levels are shown relative to the constitutively expressed RSP18 gene. Error bars indicate SD from three technical replicates. (C) Trypan blue staining of saffron roots with (i and ii) and without mycorrhization (iii and iv). Error bars = 50 μm. (D) qRT-PCR based expression analysis of the CsPSY genes in roots with or without mycorrhiza. (E) qRT-PCR based expression analysis of the CsCCD genes in roots with mycorrhiza or without. Expression levels are shown relative to the constitutively expressed saffron RSP18 gene. Error bars indicate SD from five biological replicates. (F) Orobanchol levels in roots with or without mycorrhiza. Error bars indicate SD from three biological replicates.

FIGURE 7

Functional complementation of the saffron PSY proteins. (A) Complex models of the CsPSY enzymes and their co-factor. (B) Escherichia coli cells harboring the pAC-85b vector were additionally transformed with saffron PSY constructs or empty vector. Cells carrying the pAtPSY vector confer accumulation of β-carotene and were used as a positive control. HPLC chromatograms for the extracted pigments are shown. The peak representing β-carotene was observed in cells with CsPSY1a, CsPSY1b, CsPSY2, and CsPSY3 constructs, but not in the empty vector. The inset shows the absorption spectrum of the β-carotene peak.