BACKGROUND:Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions.RESULTS:High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4).CONCLUSIONS:AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

This work presents the application of droplet-based microfluidics for the cultivation of microspores from Brassica napus using the doubled haploid technology. Under stress conditions (e.g. heat shock) or by chemical induction a certain fraction of the microspores can be reprogrammed and androgenesis can be induced. This process is an important approach for plant breeding because desired plant properties can be anchored in the germline on a genetic level. However, the reprogramming rate of the microspores is generally very low, increasing it by specific stimulation is, therefore, both a necessary and challenging task. In order to accelerate the optimisation and development process, the application of droplet-based microfluidics can be a promising tool. Here, we used a tube-based microfluidic system for the generation and cultivation of microspores inside nL-droplets. Different factors like cell density, tube material and heat shock conditions were investigated to improve the yield of vital plant organoids. Evaluation and analysis of the stimuli response were done on an image base aided by an artificial intelligence cell detection algorithm. Droplet-based microfluidics allowed us to apply large concentration programs in small test volumes and to screen the best conditions for reprogramming cells by the histone deacetylase inhibitor trichostatin A and for enhancing the yield of vital microspores in droplets. An enhanced reprogramming rate was found under the heat shock conditions at 32 °C for about 3 to 6 days. In addition, the comparative experiment with MTP showed that droplet cultivation with lower cell density (<10 cells per droplet) or adding media after 3 or 6 days significantly positively affects the microspore growth and embryo rate inside 120 nL droplets. Finally, the developed embryos could be removed from the droplets and further grown into mature plants. Overall, we demonstrated that the droplet-based tube system is suitable for implementation in an automated, miniaturized system to achieve the induction of embryogenic development in haploid microspore stem cells of Brassica napus.

Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation of the molecules in the pathway. While plant carotenoid biosynthesis has been extensively characterized, research on carotenoid degradation and catabolism into apocarotenoids is a relatively novel field. To identify apocarotenoid metabolic processes, we characterized the transcriptome of transgenic Arabidopsis (Arabidopsis thaliana) roots accumulating high levels of β-carotene and, consequently, β-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing β-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from β-carotene degradation or after secondary enzymatic derivatizations. Enzymes implicated in apocarotenoid modification reactions overlapped with detoxification enzymes of xenobiotics and reactive carbonyl species (RCS), while metabolite analysis excluded lipid stress response, a potential secondary effect of carotenoid accumulation. In agreement with structural similarities between RCS and β-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from β-carotene and from xanthophylls into apocarotenols and apocarotenoic acids in vitro. Moreover, glycosylation and glutathionylation-related processes and translocators were induced. In view of similarities to mechanisms found in crocin biosynthesis and cellular deposition in saffron (Crocus sativus), our data suggest apocarotenoid metabolization, derivatization and compartmentalization as key processes in (apo)carotenoid metabolism in plants.

Part of the studies involved in safety assessment of genetically engineered crops includes characterizing the organization, integrity, and stability of the inserted DNA and evaluating the potential allergenicity and toxicity of newly-expressed proteins. Molecular characterization of the introduced DNA in provitamin A biofortified rice event GR2E confirmed insertion of a single copy of the transfer-DNA in the genome and its inheritance as a single locus. Nucleotide sequencing of the inserted DNA confirmed it was introduced without modifications. The phytoene synthase, and carotene desaturase proteins did not display sequence similarity with allergens or toxins. Both proteins were rapidly digested in simulated gastric fluid and their enzymatic activity was inhibited upon heat treatment. Acute oral toxicity testing of the protein in mice demonstrated lack of adverse effects. These evidences substantiated the lack of any identifiable hazards for both proteins and in combination with other existing comparative analyses provided assurance that food derived from this rice is safe. This conclusion is in line with those of the regulatory agencies of US Food and Drug Administration, Health Canada and Food Standard Australia and New Zealand.

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.

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.

Provitamin A biofortification, the provision of provitamin A carotenoids through agriculture, is regarded as an effective and sustainable intervention to defeat vitamin A deficiency, representing a global health problem. This food-based intervention has been questioned in conjunction with negative outcomes for smokers and asbestos-exposed populations of the CARET and ATBC trials in which very high doses of β-carotene were supplemented. The current notion that β-carotene cleavage products (apocarotenoids) represented the harmful agents is the basis of the here-presented research. We quantitatively analyzed numerous plant food items and concluded that neither the amounts of apocarotenoids nor β-carotene provided by plant tissues, be they conventional or provitamin A-biofortified, pose an increased risk. We also investigated β-carotene degradation pathways over time. This reveals a substantial nonenzymatic proportion of carotene decay and corroborates the quantitative relevance of highly oxidized β-carotene polymers that form in all plant tissues investigated.

Phytoene desaturase (PDS) is an essential plant carotenoid biosynthetic enzyme and a prominent target of certain inhibitors, such as norflurazon, acting as bleaching herbicides. PDS catalyzes the introduction of two double bonds into 15-cis-phytoene, yielding 9,15,9'-tri-cis-ζ-carotene via the intermediate 9,15-di-cis-phytofluene. We present the necessary data to scrutinize functional implications inferred from the recently resolved crystal structure of Oryza sativa PDS in a complex with norflurazon. Using dynamic mathematical modeling of reaction time courses, we support the relevance of homotetrameric assembly of the enzyme observed in crystallo by providing evidence for substrate channeling of the intermediate phytofluene between individual subunits at membrane surfaces. Kinetic investigations are compatible with an ordered ping-pong bi-bi kinetic mechanism in which the carotene and the quinone electron acceptor successively occupy the same catalytic site. The mutagenesis of a conserved arginine that forms a hydrogen bond with norflurazon, the latter competing with plastoquinone, corroborates the possibility of engineering herbicide resistance, however, at the expense of diminished catalytic activity. This mutagenesis also supports a "flavin only" mechanism of carotene desaturation not requiring charged residues in the active site. Evidence for the role of the central 15-cis double bond of phytoene in determining regio-specificity of carotene desaturation is presented.

Retinaldehyde dehydrogenases (RALDHs) convert retinal to retinoic acid, an important chordate morphogen. Retinal also occurs in some fungi, such as Fusarium and Ustilago spp., evidenced by the presence of rhodopsins and beta-carotene cleaving, retinal-forming dioxygenases. Based on the assumption that retinoic acid may also be formed in fungi, we searched the Fusarium protein databases for RALDHs homologs, focusing on Fusarium verticillioides. Using crude lysates of Escherichia coli cells expressing the corresponding cDNAs, we checked the capability of best matches to convert retinal into retinoic acid in vitro. Thereby, we identified an aldehyde dehydrogenase, termed CarY, as a retinoic acid-forming enzyme, an activity that was also exerted by purified CarY. Targeted mutation of the carY gene in F. verticillioides resulted in alterations of mycelia development and conidia morphology in agar cultures, and reduced capacity to produce perithecia as a female in sexual crosses. Complementation of the mutant with a wild-type carY allele demonstrated that these alterations are caused by the lack of CarY. However, retinoic acid could not be detected by LC-MS analysis either in the wild type or the complemented carY strain in vivo, making elusive the connection between CarY enzymatic activity and retinoic acid formation in the fungus. (C) 2015 Elsevier Inc. All rights reserved.

splice of phytoene is subjected to a feedback-mediated, translational control via its 5’UTR to coordinate biosynthetic pathway flux with carotenoid ABSTRACT Phytoene synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of 49 the first biosynthetically formed carotene. While PSY constitutes a small gene family in most 50 plant taxa, the Brassicaceae, including Arabidopsis, predominantly possess a single PSY gene. 51 This monogenic situation is compensated by the differential expression of two alternative 52 splice variants (ASV) which differ in length and in the exon/intron retention of their 5’UTRs. 53 ASV1 contains a long 5’UTR and is involved in developmentally-regulated carotenoid 54 formation, such as during deetiolation. ASV2 contains a short 5’UTR and is preferentially 55 induced when an immediate increase in the carotenoid pathway flux is required, such as under 56 salt stress or upon sudden light intensity changes. We show that the long 5’UTR of ASV1 is 57 capable of attenuating the translational activity in response to high carotenoid pathway fluxes. 58 This function resides in a defined 5’UTR stretch with two predicted interconvertible RNA 59 conformations, as known from riboswitches, which might act as a flux sensor. The 60 translation-inhibitory structure is absent from the short 5’UTR of ASV2 allowing to bypass 61 translational inhibition under conditions requiring rapidly increased pathway fluxes. The 62 mechanism is not found in the rice PSY1 5’UTR consistent with the prevalence of 63 transcriptional control mechanisms in taxa with multiple PSY genes. The translational control 64 mechanism identified is interpreted in terms of flux adjustments needed in response to 65 retrograde signals stemming from intermediates of the plastid-localized carotenoid 66 biosynthesis pathway. dark-responsive mRNA instability dark-induced

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.

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.

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.

Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FADred, while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.

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.

The constitutive expression of the bacterial carotene desaturase (CRTI) in Arabidopsis thaliana leads to increased susceptibility of leaves to light-induced damage. Changes in the photosynthetic electron transport chain rather than alterations of the carotenoid composition in the antenna were responsible for the increased photoinhibition. A much higher level of superoxide/hydrogen peroxide was generated in the light in thylakoid membranes from the CRTI expressing lines than in wild-type while the level of singlet oxygen generation remained unchanged. The increase in reactive oxygen species was related to the activity of plastid terminal oxidase (PTOX) since their generation was inhibited by the PTOX-inhibitor octyl gallate, and since the protein level of PTOX was increased in the CRTI-expressing lines. Furthermore, cyclic electron flow was suppressed in these lines. We propose that PTOX competes efficiently with cyclic electron flow for plastoquinol in the CRTI-expressing lines and that it plays a crucial role in the control of the reduction state of the plastoquinone pool.

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.