Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron

31Citations
Citations of this article
51Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid β-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma.

Figures

  • Table 1 Primer sequences used for CsCCD2 analysis Used for 50 ? 30
  • Fig. 1 Analysis of the exon–intron structures of CsCCD2 genes. Schematic diagram of the exon–intron structures of CsCCD2 genes. Gray and black boxes indicate exons and untranslated regions (UTRs), respectively. Introns are indicated by lines. The size of each exon is indicated over the exon boxes in base pairs and the size of the protein products in presented on the left of each gene in parenthesis
  • Fig. 2 Amino acid sequences and structural comparisons of CsCCD2 and CsCCD2-t. a Amino acid alignment showing the conserved histidine residues highlighted with yellow shading and in grey shading the key amino acid residues necessary for CCDs catalytic activity. b Tridimensional models of CsCCD2-t (a, c) and CsCCD2 (b, d), with b-strands shown in cyan, a-helices in magenta, and loops in black
  • Table 2 The 50 and 30-splice sites sequences for the intronic CsCCD2 sequences, compared with the consensus sequences of the CCD1 genes from the monocot species
  • Fig. 4 Alignment of amino acid sequences of CCD truncated proteins. a Amino acid sequences of CsCCD2 truncated (CsCCD2t) homologues detected in C. ancyrensis (CaCCD2-t) and C. cartwrightianus (CcarCCD2-t). Asterisk indicates conserved amino acid residues. The hatched box denotes the deletion observed in
  • Table 3 Putative cis-elements identified in CsCCD2b promoter
  • Table 4 Commom sequences identified in CsCCD2b and CstLcyB2a promoters using PromoterWise
  • Fig. 5 Validation of CsCCD2 expression based on the elements identified by in silico promoter analysis by qRT-PCR. a Expression levels of CsCCD2 under different light and temperature conditions. Represented values were obtained after subtracting the control value. c Light stands for continuous light and C. Dark stands for continuous dark. b Expression levels of CsCCD2 in stigmas at different developmental stages (yellow, orange, and red) from plants growing in continuous dark (yellow-D, orange-D and red-D) or normal (light/ dark) conditions (yellow-L, orange-L and red-L) are plotted on the left y-axe. Crocins levels (on the right axe) for each treatment and developmental stage are also indicated. C5, trans-crocin with 5 glucose molecules; C4, trans-crocin with 4 glucose molecules; C4cis, cis-crocin with 4 glucose molecules; C3, trans-crocin with 3 glucose molecules; C2, trans-crocin with 2 glucose molecules crocins abundance on. c Expression data of CsCCD2, CsBCH2 and CsLycB2a from plants growing in field. Expression levels are plotted on the left y-axe, and temperature on the right.White box on x-axe, light interval; black box, dark interval

References Powered by Scopus

The Phyre2 web portal for protein modeling, prediction and analysis

7540Citations
N/AReaders
Get full text

PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences

5435Citations
N/AReaders
Get full text

Plant responses to drought, salinity and extreme temperatures: Towards genetic engineering for stress tolerance

2961Citations
N/AReaders
Get full text

Cited by Powered by Scopus

Carotenoid cleavage oxygenases from microbes and photosynthetic organisms: Features and functions

139Citations
N/AReaders
Get full text

Apocarotenoids: A new carotenoid-derived pathway

69Citations
N/AReaders
Get full text

Efficient production of saffron crocins and picrocrocin in Nicotiana benthamiana using a virus-driven system

57Citations
N/AReaders
Get full text

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Ahrazem, O., Rubio-Moraga, A., Argandoña-Picazo, J., Castillo, R., & Gómez-Gómez, L. (2016). Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron. Plant Molecular Biology, 91(3), 355–374. https://doi.org/10.1007/s11103-016-0473-8

Readers over time

‘16‘17‘18‘19‘20‘21‘22‘23‘24‘250481216

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 18

56%

Researcher 10

31%

Professor / Associate Prof. 4

13%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 15

54%

Biochemistry, Genetics and Molecular Bi... 10

36%

Social Sciences 2

7%

Chemical Engineering 1

4%

Save time finding and organizing research with Mendeley

Sign up for free
0