Saffron is the dried stigmas of and is the most expensive spice in the world. Its red color is due to crocins, which are apocarotenoid glycosides that accumulate in the vacuole to a level up to 10% of the stigma dry weight. Previously, we characterized the first dedicated enzyme in the crocin biosynthetic pathway, carotenoid cleavage dioxygenase2 (CsCCD2), which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative () genes expressed in stigmas. Heterologous expression in showed that only one of corresponding proteins (CsALDH3I1) was able to convert crocetin dialdehyde into the crocin precursor crocetin. CsALDH3I1 carries a carboxyl-terminal hydrophobic domain, similar to that of the membrane-associated apocarotenoid dehydrogenase YLO-1. We also characterized the UDP-glycosyltransferase CsUGT74AD1, which converts crocetin to crocins 1 and 2'. In vitro assays revealed high specificity of CsALDH3I1 for crocetin dialdehyde and long-chain apocarotenals and of CsUGT74AD1 for crocetin. Following extract fractionation, CsCCD2, CsALDH3I1, and CsUGT74AD1 were found in the insoluble fraction, suggesting their association with membranes or large insoluble complexes. Analysis of protein localization in both stigmas and following transgene expression in leaves revealed that CsCCD2, CsALDH3I, and CsUGT74AD1 were localized to the plastids, the endoplasmic reticulum, and the cytoplasm, respectively, in association with cytoskeleton-like structures. Based on these findings and current literature, we propose that the endoplasmic reticulum and cytoplasm function as transit centers for metabolites whose biosynthesis starts in the plastid and are accumulated in the vacuole.

Salvia officinalis L. (2n=2x=14) is a perennial plant from Lamiaceae family. It has a long history of medicinal and culinary use. This study was aimed to investigate the polyploidy induction in S. officinalis L. via seed treatment with colchicine concentrations of 0, 0.05, 0.1, 0.25 and 0.5% at 12, 24 and 48 h treatment time as a factorial experiment based on the completely randomized design with three replications. Polyploidy induction firstly detected by morphological, anatomical and physiological characteristics and confirmed by flow cytometry and chromosome observation from leaves. Results showed that the colchicine concentration and treatment duration have significant effects on the survival rate of plants and the percentage of tetraploidy induction. The percentage of survived plants was decreased by increasing the treatment time and colchicine concentration. Meanwhile, the highest percentage of induced tetraploid plants was obtained at 0.25 and 0.5% colchicine with a treatment time of 48 and 24 h, respectively. Tetraploid plants specified by increasing in leaf length, leaf width, plant height, number of leaves, number of nodes, internodes length, stomata size, catalase, peroxidase and polyphenol oxidase activities, total phenolic and flavonoid contents and antioxidant capacity and on the contrary, decreasing in stomata count as compared with diploid ones.

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