Patrick Schaub

Golden Indica and Japonica Rice Lines Amenable to Deregulation

As an important step toward free access and, thus, impact of GoldenRice, a freedom-to-operate situation has been achieved for developing countries for the technology involved. Specifically, to carry the invention beyond its initial “proof-of-concept” status in a Japonica rice (Oryza sativa) cultivar, we report here on two transformed elite Indica varieties (IR64 and MTL250) plus one Japonica variety Taipei 309. Indica varieties are predominantly consumed in the areas with vitamin A deficiency. To conform with regulatory constraints, we changed the vector backbone, investigated the absence of beyond-border transfer, and relied on Agrobacterium tumefaciens-mediated transformation to obtain defined integration patterns. To avoid an antibiotic selection system, we now rely exclusively on phosphomannose isomerase as the selectable marker. Single integrations were given a preference to minimize potential epigenetic effects in subsequent generations. These novel lines, now in the T3 generation, are highly valuable because they are expected to more readily receive approval for follow-up studies such as nutritional and risk assessments and for breeding approaches leading to locally adapted variety development.

Why Is Golden Rice Golden (Yellow) Instead of Red

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of β-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, ζ-carotene desaturase, carotene cis-trans-isomerase, β-lycopene cyclase, and β-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that β-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, α/β-lycopene cyclase, β-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or ζ-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the β-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

The Lycopene Cyclase CrtY from Pantoea ananatis (Formerly Erwinia uredovora) Catalyzes an FADred-dependent Non-redox Reaction

The cyclization of lycopene generates provitamin A carotenoids such as β-carotene and paves the way toward the formation of cyclic xanthophylls playing distinct roles in photosynthesis and as precursors for regulatory molecules in plants and animals. The biochemistry of lycopene cyclization has been enigmatic, as the previously proposed acid-base catalysis conflicted with the possibility of redox catalysis as predicted by the presence of a dinucleotide binding site. We show that reduced FAD is the essential lycopene cyclase (CrtY) cofactor. Using flavin analogs, mass spectrometry, and mutagenesis, evidence was obtained based on which a catalytic mechanism relying on cryptic (net) electron transfer can be refuted. The role of reduced FAD is proposed to reside in the stabilization of a transition state carrying a (partial) positive charge or of a positively charged intermediate via a charge transfer interaction, acid-base catalysis serving as the underlying catalytic principle. Lycopene cyclase, thus, ranks among the novel class of non-redox flavoproteins, such as isopentenyl diphosphate:dimethylallyl diphosphate isomerase type 2 (IDI-2) that requires the reduced form of the cofactor.

On the Structure and Function of the Phytoene Desaturase CRTI from Pantoea ananatis , a Membrane-Peripheral and FAD-Dependent Oxidase/Isomerase

CRTI-type phytoene desaturases prevailing in bacteria and fungi can form lycopene directly from phytoene while plants employ two distinct desaturases and two cis-tans isomerases for the same purpose. This property renders CRTI a valuable gene to engineer provitamin A-formation to help combat vitamin A malnutrition, such as with Golden Rice. To understand the biochemical processes involved, recombinant CRTI was produced and obtained in homogeneous form that shows high enzymatic activity with the lipophilic substrate phytoene contained in phosphatidyl-choline (PC) liposome membranes. The first crystal structure of apo-CRTI reveals that CRTI belongs to the flavoprotein superfamily comprising protoporphyrinogen IX oxidoreductase and monoamine oxidase. CRTI is a membrane-peripheral oxidoreductase which utilizes FAD as the sole redox-active cofactor. Oxygen, replaceable by quinones in its absence, is needed as the terminal electron acceptor. FAD, besides its catalytic role also displays a structural function by enabling the formation of enzymatically active CRTI membrane associates. Under anaerobic conditions the enzyme can act as a carotene cis-trans isomerase. In silico-docking experiments yielded information on substrate binding sites, potential catalytic residues and is in favor of single half-site recognition of the symmetrical C40 hydrocarbon substrate.

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Apocarotenoid glycosides contribute to carotenoid biosynthetic pathway flux compensation. 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 β-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 C13 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 β-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.

Deviation of the neurosporaxanthin pathway towards β-carotene biosynthesis in Fusarium fujikuroi by a point mutation in the phytoene desaturase gene

Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin‐producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB‐encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid‐overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21‐CarB to perform the fifth desaturation, as implied by the accumulation of γ‐carotene and β‐carotene, which arise through four‐step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five‐step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene‐producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate ζ‐carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.

Carotenogenesis Is Regulated by 5′UTR-Mediated Translation of Phytoene Synthase Splice Variants

A splice variant of Arabidopsis phytoene synthase is subjected to a feedback-mediated, translational control via its 5′UTR to coordinate biosynthetic pathway flux with carotenoid requirements. Phytoene synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of the first biosynthetically formed carotene. While PSY constitutes a small gene family in most plant taxa, the Brassicaceae, including Arabidopsis (Arabidopsis thaliana), predominantly possess a single PSY gene. This monogenic situation is compensated by the differential expression of two alternative splice variants (ASV), which differ in length and in the exon/intron retention of their 5′UTRs. ASV1 contains a long 5′UTR (untranslated region) and is involved in developmentally regulated carotenoid formation, such as during deetiolation. ASV2 contains a short 5′UTR and is preferentially induced when an immediate increase in the carotenoid pathway flux is required, such as under salt stress or upon sudden light intensity changes. We show that the long 5′UTR of ASV1 is capable of attenuating the translational activity in response to high carotenoid pathway fluxes. This function resides in a defined 5′UTR stretch with two predicted interconvertible RNA conformations, as known from riboswitches, which might act as a flux sensor. The translation-inhibitory structure is absent from the short 5′UTR of ASV2 allowing to bypass translational inhibition under conditions requiring rapidly increased pathway fluxes. The mechanism is not found in the rice (Oryza sativa) PSY1 5′UTR, consistent with the prevalence of transcriptional control mechanisms in taxa with multiple PSY genes. The translational control mechanism identified is interpreted in terms of flux adjustments needed in response to retrograde signals stemming from intermediates of the plastid-localized carotenoid biosynthesis pathway.

Enzymatic study on AtCCD4 and AtCCD7 and their potential to form acyclic regulatory metabolites

Highlight In vitro study shows that AtCCD4 claves all-trans-bicyclic-carotenoids, excludes its direct involvement in generating plastid retrograde signals supposedly derived from cis-desaturation intermediates, and demonstrates that AtCCD7 converts 9-cis-acylic carotenes.

Insights into the formation of carlactone from in‐depth analysis of the CCD8‐catalyzed reactions

Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis‐configured β‐carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used 13C and 18O‐labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC‐MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.