Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Abstract

BackgroundAs the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.Methology/Principal FindingsIn Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.ConclusionsThe sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.

Periodic root branching in Arabidopsis requires synthesis of an uncharacterized carotenoid derivative

“…PSY encodes the enzyme that catalyzes the first committed step of carotenoid synthesis. Varying expression or activity of PSY alters flux through the carotenoid biosynthesis pathway (36)(37)(38). We predicted that the expression pattern of PSY would provide functional insight into the carotenoid biosynthesis pathway in roots.…”

Carotenoids in nature: insights from plants and beyond

“…astaxanthin), which produce a deep yellow ('golden') potato tuber phenotype and a distinct pink to deep red colour compared with the typical orange colour in wild-type carrot roots (Taylor and Ramsay 2005;Diretto et al 2007;Jayaraj et al 2008). Finally, the overexpression of PSY in Arabidopsis increased carotenoid content by 10-to 100-fold in nonphotosynthetic calli and roots, predominantly b-carotene and its derivatives, which were deposited as crystals in storage plastids (Maass et al 2009). …”

Provitamin A Accumulation in Cassava (Manihot esculenta) Roots Driven by a Single Nucleotide Polymorphism in a Phytoene Synthase Gene

“…Proteins (30 mg yeast lysate; 1.5 mg E. coli lysate) were separated by SDS-PAGE and blotted onto polyvinylidene fluoride membranes. Immunodetection of PSY2 in yeast protein was performed with anti-At-PSY antibodies (Maass et al, 2009). GGPS in yeast was detected using an anti-HA-tag antibody (Cell Signaling Technology).…”

“…The Arabidopsis PSY ORF with its transit peptide sequence removed at position #58 was subcloned into pCOLDI (Takara-Clontech) to yield an N-terminal His 6 fusion as described (Maass et al, 2009). From this plasmid, a second vector coding for an Asp instead of Ala at position #187 was generated by overlapping extension PCR (for primers, see Supplemental Table 3 online).…”

“…This has been shown to be the case in canola (Brassica napus; Shewmaker et al, 1999) and Arabidopsis thaliana seeds (Lindgren et al, 2003). PSY expression can be driven to levels at which b-carotene crystals are formed, as in Arabidopsis cells and in carrot (Daucus carota) roots (Maass et al, 2009). However, there are cases where additional transgenes are needed to raise carotenoid production levels, such as in Golden Rice, in which the carotene desaturase CrtI had to be introduced along with PSY (Burkhardt et al, 1997;Ye et al, 2000;Schaub et al, 2005) or potato, in which a mini pathway of three carotenoid genes was necessary to achieve high levels of production (Diretto et al, 2007).…”