New technologies are redefining how plant biology will meet societal challenges in health, nutrition, agriculture, and energy. Rapid and inexpensive genome and transcriptome sequencing is being ...exploited to discover biochemical pathways that provide tools needed for synthetic biology in both plant and microbial systems. Metabolite detection at the cellular and subcellular levels is complementing gene sequencing for pathway discovery and metabolic engineering. The crafting of plant and microbial metabolism for the synthetic biology platforms of tomorrow will require precise gene editing and delivery of entire complex pathways. Plants sustain life and are key to discovery and development of new medicines and agricultural resources; increased research and training in plant science will accelerate efforts to harness the chemical wealth of the plant kingdom.
A study investigated the biosynthesis of carotenoids, focusing on the mechanistic aspects of the process. Carotenoids function in photosynthetic organisms such as bacteria, plants and fungi.
•There is a need to engineer carotenoids for specific physiological purposes.•Current knowledge of the carotenoid biosynthetic pathway consists of a parts list.•Elucidating localization of the ...complete pathway in different plastids is crucial.•Visualization tools to reflect localization of biosynthetic pathways are needed.
The carotenoid biosynthetic pathway serves manifold roles in plants related to photosynthesis, photoprotection, development, stress hormones, and various volatiles and signaling apocarotenoids. The pathway also produces compounds that impact human nutrition and metabolic products that contribute to fragrance and flavor of food and non-food crops. It is no surprise that the pathway has been a target of metabolic engineering, most prominently in the case of Golden Rice. The future success and predictability of metabolic engineering of carotenoids rests in the ability to target carotenoids for specific physiological purposes as well as to simultaneously modify carotenoids along with other desired traits. Here, we ask whether predictive metabolic engineering of the carotenoid pathway is indeed possible. Despite a long history of research on the pathway, at this point in time we can only describe the pathway as a parts list and have almost no knowledge of the location of the complete pathway, how it is assembled, and whether there exists any trafficking of the enzymes or the carotenoids themselves. We discuss the current state of knowledge regarding the “complete” pathway and make the argument that predictive metabolic engineering of the carotenoid pathway (and other pathways) will require investigation of the three dimensional state of the pathway as it may exist in plastids of different ultrastructures. Along with this message we point out the need to develop new types of visualization tools and resources that better reflect the dynamic nature of biosynthetic pathways.
Metabolic engineering of plant carotenoids in food crops has been a recent focus for improving human health. Pathway manipulation is predicated on comprehensive knowledge of this biosynthetic ...pathway, which has been extensively studied. However, there existed the possibility of an additional biosynthetic step thought to be dispensable because it could be compensated for by light. This step, mediated by a putative Z-ISO, was predicted to occur in the sequence of redox reactions that are coupled to an electron transport chain and convert the colorless 15-cis-phytoene to the red-colored all-trans-lycopene. The enigma of carotenogenesis in the absence of light (e.g. in endosperm, a target for improving nutritional content) argued for Z-ISO as a pathway requirement. Therefore, understanding of plant carotenoid biosynthesis was obviously incomplete. To prove the existence of Z-ISO, maize (Zea mays) and Arabidopsis (Arabidopsis thaliana) mutants were isolated and the gene identified. Functional testing of the gene product in Escherichia coli showed isomerization of the 15-cis double bond in 9,15,9'-tri-cis-ζ-carotene, proving that Z-ISO encoded the missing step. Z-ISO was found to be important for both light-exposed and "dark" tissues. Comparative genomics illuminated the origin of Z-ISO found throughout higher and lower plants, algae, diatoms, and cyanobacteria. Z-ISO evolved from an ancestor related to the NnrU (for nitrite and nitric oxide reductase U) gene required for bacterial denitrification, a pathway that produces nitrogen oxides as alternate electron acceptors for anaerobic growth. Therefore, plant carotenogenesis evolved by recruitment of genes from noncarotenogenic bacteria.
Enhancement of the carotenoid biosynthetic pathway in food crops benefits human health and adds commercial value of natural food colorants. However, predictable metabolic engineering or breeding is ...limited by the incomplete understanding of endogenous pathway regulation, including rate-controlling steps and timing of expression in carotenogenic tissues. The grass family (Poaceae) contains major crop staples, including maize (Zea mays), wheat (Triticum aestivum), rice (Oryza saliva), sorghum (Sorghum bicolor), and millet (Pennisetum glaucum). Maize carotenogenesis was investigated using a novel approach to discover genes encoding limiting biosynthetic steps in the nutritionally targeted seed endosperm. A combination of bioinformatics and cloning were first used to identify and map gene families encoding enzymes in maize and other grasses. These enzymes represented upstream pathways for isopentenyl diphosphate and geranylgeranyl diphosphate synthesis and the downstream carotenoid biosynthetic pathway, including conversion to abscisic acid. A maize germplasm collection was used for statistical testing of the correlation between carotenoid content and candidate gene transcript levels. Multiple pathway bottlenecks for isoprenoid biosynthesis and carotenoid biosynthesis were discovered in specific temporal windows of endosperm development. Transcript levels of paralogs encoding isoprenoid isopentenyl diphosphate and geranylgeranyl diphosphate-producing enzymes, DXS3, DXR, HDR, and GGPPS1, were found to positively correlate with endosperm carotenoid content. For carotenoid pathway enzymes, transcript levels for CrtISO inversely correlated with seed carotenoid content, as compared with positive correlation of PSY1 transcripts. Since zeaxanthin epoxidase (ZEP) depletes the carotenoid pool in subsequent conversion to abscisic acid, ZEP transcripts were examined. Carotenoid accumulation was found to be inversely associated with ZEP1 and ZEP2 transcript levels. Extension of the maize results using phylogenetic analysis identified orthologs in other grass species that may serve as potential metabolic engineering targets.
Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase ...(NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield.
Plant carotenoids have unique physiological roles related to specific plastid suborganellar locations. Carotenoid metabolic engineering could enhance plant adaptation to climate change and improve ...food security and nutritional value. However, lack of fundamental knowledge on carotenoid pathway localization limits targeted engineering. Phytoene synthase (PSY), a major rate-controlling carotenoid enzyme, is represented by multiple isozymes residing at unknown plastid sites. In maize (Zeamays), the three isozymes were transiently expressed and found either in plastoglobuli or in stroma and thylakoid membranes. PSY1, with one to two residue modifications of naturally occurring functional variants, exhibited altered localization, associated with distorted plastid shape and formation of a fibril phenotype. Mutating the active site of the enzyme reversed this phenotype. Discovery of differential PSY locations, linked with activity and isozyme type, advances the engineering potential for modifying carotenoid biosynthesis.
Carotenoids are essential for photosynthesis and photoprotection; they also serve as precursors to signaling molecules that influence plant development and biotic/abiotic stress responses. With ...potential to improve plant yield and nutritional quality, carotenoids are targets for metabolic breeding/engineering, particularly in the Poaceae (grass family), which includes the major food crops. Depending on genetic background, maize (Zea mays) endosperm carotenoid content varies, and therefore breeding-enhanced carotenoid levels have been of ongoing interest. The first committed step in the plastid-localized biosynthetic pathway is mediated by the nuclear-encoded phytoene synthase (PSY). The gene family in maize and other grasses contains three paralogs with specialized roles that are not well understood. Maize endosperm carotenoid accumulation requires PSY1 expression. A maize antibody was used to localize PSY1 to amyloplast envelope membranes and to determine PSY1 accumulation in relation to carotenoid accumulation in developing endosperm. To test when and if PSY transcript levels correlated with carotenoid content, advantage was taken of a maize germplasm diversity collection that exhibits genetic and chemical diversity. Total carotenoid content showed statistically significant correlation with endosperm transcript levels at 20 d after pollination for PSY1 but not PSY2 or PSY3. Timing of PSY1 transcript abundance, previously unknown, provides critical information for choosing breeding alleles or properly controlling introduced transgenes. PSY1 was unexpectedly found to have an additional role in photosynthetic tissue, where it was required for carotenogenesis in the dark and for heat stress tolerance. Leaf carotenogenesis was shown to require phytochrome-dependent and phytochrome-independent photoregulation of PSY2 plus nonphotoregulated PSY1 expression.
The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about ...the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.
A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of β-carotene hydroxylase transcript levels are linked to an increased demand for ABA.
This analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds.
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