We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5# untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.Carotenoids are lipophilic isoprenoids produced by all photosynthetic organisms as well as by some nonphotosynthetic bacteria and fungi. In animals, carotenoids come from the food chain and function as colorants and precursors for essential metabolites, such as retinal, retinol, and retinoic acid. In plants, carotenoids play their classical roles in light-harvesting complexes and photosynthetic reaction centers, where they absorb light and dissipate excess energy (for review, see Adams, 1992, 2000;Niyogi, 1999).The plant carotenoid biosynthetic pathway is localized in the plastid and has been molecularly elucidated in recent years (for review, see DellaPenna and Pogson, 2006). It diverges from C 3 carbon metabolism by the action of the enzyme deoxyxylulose phosphate synthase, followed by a series of enzymes of the socalled nonmevalonate pathway (for review, see Hunter, 2007), yielding isopentenyl diphosphate (IPP; C 5 ), the building block of all isoprenoids. IPP and its isomer dimethylallyl diphosphate (DMAPP) are then converted into carotenes through chain-elongating condensation reactions catalyzed by geranylgeranyl diphosphate synthase (GGPPS) and phytoene synthase (PSY; Fig. 1). The triene chromophore of phytoene (C 40 ), the first carotene formed, is then extended to form the colored undecaene in lycopene catalyzed by phytoene desaturase (PDS) and z-carotene desaturase (ZD...

Cassava (Manihot esculenta) is an important staple crop, especially in the arid tropics. Because roots of commercial cassava cultivars contain a limited amount of provitamin A carotenoids, both conventional breeding and genetic modification are being applied to increase their production and accumulation to fight vitamin A deficiency disorders. We show here that an allelic polymorphism in one of the two expressed phytoene synthase (PSY) genes is capable of enhancing the flux of carbon through carotenogenesis, thus leading to the accumulation of colored provitamin A carotenoids in storage roots. A single nucleotide polymorphism present only in yellow-rooted cultivars cosegregates with colored roots in a breeding pedigree. The resulting amino acid exchange in a highly conserved region of PSY provides increased catalytic activity in vitro and is able to increase carotenoid production in recombinant yeast and Escherichia coli cells. Consequently, cassava plants overexpressing a PSY transgene produce yellow-fleshed, high-carotenoid roots. This newly characterized PSY allele provides means to improve cassava provitamin A content in cassava roots through both breeding and genetic modification.

R.W. and P.B. conceived the research plan and supervised the experiments; M.C. performed most of the experiments and data analysis, A.G. contributed to project conception and performed vector construction, C.B. contributed to enzyme assay establishment and performed experiments; R.W. generated and analyzed the transgenic lines; R.W. wrote the article with support from J.K. and P.B.; R.W. agrees to serve as the author responsible for contact and ensures communication.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is assumed to lack carotenoids, which are widespread pigments fulfilling important functions as radical scavengers and as a source of apocarotenoids. In mammals, the synthesis of apocarotenoids, including retinoic acid, is initiated by the β‐carotene cleavage oxygenases I and II catalyzing either a central or an excentric cleavage of β‐carotene, respectively. The M. tuberculosis ORF Rv0654 codes for a putative carotenoid oxygenase conserved in other mycobacteria. In the present study, we investigated the corresponding enzyme, here named M. tuberculosis carotenoid cleavage oxygenase (MtCCO). Using heterologously expressed and purified protein, we show that MtCCO converts several carotenoids and apocarotenoids in vitro. Moreover, the identification of the products suggests that, in contrast to other carotenoid oxygenases, MtCCO cleaves the central C15‐C15′ and an excentric double bond at the C13‐C14 position, leading to retinal (C20), β‐apo‐14′‐carotenal (C22) and β‐apo‐13‐carotenone (C18) from β‐carotene, as well as the corresponding hydroxylated products from zeaxanthin and lutein. Moreover, the enzyme cleaves also 3,3′‐dihydroxy‐isorenieratene representing aromatic carotenoids synthesized by other mycobacteria. Quantification of the products from different substrates indicates that the preference for each of the cleavage positions is determined by the hydroxylation and the nature of the ionone ring. The data obtained in the present study reveal MtCCO to be a novel carotenoid oxygenase and indicate that M. tuberculosis may utilize carotenoids from host cells and interfere with their retinoid metabolism.

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