Differential interaction of Or proteins with the PSY enzymes in saffron

Abstract

Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.Nucleic acid purification and cDNA isolation. Total RNA was isolated from red stigmas 48 , following the manufacturer's protocols (Qiagen, Hilden, Germany). First-strand cDNAs were synthesized by RT from 2 µg of total RNA using an 18-base pair oligo dT primer and a first-strand cDNA synthesis kit (GE Healthcare Life Sciences, www.gelifesciences.com) according to manufacturer's instructions. These cDNAs were used as templates for PCR using specific primers for CsOr-a and CsOr-b (Suppl . Table 1). Thermal cycling parameters were 2 min at 95 °C, 35 cycles of 20 s at 95 °C, 20 s at 60 °C and 1 min 30 s at 72 °C, followed by a final extension of 5 min at 72 °C. The PCR products were separated in a 1% agarose gel, purified, ligated into pGEM-T (Promega, www. promega.com), and then introduced into E. coli. Scientific RepoRtS |(2020) 10:552 | https://doi.

Phytoene Synthase: The Key Rate-Limiting Enzyme of Carotenoid Biosynthesis in Plants

“…This is especially true in photosynthetically active tissues, where PSY protein amounts are extremely low and subjected to a relatively high protein turnover rate. Many PSYs are known to require chaperone proteins for maintaining their stability and carotenogenic functions within plastids ( Zhou et al, 2015 ; Park et al, 2016 ; Chayut et al, 2017 ; D’andrea et al, 2018 ; Welsch et al, 2018 ; Ahrazem et al, 2020 ).…”

Perturbations in the Carotenoid Biosynthesis Pathway in Tomato Fruit Reactivate the Leaf-Specific Phytoene Synthase 2

“…It has been established in Arabidopsis, melon, and sweet potato that the DnaJ chaperon protein ORANGE (Or) stabilizes PSY and regulates its activity by direct interaction between the two proteins (Zhou et al, 2015). Differential interaction of Or with paralogous PSY enzymes was reported in saffron (Ahrazem et al, 2020). The activation of PSY2 prompted by changes in the carotenoid composition may be enabled by a unique interaction of Or with PSY2 that either stabilizes the enzyme or interferes with its import to chromoplasts (Yuan et al, 2021).…”

Comparative transcriptome analyses shed light on carotenoid production and plastid development in melon fruit

“…OR protein was found to directly interact with PSY and posttranslationally upregulate it in melon as well as in a number of plant species 12 , 19 , 48 – 51 . OR regulates PSY activity, which in turn controls the biosynthesis of carotenoid pigments necessary for both chloroplast and chromoplast development 12 , 52 .…”