Multi-species transcriptome analyses for the regulation of crocins biosynthesis in Crocus

Abstract

Background: Crocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation.

Results: A large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation.

Conclusions: Our study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level.

Keywords: Apocarotenoids; Carotenoid cleavage dioxygenases; Carotenoids; Crocins; Stigmas; Transcription factors.

Fig. 1

Differential accumulation of crocins and accumulation in two developmental stages of stigmas from Crocus. a) Stigmas in stage I and stage II from C. sativus (i and ii), C. cartwrightianus (iii and iv), and C. ancyrensis (v and vi). b) The stigmas in stage II present a distinctive yellow colouration due to the accumulation of different apocarotenoids in this stage in all the Crocus species

Fig. 2

Expression levels of differentially expressed unigenes assigned to the MEP pathway. a) An overview of the MEP pathway. b) Homologues genes encoding for the different enzymes of the pathway were identified in the transcriptome assembly of stigmas at stages I and II in the three Crocus species

Fig. 3

Expression levels of unigenes assigned to the carotenoid and apocarotenoid biosynthetic pathways in Crocus. a) An overview of the crocins biosynthesis pathway enzymes and metabolites in Crocus. Homologues genes encoding for the different enzymes were identified in the transcriptome assembly. b) Expression analyses of genes encoding from the enzymes of the carotenoid biosynthesis pathway identified in the transcriptomes from I and II stages of the three Crocus species. c) Expression analyses of homologues to carotenoid cleavage enzymes (CCD1, CCD2, CCD4, CCD7, CCD8 and NCED), to the β-carotene isomerase D27 and UGT74AD2 genes identified in the six transcriptomes. d) Expression analyses of ALDH genes homologues identified in the six transcriptomes analysed

Fig. 4

Transcription factor families identified in the differential expression analyses of the two stigmas transcriptomes of saffron. Transcription factors (TFs) were identified by sequence comparison of saffron transcripts with the Plant Transcription Factor Database (PlnTFDB) by BLASTX, using default parameters and cut-off E-value of 1e^− 5

Fig. 5

Relative expression levels of the 11 TFs by qRT-PCR in different tissues from saffron