Transcriptome analysis in tissue sectors with contrasting crocins accumulation provides novel insights into apocarotenoid biosynthesis and regulation during chromoplast biogenesis

Abstract

Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice.

UGT709G1: a novel uridine diphosphate glycosyltransferase involved in the biosynthesis of picrocrocin, the precursor of safranal in saffron (Crocus sativus)

“…The use of plant materials with contrasting levels of secondary metabolite accumulation is essential for the characterization of specialized metabolic pathways. Based on this premise, we previously analyzed the transcriptome of C. sieberi tepals obtained from two differentially pigmented areas either devoid of (white (w)), or accumulating (yellow (y)) apocarotenoids, respectively, at two developmental stages (6 and 8) (Ahrazem et al ., ). As expected, differential expression analyses revealed the presence of CsCCD2L homologues in the highly differentially expressed transcripts in the yellow samples (Ahrazem et al ., ).…”

“…Specifically, picrocrocin was detected in the stigmas from crocuses that belong to series Crocus but was absent in the stigmas of some crocuses tested from series Versicolores , Longiflori , Koschiani , Flavi , Speciosi , and Reticulati . The sole exception found was C. sieberi (series Reticulati ), which accumulates picrocrocin in stigma and tepal pigmented localized segments (Ahrazem et al ., ). Interestingly, C. sieberi grouped in a single clade in the combined ITS/trnL‐F phylogeny with series Crocus , whereas the other species of section Nudiscapus grouped in three other different clades (Harpke et al ., ).…”

“…Coexpression and correlation network were performed by calculating pairwise Pearson correlations between each UGT gene and picrocrocin; subsequently, Fisher's Z ‐transformation was used to determine the statistical significance of the pairwise correlations. Finally, significant correlations, and with P > |0.60|, at least, were used to draw a correlation network, by using Cytoscape 3.7.0 software with a force‐directed layout, and as previously described (Gomez‐Gomez et al ., ; Ahrazem et al ., ).…”

“…stigmas HPLC-high-resolution MS (HRMS) of Crocus spp. stigmas was performed as previously described (Rambla et al, 2016;Ahrazem et al, 2018;Grosso et al, 2018), with slight modifications in the chromatographic separation and the heated electrospray ionization (HESI) parameters. More in detail, the separation was developed using 95% A (99.9% water, 0.1% formic acid) 5% B (99.9% acetonitrile, 0.1% formic acid) for 0.5 min, followed by a 10 min + 12 min (flow rates 0.25 ml min À1 and 0.125 ml min À1 , respectively) linear gradient to 75% B. HESI conditions were as follows: N was used as sheath and auxiliary gas, set to 35 units and 25 units, respectively; the vaporizer temperature was 280°C, whereas the capillary temperature was set at 280°C, the discharge current at 4.5 lA, and the S-lens radiofrequency level at 50.…”

“…The acquisition was carried out both in positive-and in negative-ion mode using a 110-1600 m/z mass range, with the following parameters: resolution 70 000, microscan 1, automatic gain control target 1 9 10 6 , and maximum injection time 50. Identification, validation, and quantification of crocins, picrocrocin, and their derivatives were performed as reported previously Research New Phytologist (D'Archivio et al, 2018) (D'Archivio et al, 2016Ahrazem et al, 2018;Moras et al, 2018).…”