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...

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.

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyA's spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.

Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly b-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C 13 apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of b-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.

Phytochromes (phy) are red/far-red-absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N-and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.Arabidopsis | basic helix-loop-helix | signal transduction P lant development is strictly regulated by the environmental light conditions. Even though light is ubiquitously present, spectral composition, intensity, and light direction varies depending on the local environment. To adapt on local light conditions plants are equipped with a highly developed repertory of photoreceptors (1, 2).Plant phytochromes are mainly involved in detection of red and far-red light wavelengths of the spectrum. A family of five members in Arabidopsis thaliana regulates the major developmental steps in the life of the plant: germination, photomorphogenesis, and flowering (3, 4). Despite the sequence similarities and identical spectroscopic features, the two major phytochromes, phyA and phyB, show overlapping as well as unique photosensory and functional characteristics (5-7). The light-stable phyB mediates red/far-red reversible low fluence response, whereas the light-labile phyA, is involved in far-red light sensing (high irradiance response) and in responses to very weak light (very low fluence response, VLFR) (8).Phytochromes are cytosolic proteins of 125 kDa that are translocated into the nucleus upon light activation (6, 9-11). Nuclear transport is an essential step in phytochrome signaling and a prerequisite for the interaction with transcription factors in the nucleus (12, 13). Concerning the kinetics of nuclear import, severe differences can be found between the two major plant phytochromes, phyA and phyB (6). PhyA cannot be detected in nuclei of dark-grown plants but is rapidly transported into the nucleus upon light irradiation. In contrast, low levels of phyB are present in the nucleus even in dark-grown seedlings. The nuclea...