Reducing PHYTOENE SYNTHASE activity fine-tunes the abundance of a cis-carotene-derived signal that regulates the PIF3/HY5 module and plastid biogenesis.

PHYTOENE SYNTHASE (PSY) is a rate-limiting enzyme catalysing the first committed step of carotenoid biosynthesis, and changes in PSY gene expression and/or protein activity alter carotenoid composition and plastid differentiation in plants. Four genetic variants of PSY (psy-4, psy-90, psy-130, and psy-145) were identified using a forward genetics approach that rescued leaf virescence phenotypes and plastid abnormalities displayed by the Arabidopsis CAROTENOID ISOMERASE (CRTISO) mutant ccr2 (carotenoid and chloroplast regulation 2) when grown under a shorter photoperiod. The four non-lethal mutations affected alternative splicing, enzyme-substrate interactions, and PSY:ORANGE multi-enzyme complex binding, constituting the dynamic post-transcriptional fine-tuning of PSY levels and activity without changing localization to the stroma and protothylakoid membranes. psy genetic variants did not alter total xanthophyll or ß-carotene accumulation in ccr2, yet they reduced specific acyclic linear cis-carotenes linked to the biosynthesis of a currently unidentified apocarotenoid signal regulating plastid biogenesis, chlorophyll biosynthesis, and photomorphogenic regulation. ccr2 psy variants modulated the PHYTOCHROME-INTERACTING FACTOR 3/ELONGATED HYPOCOTYL 5 (PIF3/HY5) ratio, and displayed a normal prolamellar body formation in etioplasts and chlorophyll accumulation during seedling photomorphogenesis. Thus, suppressing PSY activity and impairing PSY:ORANGE protein interactions revealed how cis-carotene abundance can be fine-tuned through holoenzyme-metabolon interactions to control plastid development.

Golden Rice-Lessons learned for inspiring future metabolic engineering strategies and synthetic biology solutions.

Golden Rice is a genetically modified rice variety that accumulates carotenoids with provitamin A activity in its endosperm. This biofortified crop was developed to fight provitamin A deficiency prevailing in countries with a diet based on rice as the major staple. Since its first version in 2000, Golden Rice was continuously developed to increase endosperm carotenoids, to achieve regulatory compliance, and to introgress the trait into regional rice varieties. Golden Rice was recently approved for planting in the Philippines. In this review, we recapitulate major steps leading to the current version of Golden Rice. We examine the knowledge regarding the carotenoid pathway and the technical capabilities to generate transgenic plants at each development stage. Moreover, we discuss whether the scientific approaches taken at the time would be any different today with the increased knowledge and analytical capacities available. We conclude that most decisions for each Golden Rice version would be very similar which is due to the peculiarities and the complexity of the carotenoid biosynthetic pathway. However, each step taken in the development of Golden Rice yielded much insight about regulation of the carotenoid pathway in plants as well as into specific requirements in rice endosperm. The knowledge gained and the recent advances in the field can provide strategies and molecular tools, including synthetic biology approaches, to help develop future versions of Golden Rice with improved provitamin A levels and carotenoid storage capabilities of the rice endosperm.

Determination of protoplast growth properties using quantitative single-cell tracking analysis.

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.

Phytoene Synthase: The Key Rate-Limiting Enzyme of Carotenoid Biosynthesis in Plants.

Phytoene synthase (PSY) catalyzes the first committed step in the carotenoid biosynthesis pathway and is a major rate-limiting enzyme of carotenogenesis. PSY is highly regulated by various regulators and factors to modulate carotenoid biosynthesis in response to diverse developmental and environmental cues. Because of its critical role in controlling the total amount of synthesized carotenoids, PSY has been extensively investigated and engineered in plant species. However, much remains to be learned on its multifaceted regulatory control and its catalytic efficiency for carotenoid enrichment in crops. Here, we present current knowledge on the basic biology, the functional evolution, the dynamic regulation, and the metabolic engineering of PSY. We also discuss the open questions and gaps to stimulate additional research on this most studied gene/enzyme in the carotenogenic pathway.

Characterization of cassava ORANGE proteins and their capability to increase provitamin A carotenoids accumulation.

Cassava (Manihot esculenta Crantz) biofortification with provitamin A carotenoids is an ongoing process that aims to alleviate vitamin A deficiency. The moderate content of provitamin A carotenoids achieved so far limits the contribution to providing adequate dietary vitamin A levels. Strategies to increase carotenoid content focused on genes from the carotenoids biosynthesis pathway. In recent years, special emphasis was given to ORANGE protein (OR), which promotes the accumulation of carotenoids and their stability in several plants. The aim of this work was to identify, characterize and investigate the role of OR in the biosynthesis and stabilization of carotenoids in cassava and its relationship with phytoene synthase (PSY), the rate-limiting enzyme of the carotenoids biosynthesis pathway. Gene and protein characterization of OR, expression levels, protein amounts and carotenoids levels were evaluated in roots of one white (60444) and two yellow cassava cultivars (GM5309-57 and GM3736-37). Four OR variants were found in yellow cassava roots. Although comparable expression was found for three variants, significantly higher OR protein amounts were observed in the yellow varieties. In contrast, cassava PSY1 expression was significantly higher in the yellow cultivars, but PSY protein amount did not vary. Furthermore, we evaluated whether expression of one of the variants, MeOR_X1, affected carotenoid accumulation in cassava Friable Embryogenic Callus (FEC). Overexpression of maize PSY1 alone resulted in carotenoids accumulation and induced crystal formation. Co-expression with MeOR_X1 led to greatly increase of carotenoids although PSY1 expression was high in the co-expressed FEC. Our data suggest that posttranslational mechanisms controlling OR and PSY protein stability contribute to higher carotenoid levels in yellow cassava. Moreover, we showed that cassava FEC can be used to study the efficiency of single and combinatorial gene expression in increasing the carotenoid content prior to its application for the generation of biofortified cassava with enhanced carotenoids levels.

Color recycling: metabolization of apocarotenoid degradation products suggests carbon regeneration via primary metabolic pathways.

KEY MESSAGE: Analysis of carotenoid-accumulating roots revealed that oxidative carotenoid degradation yields glyoxal and methylglyoxal. Our data suggest that these compounds are detoxified via the glyoxalase system and re-enter primary metabolic pathways. Carotenoid levels in plant tissues depend on the relative rates of synthesis and degradation. We recently identified redox enzymes previously known to be involved in the detoxification of fatty acid-derived reactive carbonyl species which were able to convert apocarotenoids into corresponding alcohols and carboxylic acids. However, their subsequent metabolization pathways remain unresolved. Interestingly, we found that carotenoid-accumulating roots have increased levels of glutathione, suggesting apocarotenoid glutathionylation to occur. In vitro and in planta investigations did not, however, support the occurrence of non-enzymatic or enzymatic glutathionylation of ß-apocarotenoids. An alternative breakdown pathway is the continued oxidative degradation of primary apocarotenoids or their derivatives into the shortest possible oxidation products, namely glyoxal and methylglyoxal, which also accumulated in carotenoid-accumulating roots. In fact, combined transcriptome and metabolome analysis suggest that the high levels of glutathione are most probably required for detoxifying apocarotenoid-derived glyoxal and methylglyoxal via the glyoxalase pathway, yielding glycolate and D-lactate, respectively. Further transcriptome analysis suggested subsequent reactions involving activities associated with photorespiration and the peroxisome-specific glycolate/glyoxylate transporter. Finally, detoxified primary apocarotenoid degradation products might be converted into pyruvate which is possibly re-used for the synthesis of carotenoid biosynthesis precursors. Our findings allow to envision carbon recycling during carotenoid biosynthesis, degradation and re-synthesis which consumes energy, but partially maintains initially fixed carbon via re-introducing reactive carotenoid degradation products into primary metabolic pathways.

Small molecules mediate cellular reprogramming across two kingdoms.

The fertilized egg is the single totipotent cell from which multicellular organisms arise through the processes of cell division and differentiation. While animals typically lose their capacity to redifferentiate cells that are already fully differentiated, plant cells are thought to remain totipotent (Su et al., 2020). Every gardener knows well that plants can regenerate a full array of plant tissues from already differentiated organs. This also seems to be true for single plant cells such as protoplasts, which, under proper in vitro culture conditions, served as the initial source for generation of transgenic plants (Skoog and Miller, 1957; Birnbaum and Sánchez Alvarado, 2008). However, the mechanisms behind the totipotency of plant cells remain elusive, with the exception of the knowledge that the developmental fate of regenerating tissues can be directed by the ratio of two plant hormones, auxin and cytokinin (Skoog and Miller, 1957).

Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics.

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 ß-carotene and, consequently, ß-apocarotenoids. Transcriptome analysis revealed feedback regulation on carotenogenic gene transcripts suitable for reducing ß-carotene levels, suggesting involvement of specific apocarotenoid signaling molecules originating directly from ß-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 ß-apocarotenoids, RCS detoxification enzymes also converted apocarotenoids derived from ß-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.

Molecular Control of Sporophyte-Gametophyte Ontogeny and Transition in Plants.

Alternation of generations between a sporophytic and gametophytic developmental stage is a feature common to all land plants. This review will discuss the evolutionary origins of these two developmental programs from unicellular eukaryotic progenitors establishing the ability to switch between haploid and diploid states. We will compare the various genetic factors that regulate this switch and highlight the mechanisms which are involved in maintaining the separation of sporophytic and gametophytic developmental programs. While haploid and diploid stages were morphologically similar at early evolutionary stages, largely different gametophyte and sporophyte developments prevail in land plants and finally allowed the development of pollen as the male gametes with specialized structures providing desiccation tolerance and allowing long-distance dispersal. Moreover, plant gametes can be reprogrammed to execute the sporophytic development prior to the formation of the diploid stage achieved with the fusion of gametes and thus initially maintain the haploid stage. Upon diploidization, doubled haploids can be generated which accelerate modern plant breeding as homozygous plants are obtained within one generation. Thus, knowledge of the major signaling pathways governing this dual ontogeny in land plants is not only required for basic research but also for biotechnological applications to develop novel breeding methods accelerating trait development.

Molecular characterization and safety assessment of biofortified provitamin A rice.

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.

Expression, Purification, and Enzyme Activity Assay of Phytoene Synthase In Vitro.

Phytoene synthase (PSY) is the rate-limiting step in carotenoid biosynthesis, and accordingly subjected to a number of regulatory mechanisms at various levels, including transcriptional, posttranscriptional, and posttranslational. Several PSY genes are present in most taxa and show various degrees of tissue and/or stress-specific responses providing an additional layer of regulating carotenogenesis. Moreover, only a small number of amino acid differences between paralogs or even single nucleotide polymorphisms distinguishing orthologs greatly affect enzyme properties, suggesting that different enzymatic parameters determined by intrinsic properties of PSY protein sequences also determine pathway flux. The characterization of enzyme properties of PSY variants from different origins requires in vitro enzyme assays with recombinant PSY. In this protocol, we present detailed instructions how to purify several milligrams of active PSY enzyme from bacterial lysates, which includes initial recombinant PSY enrichment through inclusion body purification, chaotropic unfolding, refolding in presence of detergents and purification through immobilized metal affinity chromatography. In addition, we provide a protocol to obtain active geranylgeranyl pyrophosphate (GGPP) synthase as active supply of GGPP substrate is a requirement for high in vitro PSY activity. The activity assay requires 14C-labeled substrate and allows to determine its incorporation into phytoene as well as GGPP. The protocol described here was successfully applied to a variety of PSY and GGPP synthase homologs from various plant species.

Quantification of Carotenoid Pathway Flux in Green and Nongreen Systems.

Metabolite accumulation in plant tissues represents the transient net result of their constant biosynthesis and degradation. For carotenoids, degradation might occur enzymatically by carotenoid cleavage producing plant hormones and volatiles or by nonenzymatic oxidation, both depending on environmental and developmental conditions. Carotenoid biosynthesis is therefore constantly regulated at various levels to attain sufficient carotenoid accumulation, mainly for photosynthesis and photoprotection. Due to the plenitude of carotenoids and their degradation products, it is not feasible to investigate overall carotenoid biosynthetic activity and its regulation by the quantification of all carotenoids including their derivatives. This is an issue encountered in investigations on many other highly branched pathways. We therefore present protocols to determine carotenoid biosynthesis flux in a given plant tissue by HPLC quantification of phytoene, the first pathway-specific intermediate and precursor of all carotenoids synthesized by phytoene synthase (PSY). For this purpose, enzymatic metabolization of phytoene in the tissue under investigation is prevented by treatment with the bleaching herbicide norflurazon, blocking the carotenogenic pathway downstream of PSY. As phytoene is more resistant to oxidation than desaturated carotenoids, the rate of phytoene biosynthesis serves as a good measure for total carotenogenic flux in a given tissue. The method is described for Arabidopsis for two photosynthetically active sample types, namely, seedlings and leaves, as well as for seed-derived callus as nongreen tissue. It should be realizable using only a relatively simple experimental setup and is applicable to other plant tissues as well as to different plant species. Additionally, similar experimental setups could be a useful tool to investigate total flux and turnover rates in other biosynthetic pathways.

A Neighboring Aromatic-Aromatic Amino Acid Combination Governs Activity Divergence between Tomato Phytoene Synthases.

Carotenoids exert multifaceted roles to plants and are critically important to humans. Phytoene synthase (PSY) is a major rate-limiting enzyme in the carotenoid biosynthetic pathway. PSY in plants is normally found as a small enzyme family with up to three members. However, knowledge of PSY isoforms in relation to their respective enzyme activities and amino acid residues that are important for PSY activity is limited. In this study, we focused on two tomato (Solanum lycopersicum) PSY isoforms, PSY1 and PSY2, and investigated their abilities to catalyze carotenogenesis via heterologous expression in transgenic Arabidopsis (Arabidopsis thaliana) and bacterial systems. We found that the fruit-specific PSY1 was less effective in promoting carotenoid biosynthesis than the green tissue-specific PSY2. Examination of the PSY proteins by site-directed mutagenesis analysis and three-dimensional structure modeling revealed two key amino acid residues responsible for this activity difference and identified a neighboring aromatic-aromatic combination in one of the PSY core structures as being crucial for high PSY activity. Remarkably, this neighboring aromatic-aromatic combination is evolutionarily conserved among land plant PSYs except PSY1 of tomato and potato (Solanum tuberosum). Strong transcription of tomato PSY1 likely evolved as compensation for its weak enzyme activity to allow for the massive carotenoid biosynthesis in ripe fruit. This study provides insights into the functional divergence of PSY isoforms and highlights the potential to rationally design PSY for the effective development of carotenoid-enriched crops.

Characterization of Cauliflower OR Mutant Variants.

Cauliflower Orange (Or) mutant is characterized by high level of ß-carotene in its curd. Or mutation affects the OR protein that was shown to be involved in the posttranslational control of phytoene synthase (PSY), a major rate-limiting enzyme of carotenoid biosynthesis, and in maintaining PSY proteostasis with the plastid Clp protease system. A transposon integration into the cauliflower wild-type Or gene (BoOR-wt) results in the formation of three differently spliced transcripts. One of them is characterized by insertion (BoOR-Ins), while the other two have exon-skipping deletions (BoOR-Del and BoOR-LD). We investigated the properties of individual BoOR variants and examined their effects on carotenoid accumulation. Using the yeast split-ubiquitin system, we showed that all variants were able to form OR dimers except BoOR-LD. The deletion in BoOR-LD eliminated the first of two adjacent transmembrane domains and was predicted to result in a misplacement of the C-terminal zinc finger domain to the opposite side of membrane, thus preventing OR dimerization. As interaction with PSY is mediated by the N-terminus of BoOR, which remains unaffected after splicing, all BoOR variants including BoOR-LD maintained interactions with PSY. Expression of individual BoOR mutant variants in Arabidopsis revealed that their protein stability varied greatly. While expression of BoOR-Del and BoOR-Ins resulted in increased BoOR protein levels as BoOR-wt, minimal amounts of BoOR-LD protein accumulated. Carotenoid accumulation showed correlated changes in calli of Arabidopsis expressing these variants. Furthermore, we found that OR also functions in E. coli to increase the proportion of native, enzymatically active PSY from plants upon co-expression, but not of bacterial phytoene synthase CrtB. Taken together, these results suggest that OR dimerization is required for OR stability in planta and that the simultaneous presence of PSY interaction-domains in both OR and PSY proteins is required for the holdase function of OR. The more pronounced effect of simultaneous expression of all BoOR variants in cauliflower Or mutant compared with individual overexpression on carotenoid accumulation suggests an enhanced activity with possible formation of various BoOR heterodimers.

Construction of a fusion enzyme for astaxanthin formation and its characterisation in microbial and plant hosts: A new tool for engineering ketocarotenoids.

The high-value ketocarotenoid astaxanthin, a natural red colorant with powerful antioxidant activity, is synthesised from ß-carotene by a hydroxylase and an oxygenase enzyme, which perform the addition of two hydroxyl and keto moieties, respectively. Several routes of intermediates, depending on the sequence of action of these enzymes, lead to the formation of astaxanthin. In the present study, the enzyme activities of 3, 3' ß-carotene hydroxylase (CRTZ) and 4, 4' ß-carotene oxygenase (CRTW) have been combined through the creation of "new to nature" enzyme fusions in order to overcome leakage of non-endogenous intermediates and pleotropic effects associated with their high levels in plants. The utility of flexible linker sequences of varying size has been assessed in the construction of pZ-W enzyme fusions. Frist, in vivo color complementation assays in Escherichia coli have been used to evaluate the potential of the fusion enzymes. Analysis of the carotenoid pigments present in strains generated indicated that the enzyme fusions only possess both catalytic activities when CRTZ is attached as the N-terminal module. Astaxanthin levels in E. coli cells were increased by 1.4-fold when the CRTZ and CRTW enzymes were fused compared to the individual enzymes. Transient expression in Nicotiana benthamiana was then performed in order to assess the potential of the fusions in a plant system. The production of valuable ketocarotenoids was achieved using this plant-based transient expression system. This revealed that CRTZ and CRTW, transiently expressed as a fusion, accumulated similar levels of astaxanthin compared to the expression of the individual enzymes whilst being associated with reduced ketocarotenoid intermediate levels (e.g. phoenicoxanthin, canthaxanthin and 3-OH-echinenone) and a reduced rate of leaf senescence after transformation. Therefore, the quality of the plant material producing the ketocarotenoids was enhanced due to a reduction in the stress induced by the accumulation of high levels of heterologous ketocarotenoid intermediates. The size of the linkers appeared to have no effect upon activity. The potential of the approach to production of valuable plant derived products is discussed.

Enzyme Fusion Removes Competition for Geranylgeranyl Diphosphate in Carotenogenesis.

Geranylgeranyl diphosphate (GGPP), a prenyl diphosphate synthesized by GGPP synthase (GGPS), represents a metabolic hub for the synthesis of key isoprenoids, such as chlorophylls, tocopherols, phylloquinone, gibberellins, and carotenoids. Protein-protein interactions and the amphipathic nature of GGPP suggest metabolite channeling and/or competition for GGPP among enzymes that function in independent branches of the isoprenoid pathway. To investigate substrate conversion efficiency between the plastid-localized GGPS isoform GGPS11 and phytoene synthase (PSY), the first enzyme of the carotenoid pathway, we used recombinant enzymes and determined their in vitro properties. Efficient phytoene biosynthesis via PSY strictly depended on simultaneous GGPP supply via GGPS11. In contrast, PSY could not access freely diffusible GGPP or time-displaced GGPP supply via GGPS11, presumably due to liposomal sequestration. To optimize phytoene biosynthesis, we applied a synthetic biology approach and constructed a chimeric GGPS11-PSY metabolon (PYGG). PYGG converted GGPP to phytoene almost quantitatively in vitro and did not show the GGPP leakage typical of the individual enzymes. PYGG expression in Arabidopsis resulted in orange-colored cotyledons, which are not observed if PSY or GGPS11 are overexpressed individually. This suggests insufficient GGPP substrate availability for chlorophyll biosynthesis achieved through GGPP flux redirection to carotenogenesis. Similarly, carotenoid levels in PYGG-expressing callus exceeded that in PSY- or GGPS11-overexpression lines. The PYGG chimeric protein may assist in provitamin A biofortification of edible plant parts. Moreover, other GGPS fusions may be used to redirect metabolic flux into the synthesis of other isoprenoids of nutritional and industrial interest.

Strigolactone Levels in Dicot Roots Are Determined by an Ancestral Symbiosis-Regulated Clade of the PHYTOENE SYNTHASE Gene Family.

Strigolactones (SLs) are apocarotenoid phytohormones synthesized from carotenoid precursors. They are produced most abundantly in roots for exudation into the rhizosphere to cope with mineral nutrient starvation through support of root symbionts. Abscisic acid (ABA) is another apocarotenoid phytohormone synthesized in roots, which is involved in responses to abiotic stress. Typically low carotenoid levels in roots raise the issue of precursor supply for the biosynthesis of these two apocarotenoids in this organ. Increased ABA levels upon abiotic stress in Poaceae roots are known to be supported by a particular isoform of phytoene synthase (PSY), catalyzing the rate-limiting step in carotenogenesis. Here we report on novel PSY3 isogenes from Medicago truncatula (MtPSY3) and Solanum lycopersicum (SlPSY3) strongly expressed exclusively upon root interaction with symbiotic arbuscular mycorrhizal (AM) fungi and moderately in response to phosphate starvation. They belong to a widespread clade of conserved PSYs restricted to dicots (dPSY3) distinct from the Poaceae-PSY3s involved in ABA formation. An ancient origin of dPSY3s and a potential co-evolution with the AM symbiosis is discussed in the context of PSY evolution. Knockdown of MtPSY3 in hairy roots of M. truncatula strongly reduced SL and AM-induced C13 α-ionol/C14 mycorradicin apocarotenoids. Inhibition of the reaction subsequent to phytoene synthesis revealed strongly elevated levels of phytoene indicating induced flux through the carotenoid pathway in roots upon mycorrhization. dPSY3 isogenes are coregulated with upstream isogenes and downstream carotenoid cleavage steps toward SLs (D27, CCD7, CCD8) suggesting a combined carotenoid/apocarotenoid pathway, which provides "just in time"-delivery of precursors for apocarotenoid formation.

Establishment of an Arabidopsis callus system to study the interrelations of biosynthesis, degradation and accumulation of carotenoids.

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 ß-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 ß-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in ß-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state ß-carotene amounts to up to 500 µg g-1 dry mass. Furthermore, we identified oxidative ß-carotene degradation products which correlated with pathway activities, yielding ß-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 ß-carotene sequestration by co-localization experiments which revealed that ß-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 ß-carotene levels prior to their application in crops.

Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants.

Carotenoid gene expression explains the difference of carotenoid accumulation in carrot root tissues.

Main conclusion Variations in gene expression can partially explain the difference of carotenoid accumulation in secondary phloem and xylem of fleshy carrot roots. The carrot root is well divided into two different tissues separated by vascular cambium: the secondary phloem and xylem. The equilibrium between these two tissues represents an important issue for carrot quality, but the knowledge about the respective carotenoid accumulation is sparse. The aim of this work was (i) to investigate if variation in carotenoid biosynthesis gene expression could explain differences in carotenoid content in phloem and xylem tissues and (ii) to investigate if this regulation is differentially modulated in the respective tissues by water-restricted growing conditions. In this work, five carrot genotypes contrasting by their root color were studied in control and water-restricted conditions. Carotenoid content and the relative expression of 13 genes along the carotenoid biosynthesis pathway were measured in the respective tissues. Results showed that in orange genotypes and the purple one, carotenoid content was higher in phloem compared to xylem. For the red one, no differences were observed. Moreover, in control condition, variations in gene expression explained the different carotenoid accumulations in both tissues, while in water-restricted condition, no clear association between gene expression pattern and variations in carotenoid content could be detected except in orange-rooted genotypes. This work shows that the structural aspect of carrot root is more important for carotenoid accumulation in relation with gene expression levels than the consequences of expression changes upon water restriction.