Identification and Cloning of Differentially Expressed SOUL and ELIP Genes in Saffron Stigmas Using a Subtractive Hybridization Approach

Abstract

Using a subtractive hybridization approach, differentially expressed genes involved in the light response in saffron stigmas were identified. Twenty-two differentially expressed transcript-derived fragments were cloned and sequenced. Two of them were highly induced by light and had sequence similarity to early inducible proteins (ELIP) and SOUL heme-binding proteins. Using these sequences, we searched for other family members expressed in saffron stigma. ELIP and SOUL are represented by small gene families in saffron, with four and five members, respectively. The expression of these genes was analyzed during the development of the stigma and in light and dark conditions. ELIP transcripts were detected in all the developmental stages showing much higher expression levels in the developed stigmas of saffron and all were up-regulated by light but at different levels. By contrast, only one SOUL gene was up-regulated by light and was highly expressed in the stigma at anthesis. Both the ELIP and SOUL genes induced by light in saffron stigmas might be associated with the structural changes affecting the chromoplast of the stigma, as a result of light exposure, which promotes the development and increases the number of plastoglobules, specialized in the recruitment of specific proteins, which enables them to act in metabolite synthesis and disposal under changing environmental conditions and developmental stages.

Morphological, molecular and phytochemical variations induced by colchicine and EMS chemical mutagens in Crocus sativus L.

“…The biosynthesis pathway of the C. sativus apocarotenoids, which makes it an important crop ( Wani et al, 2017 ), begins by the oxidative cleavage of zeaxanthin in several steps, which are catalysed by the carotenoid cleavage dioxygenase (CCD2), aldehyde dehydrogenase (ALDH) and UDP-glycosyltransferases (UGTs) enzymes. Finally, due to the combined effect of heat and β-glucosidase (BGL), picrocrocin is transformed into safranal ( Ahrazem et al, 2016 , Frusciante et al, 2014 , Jain et al, 2016 ). According to RNA-seq data, the analysis of expression patterns of transcripts involved in key enzymatic steps of apocarotenoids biosynthesis, including carotenoid cleavage dioxygenase, glucosyltransferases, aldehyde dehydrogenases, and beta glucosidases, demonstrate their higher expression levels in stigma than in other tissues ( Jain et al, 2016 ).…”

“…LINK_LOC=BlastHome ). According to the Ahrazem et al (2016) , the 18S rRNA gene was used as the reference gene ( Supplementary File 2 . Table S1).…”

“…Then, the 3-hydroxy-cyclocitral and crocetin dialdehyde undergo dehydrogenation and glucosylation to make crocin and picrocrocin. Finally, after harvesting, when stigma is dried, picrocrocin will produce safranal through the combined activity of heat and β-glucosidase ( Ahrazem et al, 2016 , Frusciante et al, 2014 , Jain et al, 2016 ). It is worth noting that there is an alternative pathway where the inter-conversion of zeaxanthin and violaxanthin occur by zeaxanthin epoxidase (ZEP) and violaxanthin de-epoxidase (VDE), depending on the light intensity ( Jain et al, 2016 ) ( Supplementary File 1 .…”

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

“…The accumulation of apocarotenoids and flower development in saffron are associated with cold [3, 20], and both autumn and spring Crocus species produce and accumulate crocins during autumn-winter conditions in the Mediterranean area [25, 72]. In addition, in saffron and other Crocus species, synthesis and accumulation of crocins occurs in dark conditions [20, 82], as does the accumulation of β-carotene in carrot [83], and lycopene in some cultivars of red grapefruits kept in the dark [84], confirming how light modulates a complex network of signals connecting carotenoid biosynthesis, storage, and plastid development [85]. In addition, in this study TFs involved in retrograde signaling and epigenetic development have been identified.…”