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Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.arotenoids are a group of C40 isoprenoids synthesized in chloroplasts, chromoplasts, and other plastids in plants. Carotenoids serve as components of photosynthetic machinery, precursors for phytohormones, and important contributors to fruit nutritional quality and flower color (1, 2). The carotenoid biosynthetic pathway in higher plants has been well defined. However, identification of the regulatory mechanisms underlying carotenoid biosynthesis remains a challenge.Phytoene synthase (PSY) catalyzes the first committed step in carotenoid biosynthesis and controls carbon flux into the carotenoid biosynthetic pathway (1-5). Alteration of PSY expression exerts profound effects on carotenoid content (6-11). A number of factors are known to affect PSY gene expression (12)(13)(14)(15)(16)(17)(18). PSY is found to be repressed by phytochrome-interacting factors in etiolated Arabidopsis seedlings (16). PSY1 expression in tomato fruits is reported to be regulated by cis-carotenoids (14) and requires the MADS-Box transcription factor RIPENING INHIBITOR (18). Recently, it was discovered that PSY protein levels in carrot roots are modulated by a negative feedback emerging from carotenoids (19). The crucial role of PSY in carotenogenesis and the multiple factors affecting its expression suggest a complex regulatory system involved in controlling PSY. However, the factors involved in posttranscriptional regulation of PSY within plastids remain a mystery. No proteins have been re...

Yellow dwarf viruses in the family Luteoviridae, which are the causal agents of yellow dwarf disease in cereal crops, are each transmitted most efficiently by different species of aphids in a circulative manner that requires the virus to interact with a multitude of aphid proteins. Aphid proteins differentially expressed in F2 Schizaphis graminum genotypes segregating for the ability to transmit Cereal yellow dwarf virus-RPV (CYDV-RPV) were identified using two-dimensional difference gel electrophoresis (DIGE) coupled to either matrix-assisted laser desorption ionization-tandem mass spectrometry or online nanoscale liquid chromatography coupled to electrospray tandem mass spectrometry. A total of 50 protein spots, containing aphid proteins and proteins from the aphid's obligate and maternally inherited bacterial endosymbiont, Buchnera, were identified as differentially expressed between transmission-competent and refractive aphids. Surprisingly, in virus transmission-competent F2 genotypes, the isoelectric points of the Buchnera proteins did not match those in the maternal Buchnera proteome as expected, but instead they aligned with the Buchnera proteome of the transmission-competent paternal parent. Among the aphid proteins identified, many were involved in energy metabolism, membrane trafficking, lipid signaling, and the cytoskeleton. At least eight aphid proteins were expressed as heritable, isoelectric point isoform pairs, one derived from each parental lineage. In the F2 genotypes, the expression of aphid protein isoforms derived from the competent parental lineage aligned with the virus transmission phenotype with high precision. Thus, these isoforms are candidate biomarkers for CYDV-RPV transmission in S. graminum. Our combined genetic and DIGE approach also made it possible to predict where several of the proteins may be expressed in refractive aphids with different barriers to transmission. Twelve proteins were predicted to act in the hindgut of the aphid, while six proteins were predicted to be associated with the accessory salivary glands or hemolymph. Knowledge of the proteins that regulate virus transmission and their predicted locations will aid in understanding the biochemical mechanisms regulating circulative virus transmission in aphids, as well as in identifying new targets to block transmission.

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953–2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.