Abstract
Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.
Highlights
A fascinating feature of vascular plants is their capacity to produce extremely diverse specialized metabolites (Pichersky and Lewinsohn, 2011) and to accumulate some of them to extremely high concentrations: steviol glycosides in Stevia rebaudiana leaves (Brandle and Telmer, 2007) and crocins in Crocus sativus stigmas (Bouvier et al, 2003), for instance, can make up to 10% of the tissue’s dry weight
We study the in vitro transport of multiple metabolites from C. sativus stigma extracts, through a transportomic assay based on liquid chromatography-photodiode array-high resolution mass spectrometry (LC-PDA-HRMS) and its use to identify and characterize C. sativus tonoplast transporters involved in vacuolar crocin accumulation
A C. sativus stigma transcriptome (Supplemental Data Set 1) was searched for expressed genes belonging to the ATP binding cassette C (ABCC) and MATE classes of tonoplast transporters
Summary
A fascinating feature of vascular plants is their capacity to produce extremely diverse specialized metabolites (Pichersky and Lewinsohn, 2011) and to accumulate some of them to extremely high concentrations: steviol glycosides in Stevia rebaudiana leaves (Brandle and Telmer, 2007) and crocins in Crocus sativus stigmas (Bouvier et al, 2003), for instance, can make up to 10% of the tissue’s dry weight. Well-known examples of the latter strategy are the vacuolar sequestration of anthocyanins in grape (Vitis vinifera) berries (Francisco et al, 2013), nicotine in Nicotiana tabacum leaves (Morita et al, 2009), steviol glycosides in S. rebaudiana leaves (Brandle and Telmer, 2007), and crocins in C. sativus stigmas (Bouvier et al, 2003). More highly glycosylated crocins are synthesized by an unidentified UDP-glycosyl transferase (UGT), probably localized in the cytosol (Figure 1D). Given their polar nature, crocins synthesized in the cytosol must be transported to the vacuole by one or more tonoplast transporters (Figure 1D).
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