Implications of carotenoid biosynthetic genes in apocarotenoid formation during the stigma development of Crocus sativus and its closer relatives

Abstract

Crocus sativus is a triploid sterile plant characterized by its long red stigmas, which produce and store significant quantities of the apocarotenoids crocetin and crocin, formed from the oxidative cleavage of zeaxanthin. Here, we investigate the accumulation and the molecular mechanisms that regulate the synthesis of these apocarotenoids during stigma development in C. sativus. We cloned the cDNAs for phytoene synthase, lycopene-beta-cyclase, and beta-ring hydroxylase from C. sativus. With the transition of yellow undeveloped to red fully developed stigmas, an accumulation of zeaxanthin was observed, accompanying the expression of CsPSY, phytoene desaturase, and CsLYCb, and the massive accumulation of CsBCH and CsZCD transcripts. We analyzed the expression of these two transcripts in relation to zeaxanthin and apocarotenoid accumulation in other Crocus species. We observed that only the relative levels of zeaxanthin in the stigma of each cultivar were correlated with the level of CsBCH transcripts. By contrast, the expression levels of CsZCD were not mirrored by changes in the apocarotenoid content, suggesting that the reaction catalyzed by the CsBCH enzyme could be the limiting step in the formation of saffron apocarotenoids in the stigma tissue. Phylogenetic analysis of the CsBCH intron sequences allowed us to determine the relationships among 19 Crocus species and to identify the closely related diploids of C. sativus. In addition, we examined the levels of the carotenoid and apocarotenoid biosynthetic genes in the triploid C. sativus and its closer relatives to determine whether the quantities of these specific mRNAs were additive or not in C. sativus. Transcript levels in saffron were clearly higher and nonadditive, suggesting that, in the triploid gene, regulatory interactions that produce novel effects on carotenoid biosynthesis genes are involved.

Figure 1.

HPLC profile of the chloroform extract of C. sativus stigmas in different developmental stages. The HPLC profile of fully developed stigmas is shown in blue: the main peak detected corresponds to saffron apocarotenoids (1). The inset shows an enlargement of the HPLC trace between 23 and 38 min; 2, online diode array spectrum of zeaxanthin; 3, online diode array spectrum of β-cryptoxanthin; 4, online diode array spectrum of ζ-carotene; 5, online diode array spectrum of β-carotene. Lycopene and phytofluene were also observed, but at low amounts. The HPLC profiles of yellow and orange stigmas are shown in the inset in gray and black, respectively. The HPLC conditions used for separation of the extracted carotenoids were the same in both cases.

Figure 2.

A Phylogenetic tree based on carotene hydroxylase amino acid sequences deduced and obtained from the GenBank. The neighbor-joining tree was constructed by using the putative carotene hydroxylase enzyme from Haematococcus pluvialis as an outgroup. Values indicate the percent bootstrap support for each branch. The horizontal scale shows the number of differences per 100 residues derived from the ClustalW alignment. For accession numbers of the BCH sequences, see “Materials and Methods.”

Figure 3.

Intron distribution within the BCH genes from several plant species. A, Diagrammatic representation of the BCH genes of Arabidopsis, maize, rice, and C. sativus. The position of the ATG translation start and the translation stop are indicated. Boxes and lines denote coding regions and introns, respectively. The position of the primers used for intron analysis and sequencing are indicated on the diagrammatic representation of the genomic clone CsBCH1. B, The BCH-300s and BCHCs-r primer combination was used to amplify the BCH product in several Crocus species. 1, C. speciosus; 2, C. biflourus; 3, C. sieberi; 4, C. niveus; 5 C. goulimyi; 6, C. vernus; 7, C. chrysanthus; 8, C. kotschyanus; 9, C. kosaninii; 10, C. hadriaticus; 11, C. imperati; and 12, C. cartwrightianus.

Figure 4.

Phylogenetic tree resulting from GeneBee analysis of BCH1 sequences from the 5′ end of intron I to the 3′ end of intron II from the Crocus populations described in Table II. Symbols next to each name denote populations of the same species: •, C. cartwrightianus; C, C. hadriaticus; ▴, C. oreocreticus; and 8, C. pallasii. On the right side of the tree, representative HPLC profiles of aqueous extracts (apocarotenoid extracts) obtained from fully developed stigmas from several Crocus species, which are indicated in the right side of each profile, are shown. The species inside the group Crocus are characterized by the highest levels of apocarotenoids accumulated in their stigma tissue.

Figure 5.

Expression levels of saffron carotenogenic genes during stigma development. The mRNA levels were determined by RT-PCR amplification using specific oligonucleotides for each carotenogenic gene. A, Stigma tissue of C. sativus in different developmental stages. B, CsBCH1 and CsBCH2 transcript expression in undeveloped (StII and StIV) and fully developed (StVIII) stigma, petals, and leaves. The expression pattern of CsPSY, CsPDS, CsLYCb, CsCCD, and CsZDS was monitored during stigma development. Equal amounts of total RNA were used in each reaction. The levels of constitutively expressed RPS18 coding genes were assayed as controls. The PCR products were separated by 2% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining.

Figure 6.

Pigmentation levels in Crocus species are associated with carotenoid and BCH transcript accumulation. A, External pigmentation of fully developed stigmas of several Crocus species. From left to right, C. hadriaticus, C. cartwrightianus, C. imperati, C. goulimyi, and C. sativus. B, HPLC profile of carotenoids extracted form stigma tissues. C, Detection of BCH transcripts by RT-PCR in fully developed stigma at the time of anthesis in different Crocus species. The levels of constitutively expressed RPS18 coding genes were assayed as controls. The PCR products were separated by 2% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining. At the bottom, the crocin concentration for each of the species analyzed is shown.