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Creating plant molecular factories for industrial and nutritional isoprenoid production
... However, barriers, such as the lack of access to healthy foods, limit the availability of these essential nutrients for human consumption (Hendrickson et al., 2006). Plants also produce a wealth of therapeutic phytochemicals, both pharmaceuticals and nutraceuticals (bioactive natural health-enhancing compounds in food), that are difficult to chemically synthesize, leaving consumption of medicinal plants or plant extracts as the sole source of these important chemicals (Nogueira et al., 2018). Additionally, many important phytochemicals are expressed in plants that are difficult to cultivate or produce insignificant amounts of the desired phytochemical (Nogueira et al., 2018). ...
... Plants also produce a wealth of therapeutic phytochemicals, both pharmaceuticals and nutraceuticals (bioactive natural health-enhancing compounds in food), that are difficult to chemically synthesize, leaving consumption of medicinal plants or plant extracts as the sole source of these important chemicals (Nogueira et al., 2018). Additionally, many important phytochemicals are expressed in plants that are difficult to cultivate or produce insignificant amounts of the desired phytochemical (Nogueira et al., 2018). With an increasing global population, new tools are needed to provide highly nutritive foods and low-cost therapeutics to a larger populace. ...
... Opioids from Papaver somniferum, taxol from Taxus brevifolia, and QS-21 adjuvants from Quillaja saponaria, are several notable examples of therapeutic molecules that are produced and extracted from their native plant host (Jennings et al., 1992;Labanca et al., 2018;Qi and Fox, 2021). While many therapeutic chemicals can be extracted from their native producer, many species are difficult to cultivate or suffer from limited yields (Nogueira et al., 2018). Past efforts have focused on the production of nutrients and phytochemicals in microbes or with the use of chemical synthesis, but plants are an ideal platform for the production of small molecules at a large scale, as they are autotrophic and can be grown in non-axenic conditions like an open field. ...
Plants offer a vast source of bioactive chemicals with the potential to improve human health through the prevention and treatment of disease. However, many potential therapeutics are produced in small amounts or in species that are difficult to cultivate. The rapidly evolving field of plant synthetic biology provides tools to capitalize on the inventive chemistry of plants by transferring metabolic pathways for therapeutics into far more tenable plants, increasing our ability to produce complex pharmaceuticals in well-studied plant systems. Plant synthetic biology also provides methods to enhance the ability to fortify crops with nutrients and nutraceuticals. In this review, we discuss (1) the potential of plant synthetic biology to improve human health by generating plants that produce pharmaceuticals, nutrients, and nutraceuticals and (2) the technological challenges hindering our ability to generate plants producing health-promoting small molecules.
... Nevertheless, as mentioned earlier, the high costs associated with this technology makes it uncompetitive when compared to less expensive but environmentally unsustainable processes such as collection of wild plants or when compared to chemical synthesis. These culture methods also still require the use of sterilizable bioreactors, so their scale-up is limited (Nogueira et al. 2018;Buyel et al. 2017). ...
... Chief among these is the use of targeted genome engineering, particularly the previously mentioned genome editing mediated by CRISPR/ Cas9. The use of this technological approach creates the possibility of producing new plant varieties without the introduction of foreign genes (Doudna and Charpentier 2014;Baltes and Voytas 2015;Nogueira et al. 2018). Gene editing could potentially be used for the introduction of new alleles, promoter replacement or the introduction of new pathways, all of which could result in the creation of plant-based systems capable of novel expression of useful bioactive molecules (Nogueira et al. 2018). ...
... The use of this technological approach creates the possibility of producing new plant varieties without the introduction of foreign genes (Doudna and Charpentier 2014;Baltes and Voytas 2015;Nogueira et al. 2018). Gene editing could potentially be used for the introduction of new alleles, promoter replacement or the introduction of new pathways, all of which could result in the creation of plant-based systems capable of novel expression of useful bioactive molecules (Nogueira et al. 2018). ...
Main conclusion:
Plant tissue culture as an important tool for the continuous production of active compounds including secondary metabolites and engineered molecules. Novel methods (gene editing, abiotic stress) can improve the technique. Humans have a long history of reliance on plants for a supply of food, shelter and, most importantly, medicine. Current-day pharmaceuticals are typically based on plant-derived metabolites, with new products being discovered constantly. Nevertheless, the consistent and uniform supply of plant pharmaceuticals has often been compromised. One alternative for the production of important plant active compounds is in vitro plant tissue culture, as it assures independence from geographical conditions by eliminating the need to rely on wild plants. Plant transformation also allows the further use of plants for the production of engineered compounds, such as vaccines and multiple pharmaceuticals. This review summarizes the important bioactive compounds currently produced by plant tissue culture and the fundamental methods and plants employed for their production.
... However, as previously stated, the high costs of this technology render it uncompetitive when compared to less priced but environmentally unsustainable procedures like wild plant gathering or chemical synthesis. Because these culture methods still require bioreactors, their scale-up potential is limited (Nogueira et al. 2018;Buyel et al. 2017). New molecular technologies have opened up new avenues for the production of important metabolites using plant systems. ...
... New molecular technologies have opened up new avenues for the production of important metabolites using plant systems. Gene editing could be used to introduce new alleles, replace promoters, or create new pathways, all of which could lead to the development of plant-based systems capable of novel expression of useful bioactive chemicals (Nogueira et al. 2018). Once the plant has been designed, it can be utilized to produce the desired metabolites on a massive scale, including plantmade medications. ...
The in vitro production of phenolic compounds in different plant culture techniques offers an attractive substitute rather than separation from plant materials. The variability and instability of phenolic compound composition hinders the standardization and quality assurance of crude materials. Therefore, in vitro studies offer several allied advantages, such as little or no seasonal variability, the use of high metabolite yielding cell lines for scaling-up production, and reduced production time. Despite the fact that various strategies have been used to increase phenolic compound content, elicitation, along with hairy root culture, is one of the most feasible strategies currently in use. The present work intends to provide an illustrative understanding of the factors having conspicuous impacts on phenolic compound production in in vitro culture conditions. In addition, emphasis was placed on highlighting important phenolic compounds with broad implications for human health and contrasting metabolic pathways involved. In conclusion, we further postulate in-depth research on specific compound/compounds and their optimal extraction techniques rather than studies encircling the phenolic extract as a whole and their corresponding bioactivities. Such a strategy finds wide acceptability and application, considering the needs of the pharmaceutical industry or for encapsulation in functional food.KeywordsHairy root cultureIn vitro culturePhenolic compoundsSecondary metabolite
... Today over 50,000 plant species are utilized by us for their bioactive compounds (Chakraborty 2018). They may be correctly called "green chemical factories" for their huge variety of resources that supply food, feed, pharmaceuticals, aromatic compounds and industrial raw materials (Nogueira et al. 2018). Analgesics like morphine, cardiotonic like digoxin, anticancer drugs like vinblastine and paclitaxel, etc. are only a few of the wide diversity of medicines derived from plants (Nessler 1994). ...
... Development of cisgenic crops, by overexpressing "cis"-genes in our cultivated varieties from its wild relatives, could be a possible solution to this problem. Genetic modification and free targeted mutation using TILLING is another possible approach of increasing valuable secondary metabolite production (Nogueira et al. 2018). ...
As suggested by the reports of the World Health Organization, the ancient knowledge of medicinal and aromatic plants still forms the basis of traditional and indigenous health system in most developing countries. Almost 80% of the world population depends on plant secondary metabolites (such as alkaloids, phenolics, terpenes, etc.) as a safer alternative than synthetic medicines. Traditional breeding method falls short to meet the huge demands, and technological intervention like biotechnology-based breeding methods (BBBMs) becomes a necessity. Plant tissue culture techniques like Agrobacterium-mediated gene transfer, bioreactor technology, etc. helps to improve yield to satisfactory levels, while methods like cryopreservation, micropropagation, synthetic seeds, etc. help in conservation and storage of the products. Formation of hairy roots from Agrobacterium rhizogenes transformation is one of the most exploited methods for mass production of secondary metabolites. Use of elicitors, improved media constitution, precursor feeding and engineering metabolic pathways helps to drive the flux towards our desired products. Emergence of medicinal plant genomics consortium and functional genomics technologies such as expressed sequence tag (EST) databases, microarrays and restriction site-associated DNA sequencing (RAD-seq) allows transcriptome profiling and study of yet unexcavated wild plant resources. Biotechnological intervention is also used to impart stress tolerance and reduce the toxicity of the metabolic products. Specific gene editing techniques like CRISPR/Cas9, COSTREL, TALENs and polyploidy generation are also being applied to medicinal and aromatic plants for improved industrial strain generation. The present book chapter represents an overview of the latest technological advancements in improving the quality of the medicinal plants to exploit maximum benefit out of them in a sustainable way.
... They are the largest and most diverse class of metabolites, containing over 40,000 substances. The molecules are industrially relevant and are used as flavors, pigments, polymers and drugs [1,2]. ...
... (1 + E) −∆∆Ct (2) where E = primer efficiency, ∆Ct = Ct target gene -Ct reference gene and ∆∆Ct = ∆Ct target -∆Ct internal calibrator , as previously described [62]. In all cases, GAPDH was the normalizer, as recommended for coffee plants [62,65]. ...
Terpenoids are a diverse class of metabolites that impact plant metabolism in response to environmental cues. They are synthesized either via a predominantly cytosolic (MVA) pathway or a plastidic pathway (MEP). In Arabidopsis, several enzymes from the MVA and MEP pathways are encoded by gene families, excluding MVK and DXR, which are single-copy genes. In this study, we assess the diversity, evolution and expression of DXR and MVK genes in selected angiosperms and Coffea arabica in particular. Evolutionary analysis revealed that DXR and MVK underwent purifying selection, but the selection effect for DXR was stronger than it was for MVK. Digital gene expression (DGE) profile analysis of six species revealed that expression levels of MVK in flowers and roots were high, whereas for DXR peak values were observed in leaves. In C. arabica, both genes were highly expressed in flowers, and CaDXR was upregulated in response to methyl jasmonate. C. arabica DGE data were validated by assessing gene expression in selected organs, and by plants treated with hexanoic acid (Hx) using RT-qPCR. MVK expression was upregulated in roots treated with Hx. CaDXR was downregulated in leaves by Hx treatment in a genotype-specific manner, indicating a differential response to priming.
... Metabolic pathways are immensely complex systems that involve overlapping, competing, branched and redundant regulatory mechanisms which have evolved by natural selection over the course of hundreds of millions of years [230]. Early attempts at metabolic engineering either modulated singular key enzymatic steps within a pathway or the first committed step in the target pathway to sequester necessary precursors [231]. Unfortunately, such strategies tend to result in pleiotropic effects, such as genetic co-suppression, depletion of precursor pools, allosteric regulation or other undesired biochemical and phenotypic effects [231]. ...
... Early attempts at metabolic engineering either modulated singular key enzymatic steps within a pathway or the first committed step in the target pathway to sequester necessary precursors [231]. Unfortunately, such strategies tend to result in pleiotropic effects, such as genetic co-suppression, depletion of precursor pools, allosteric regulation or other undesired biochemical and phenotypic effects [231]. To solve these issues, ideally one would be able to sequester a desired metabolite in a protective pocket that would shield the rest of the cell from its accumulation. ...
The dynamic nutrient availability and photon flux density of diatom habitats necessitate buffering capabilities in order to maintain metabolic homeostasis. This is accomplished by the biosynthesis and turnover of storage lipids, which are sequestered in lipid droplets (LDs). LDs are an organelle conserved among eukaryotes, composed of a neutral lipid core surrounded by a polar lipid monolayer. LDs shield the intracellular environment from the accumulation of hydrophobic compounds and function as a carbon and electron sink. These functions are implemented by interconnections with other intracellular systems, including photosynthesis and autophagy. Since diatom lipid production may be a promising objective for biotechnological exploitation, a deeper understanding of LDs may offer targets for metabolic engineering. In this review, we provide an overview of diatom LD biology and biotechnological potential.
... Constitutive overexpression of Arabidopsis PDS gene also effectively enhanced carotenoids accumulation in tomato fruit [201]. However, constitutive overexpression of carotenogenic enzymes can cause unwanted pleiotropic developmental effects, such as dwarfism, loss of vigor and senescence [202][203][204][205]. These developmental phenotypes are likely the result of competition between metabolic routes and of alterations in the content of plant hormones, particularly gibberellins, which derive from carotenoids or their isoprenoid precursors. ...
... Gene editing tools, such as the CRISPR/Cas system, allow the production of biofortified, non-transgenic crops that do not contain foreign genes [266][267][268][269][270][271]. Such an approach may find a better public acceptance and accelerate the regulatory approval process, thus bringing biotech crops faster to market [205,270]. There are already examples for employing the CRISPR/Cas9 technology to alter carotenoid content in flower, callus and fruit [159,196,272]. ...
Carotenoids are indispensable for human health, required as precursors of vitamin A and efficient antioxidants. However, these plant pigments that play a vital role in photosynthesis are represented at insufficient levels in edible parts of several crops, which creates a need for increasing their content or optimizing their composition through biofortification. In particular, vitamin A deficiency, a severe health problem affecting the lives of millions in developing countries, has triggered the development of a series of high-provitamin A crops, including Golden Rice as the best-known example. Further carotenoid-biofortified crops have been generated by using genetic engineering approaches or through classical breeding. In this review, we depict carotenoid metabolism in plants and provide an update on the development of carotenoid-biofortified plants and their potential to meet needs and expectations. Furthermore, we discuss the possibility of using natural variation for carotenoid biofortification and the potential of gene editing tools.
... 1 3 the development of medicines, agricultural products, and industrial raw materials (Capell and Christou 2004;Zhu et al. 2013;Nogueira et al. 2018). In plants, isoprenoids can function as primary or secondary metabolites, and they participate in a wide range of physiological processes, where they act synergistically, such as chlorophylls and carotenoids during photosynthesis, or antagonistically, such as gibberellic acid and abscisic acid during seed germination (Vranova et al. 2012). ...
... Some bacteria as well as all higher plants have the ability to use both pathways (Vranova et al. 2013). Some isoprenoids are derived solely from the MVA pathway, including phytosterols, sesquiterpenes, triterpenes, and the side-chain of ubiquinone, whereas monoterpenes, diterpenes, carotenoids, and the side chains of chlorophylls and tocopherols are all derived from the MEP pathway (Vranova et al. 2013;Zhu et al. 2013;Nogueira et al. 2018). The MEP pathway in plants is compartmentalized in the plastids, whereas the MVA pathway operates in the rest of the cytoplasm, thus allowing the optimization of isoprenoid biosynthesis and regulation according to the availability of fixed carbon and ATP (Vranova et al. 2013). ...
Key message
Both OsIPPI1 and OsIPPI2 enzymes are found in the endoplasmic reticulum, providing novel important insights into the role of this compartment in the synthesis of MVA pathway isoprenoids.
Abstract
Isoprenoids are synthesized from the precursor’s isopentenyl diphosphate (IPP) and dimethylallyl diphosphosphate (DMAPP), which are interconverted by the enzyme isopentenyl diphosphate isomerase (IPPI). Many plants express multiple isoforms of IPPI, the only enzyme shared by the mevalonate (MVA) and non-mevalonate (MEP) pathways, but little is known about their specific roles. Rice (Oryza sativa) has two IPPI isoforms (OsIPPI1 and OsIPPI2). We, therefore, carried out a comprehensive comparison of IPPI gene expression, protein localization, and isoprenoid biosynthesis in this species. We found that OsIPPI1 mRNA was more abundant than OsIPPI2 mRNA in all tissues, and its expression in de-etiolated leaves mirrored the accumulation of phytosterols, suggesting a key role in the synthesis of MVA pathway isoprenoids. We investigated the subcellular localization of both isoforms by constitutively expressing them as fusions with synthetic green fluorescent protein. Both proteins localized to the endoplasmic reticulum (ER) as well as peroxisomes and mitochondria, whereas only OsIPPI2 was detected in plastids, due to an N-terminal transit peptide which is not present in OsIPPI1. Despite the plastidial location of OsIPPI2, the expression of OsIPPI2 mRNA did not mirror the accumulation of chlorophylls or carotenoids, indicating that OsIPPI2 may be a redundant component of the MEP pathway. The detection of both OsIPPI isoforms in the ER indicates that DMAPP can be synthesized de novo in this compartment. Our work shows that the ER plays an as yet unknown role in the synthesis of MVA-derived isoprenoids, with important implications for the metabolic engineering of isoprenoid biosynthesis in higher plants.
... Plants are the "green chemical factories" with a wide range of chemical diversity that can support food, feed, medicines, and biomaterial industries (Nogueira et al. 2018). The safe application of organic materials leads to more popularity of plant-derivate medicines in the recent years (Niazian et al. 2017c;Soltani Howyzeh et al. 2018). ...
... The modified gene transformation method of COSTREL has great potential for significant enhancement in the production of useful secondary metabolites of medicinal plants (Fuentes et al. 2016). GM-free targeted mutation induction through TILLING is a promising way to enhance the production of valuable secondary metabolites (Nogueira et al. 2018) or produce new useful products from different medicinal plants. Genome editing method of CRISPR/Cas9 emerged as a powerful and promising method for targeted mutation induction in the genome of medicinal plants and subsequent purposeful alteration of their biochemical profile (Feng et al. 2018;Li et al. 2017;Zhou et al. 2018). ...
Main conclusion
Plant tissue culture has been used for conservation, micropropagation, and in planta overproduction of some pharma molecules of medicinal plants. New biotechnology-based breeding methods such as targeted genome editing methods are able to create custom-designed medicinal plants with different secondary metabolite profiles.
For a long time, humans have used medicinal plants for therapeutic purposes and in food and other industries. Classical biotechnology techniques have been exploited in breeding medicinal plants. Now, it is time to apply faster biotechnology-based breeding methods (BBBMs) to these valuable plants. Assessment of the genetic diversity, conservation, proliferation, and overproduction are the main ways by which genetics and biotechnology can help to improve medicinal plants faster. Plant tissue culture (PTC) plays an important role as a platform to apply other BBBMs in medicinal plants. Agrobacterium-mediated gene transformation and artificial polyploidy induction are the main BBBMs that are directly dependent on PTC. Manageable regulation of endogens and/or transferred genes via engineered zinc-finger proteins or transcription activator-like effectors can help targeted manipulation of secondary metabolite pathways in medicinal plants. The next-generation sequencing techniques have great potential to study the genetic diversity of medicinal plants through restriction-site-associated DNA sequencing (RAD-seq) technique and also to identify the genes and enzymes that are involved in the biosynthetic pathway of secondary metabolites through precise transcriptome profiling (RNA-seq). The sequence-specific nucleases of transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, and clustered regularly interspaced short palindromic repeats-associated (Cas) are the genome editing methods that can produce user-designed medicinal plants. These current targeted genome editing methods are able to manage plant synthetic biology and open new gates to medicinal plants to be introduced into appropriate industries.
... compounds, including heterologous expression of the biosynthetic pathway, improved transcription and translation, regulation of transcription factors, enrichment of precursor availability, and metabolite modification. 81 New methods of CoQ10 production also have to be taken into account. The biosynthesis pathway has been deciphered in model organisms, providing an opportunity for precise manipulations in the genome for enhanced CoQ10 yield. ...
Ubiquinones (CoQ10) are produced in the mitochondrial membrane, which executes bioenergetics as electron and proton carriers, and have demonstrated such extensive health benefits that they are considered ''super vitamin.'' Currently, wild-type and genetically modified microorganisms (Agro-bacterium tumefaciens, Paracoccus denitrificans, Rhodo-bacter sphaeroides, and Escherichia coli) are being explored for CoQ10 production. However, a poor production rate limits commercial production by bacterial biosynthesis. Hence, further process improvement and identification of challenges in CoQ10 bioproduction require review. Researchers have used gene editing and metabolic engineering to genetically modulate the CoQ10 biosynthesis pathway to develop engineered microorganisms that efficiently produce CoQ10. Site-directed mutagenesis has emerged as a promising approach for the enhancement of microbial strains toward CoQ10 production. Moreover, various precursor supplemen-tation in media and the development of mutant strains have resulted in improved CoQ10 yields. This review focuses on future strategies such as modification/overexpressing key enzymes, mutagenesis, and media optimization for enhanced CoQ10 production.
... FBNs are best known for their role in chromoplasts, the coloured type of plastids. Typically occurring in flowers and fruits, chromoplasts remodels their metabolism to synthesise and store large amounts of carotenoids, isoprenoid-derived compounds with high nutritional and industrial value (16,17) . FBN1/PAP and its homologs in different species are required for the formation of carotenoid-sequestering structures occurring during fruit ripening as PGs and fibrils (13,(18)(19)(20)(21). Enhanced-carotenoid plant genotypes produced by biotechnological interventions typically exhibited altered chromoplast structure with increased PG number, highlighting the metabolic composition influence on plastid morphology (22)(23)(24)(25). ...
Plastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBN) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs to isoprenoid sequestration and PG function, a multiplex gene editing approach was undertaken. Analysis of single and high-order fbn mutants for the major PG-related FBNs in tomato, namely SlFBN 1, SlFBN 2a, SlFBN 4, SlFBN 7a, revealed functional redundancy. High order fbn mutants displayed phenotypes associated with abnormal isoprenoid accumulation, and aberrant PG formation and morphology. Lipidomic analysis highlighted broader changes in lipid metabolism. Paralog-specific roles were also observed and included the regulation of specific isoprenoids (e.g., plastochromanol) and control of plastidial esterification capability by SlFBN7a. Collectively, the results support both structural and regulatory roles of SlFBNs in PGs. Our findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for their plastid metabolism/physiology.
Significance statement
In the chromoplast of ripe tomato fruit and flower, plastoglobuli (PGs) are associated with several important biotechnological traits, due to their functional involvement in metabolism, developmental transitions, and environmental adaption. FIBRILLINS (FBN) are a multigene family of proteins that are collectively major components of the PG. Using a multiplex CRISPR-Cas9 approach single and high-order fbn mutants have been developed. Functional redundancy amongst the members of the FBN multigene family was evident, but also paralog specific functions/influence. Aberrant plastoglobuli formation and altered lipid metabolism are evident among fbn mutants. Characterisation of this resource has shed light on the functional role of FBN and their role in PG formation. This strategy offers new potential for the development of nutritional enhanced and climate resilient crops.
... Plants are "green chemical factories" with a wide variety of chemicals that can support the food, feed, pharmaceutical and biomaterials industries (Nogueira et al., 2018). Plants that are used in the treatment of diseases internally or externally from various parts of plants and the substances obtained from these parts are called 'medicinal plants'. ...
... Plants help the food, feed, medicinal, and biomaterial sectors by containing a wide range of chemicals [84]. Herbal medicinal plants (HMPs) are important source of active secondary metabolites and play an important role in human health. ...
Herbal medicinal plants constitute valuable resources of natural secondary active biomolecules that supplement human nourishment, support health care, and are the backbone of herbal industry. Plant breeders are faced with challenging task to realize qualitative and quantitative improvement in their secondary content using traditional breeding methods. Plant genomics research is fast progressing providing better understanding of the complex genetics and biological chemistry involved in the synthesis of natural secondary biomolecules, thus complementing valuable tools to facilitate genetic enhancement. The goal of the genomics study is to build high-throughput sequencing and genotyping technologies to generate huge genomic resources for gene/QTL mapping and marker-trait association for marker-assisted breeding. It also makes easier to find and isolate genes that are involved in various phases of several biosynthetic processes. Efforts are also being made to construct EST and microarray-based functional databases, which will open up the new possibilities for the rapid enrichment of herbal medicinal plants utilizing smart markers and genetic transformation. Metabolic engineering of endogenous and exotic genes based on genome editing could aid in the modification of biosynthetic pathways in these plants. The present review provides an overview of recent developments, limitations, and future possibilities in herbal medicinal plant molecular breeding, including marker-assisted selection and genetic engineering approaches for genetic improvement.
... Nicotiana tabacum L. is not only an important agricultural crop but also among one of the widely studied model plant systems worldwide [27,28]. Despite its production for human tobacco consumption, it holds huge potential to be used for vaccines, bio-factories, medicine, and other beneficial metabolites [29,30]. Moreover, it is extensively used in several basic as well as applied plant research due to its easy manipulation in cell culture, rapidly and easily flourish in both greenhouse and field and readily genetic transformation and regeneration. ...
Background
12-oxophytodienoic acid (OPDA) is a signaling molecule involved in defense and stress responses in plants. 12-oxophytodienoate reductase (OPR) is involved in the biosynthesis of jasmonic acid and trigger the conversion of OPDA into 3-oxo-2(2′[Z]-pentenyl)-cyclopentane-1-octanoic acid (OPC-8:0).
Methods and results
Sequence analysis revealed that Nicotiana tabacum 12-oxophytodienoate reductase 1 (OPR1) and OPR2 encoded polypeptides of 375 and 349 amino acids with molecular masses of 41.67 and 39.04 kilodaltons (kDa), respectively, while the deduced protein sequences of NtOPR1 and NtOPR2 showed high homology with other 12-oxophytodienoate reductases. BLAST (Basic local alignment search tool) analysis revealed that both NtOPRs belong to the family of Old Yellow Enzymes (OYE), and analysis of genomic DNA structure indicated that both genes include 5 exons and 4 introns. Phylogenetic analysis using MEGA X showed that NtOPR1 and NtOPR2 shared a close evolutionary relationship with Nicotiana attenuata 12-oxophytodienoate reductases. In silico analysis of subcellular localization indicated the probable locations of NtOPR1 and NtOPR2 to be the cytoplasm and the peroxisome, respectively. Tissue-specific expression assays via qRT-PCR revealed that NtOPR1 and NtOPR2 genes were highly expressed in Nicotiana tabacum roots, temperately expressed in leaves and flowers, while low expression was observed in stem tissue.
Conclusions
Presently, two 12-oxophytodienoate reductase genes (NtOPR1 and NtOPR2) were cloned and comprehensively characterized. Our findings provide comprehensive analyses that may guide future deep molecular studies of 12-oxophytodienoate reductases in Nicotiana tabacum.
... It was reported that the intermediates of the MVA pathway could most likely not be transported out of mitochondria, thus leading to intermediate accumulation and growth inhibition (Lv et al., 2016). Secondly, IPP and its isomer DMAPP could be transported from the mitochondria to the cytoplasm for the biosynthesis of squalene, as reported in the previous studies (Farhi et al., 2011;Nogueira et al., 2018). Interestingly, compared to SquM5, SquM2, SquM3, and SquM4 had different cell growth phenotypes ( Fig. 2A). ...
Harnessing mitochondria is considered as a promising method for biosynthesis of terpenes due to the adequate supply of acetyl-CoA and redox equivalents in mitochondria. However, mitochondrial engineering often causes serious metabolic burden indicated by poor cell growth. Here, we systematically analyzed the metabolic burden caused by the compartmentalization of the MVA pathway in yeast mitochondria for squalene synthesis. The phosphorylated intermediates of the MVA pathway, especially mevalonate-5-P and mevalonate-5-PP, conferred serious toxicity within mitochondria, which significantly compromised its possible advantages for squalene synthesis and was difficult to be significantly improved by routine pathway optimization. These phosphorylated intermediates were converted into ATP analogues, which strongly inhibited ATP-related cell function, such as mitochondrial oxidative respiration. Fortunately, the introduction of a partial MVA pathway from acetyl-CoA to mevalonate in mitochondria as well as the augmentation of the synthesis of mevalonate in cytosol could significantly promote the growth of yeasts. Accordingly, a combinatorial strategy of cytoplasmic and mitochondrial engineering was proposed to alleviate the metabolic burden caused by the compartmentalized MVA pathway in mitochondria and improve cell growth. The strategy also displayed the superimposed effect of cytoplasmic engineering and mitochondrial engineering on squalene production. Through a two-stage fermentation process, the squalene titer reached 21.1 g/L with a specific squalene titer of 437.1 mg/g dcw, which was the highest at present. This provides new insight into the production of squalene and other terpenes in yeasts based on the advantages of mitochondrial engineering.
... Even though we are aware of the advantages and diverse applications of medicinal plants, they are still placed at the bottom wave of domestication (Nogueira et al., 2018). With advances in time and technologies, researchers became aware of the importance and use of medicinal plants. ...
With the ever-increasing population, the plant cover is decreasing at an alarming rate. The medicinal plants are most affected by this because they are present in the last tier of cultivation. Let it be pharmaceutical companies or people using it for herbalism, medicinal plants have been exploited without getting a chance to flourish in their natural environment. Modern biotechnology acts as a bridge between the cultivation and utilization of medicinal plants. Next Generation Sequencing (NGS) technology which is a decade old but emerging field helps to unveil the importance of medicinal plants. Thus, it paves the way for sustenance of medicinal plants by molecular breeding, micropropagation, large-scale tissue culture, and other methods to conserve the plants with great medicinal value. Various NGS technologies can be found in the market like Ilumina, PacBio, Ion Torrent, and others. The present review will summarize the NGS technologies and their potential use to study the genomes, transcriptome, epigenome, and interactome of medicinal plants towards the identification of bioactive compounds.
... Instead of its use for smoking, cultivated tobacco, Nicotiana tabacum, as well as Nicotiana benthamiana hold great potential to be used as bio-factories for the production of medicines, vaccines or other valuable small metabolites (Capell et al., 2020;Ma et al., 2005;Nogueira et al., 2018;Stoger et al., 2014;Tschofen et al., 2016). To approach this goal, molecular tools for genome editing are necessary for building this 'molecular farm'. ...
Nicotiana tabacum is a non‐food herb that has the potential to be utilized as bio‐factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre‐designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site‐specific double‐strand breaks to enhance homologous recombination‐mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas‐based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature‐insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium‐mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T‐DNA integration. Thus, we were not only able to obtain CRISPR/Cas‐based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.
... It also suggests that the DXS expression is linked with the plant parts in a tissue-specific manner and the accumulation of the final products [13,14]. The enhanced production of these compounds, particularly diterpene lactones can be achieved through genetic manipulation of biosynthetic pathway [15,16]. The DXS gene has made known to be a significant target for the manipulation of terpenoids biosynthesis [17][18][19][20][21]. ...
Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1) was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata 3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 µm JA elicitation followed by 50 µm. HPLC data indicated that the transcription levels were correlated with increased andrographolide accumulation. The peak level of andrographolide accumulation was recorded at 25 μM JA (9.38-fold) followed by 50 µM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further, prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata.
... It also suggests that the DXS expression is linked with the plant parts in a tissue-specific manner and the accumulation of the final products [13,14]. The enhanced production of these compounds, particularly diterpene lactones can be achieved through genetic manipulation of biosynthetic pathway [15,16]. The DXS gene has made known to be a significant target for the manipulation of terpenoids biosynthesis [17][18][19][20][21]. ...
Abstract
Andrographis paniculata 1-deoxy-D-xylulose-5-phosphate synthase (ApDXS) gene (GenBank Accession No MG271749.1)
was isolated and cloned from leaves for the first time. Expression of ApDXS gene was carried out in Escherichia coli Rosetta
cells. Tissue-specific ApDXS gene expression by quantitative RT-PCR (qRT-PCR) revealed maximum fold expression
in the leaves followed by stem and roots. Further, the differential gene expression profile of Jasmonic acid (JA)-elicited
in vitro adventitious root cultures showed enhanced ApDXS expression compared to untreated control cultures. A. paniculata
3-hydroxy-3-methylglutaryl-coenzyme A reductase (ApHMGR) gene expression was also studied where it was up-regulated
by JA elicitation but showed lower expression compared to ApDXS. The highest expression of both genes was found at 25 μm
JA elicitation followed by 50 μm. HPLC data indicated that the transcription levels were correlated with increased andrographolide
accumulation. The peak level of andrographolide accumulation was recorded at 25 μM JA (9.38-fold) followed
by 50 μM JA (7.58-fold) in elicitation treatments. The in silico generated ApDXS 3D model revealed 98% expected amino
acid residues in the favored and 2% in the allowed regions of the Ramachandran plot with 92% structural reliability. Further,
prediction of conserved domains and essential amino acids [Arg (249, 252, 255), Asn (307) and Ser (247)] involved in ligand/
inhibitor binding was carried out by in silico docking studies. Our present findings will generate genomic information and
provide a blueprint for future studies of ApDXS and its role in diterpenoid biosynthesis in A. paniculata.
... In plant, carotenoids are derived from isopentenyl pyrophosphate (IPP) formed from the methylerythritol 4-phosphate (MEP) pathway in the plastid. IPP is condensed into geranylgeranyl diphosphate (GGPP; C 20 ), a common precursor for several groups of plastidial isoprenoids (Nogueira et al., 2018;Ruiz-Sola and Rodríguez-Concepción, 2012). The tail-to-tail condensation of two molecules of GGPP form 15-cis-phytoene (C 40 ), which represents the first dedicated step in carotenoid biosynthesis and is catalyzed by phytoene synthase (PSY) (Fig. 1). ...
This work reports the development of suspension culture system of transgenic Marsh grapefruit (Citrus paradisi Macf., Rutaceae) callus overexpressing bacterial phytoene synthase; and the use of this suspension culture to investigate the effects of β-cyclocitral on carotenoid content and composition. At a β-cyclocitral concentration of 0.5 mM and after ten days cultivation, analysis of the carotenoids showed a significant increase in the content of β-, α-carotene, and phytoene predominantly. The maximal increase in total provitamin A carotenoids content following β-cyclocitral application was ~2-fold higher than the control, reaching 245.8 μg/g DW. The trend for increased transcript levels of biosynthetic genes PSY and ZDS correlated with the enhancement of the content of these carotenes following β-cyclocitral treatment and GC-MS based metabolite profiling showed significant changes of metabolite levels across intermediary metabolism. These findings suggest that β-cyclocitral can act as a chemical elicitor, to enhance the formation of carotenes in citrus suspension-cultured cells (SCC), which could be utilized in studying the regulation of carotenoid biosynthesis and biotechnological application to the renewable production of nutritional carotenoids.
... Secondly, efficient carotenoid desaturation conducted by phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS) depends on the redox status of plastoquinone/plastoquinol pool dependent on the activity of PTOX (Shahbazi et al., 2007) whose transcripts were found heatsensitive. Finally, methyl-erythritol phosphate (MEP)-derived precursors for carotenogenesis (Almeida et al., 2015;Nogueira et al., 2018) might be altered as the expression of ISPE, GGPPS3 and the ripening- , 2018). Therefore, it is expected some contribution of post-translational mechanisms curbing the activity of carotenoidrelated enzymes under heat stress. ...
High temperatures can negatively influence plant growth and development. Besides yield, the effects of heat stress on fruit quality traits remain poorly characterised. In tomato, insights into how fruits regulate cellular metabolism in response to heat stress could contribute to the development of heat‐tolerant varieties, without detrimental effects on quality. In the present study, the changes occurring in wild type tomato fruits after exposure to transient heat stress have been elucidated at the transcriptome, cellular and metabolite level. An impact on fruit quality was evident as nutritional attributes changed in response to heat stress. Fruit carotenogenesis was affected, predominantly at the stage of phytoene formation, although altered desaturation/isomerisation arose during the transient exposure to high temperatures. Plastidial isoprenoid compounds showed subtle alterations in their distribution within chromoplast sub‐compartments. Metabolite profiling suggests limited effects on primary/intermediary metabolism but lipid remodelling was evident. The heat‐induced molecular signatures included the accumulation of sucrose and triacylglycerols, and a decrease in the degree of membrane lipid unsaturation, which influenced the volatile profile. Collectively, these data provide valuable insights into the underlying biochemical and molecular adaptation of fruit to heat stress and will impact on our ability to develop future climate resilient tomato varieties.
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... With recent advancements in plant synthetic biology, implementation in crops of multigene isoprenoid metabolic pathways to develop biofuels and bioproducts at industrial scale is becoming attainable [200][201][202]. The wondrous diversity and structural richness of isoprenoid molecules from plants provide a plethora of potent and useful target compounds, especially for advanced biofuels and value-added bioproducts [152,[203][204][205][206]. In relation to bioenergy crops, several efforts have been made to elucidate the genetic components that influence terpene yields, as well as to develop biomass pretreatment methods for simultaneous extraction of both terpenes and fermentable sugars [207,208]. ...
Abstract Industrial crops are grown to produce goods for manufacturing. Rather than food and feed, they supply raw materials for making biofuels, pharmaceuticals, and specialty chemicals, as well as feedstocks for fabricating fiber, biopolymer, and construction materials. Therefore, such crops offer the potential to reduce our dependency on petrochemicals that currently serve as building blocks for manufacturing the majority of our industrial and consumer products. In this review, we are providing examples of metabolites synthesized in plants that can be used as bio-based platform chemicals for partial replacement of their petroleum-derived counterparts. Plant metabolic engineering approaches aiming at increasing the content of these metabolites in biomass are presented. In particular, we emphasize on recent advances in the manipulation of the shikimate and isoprenoid biosynthetic pathways, both of which being the source of multiple valuable compounds. Implementing and optimizing engineered metabolic pathways for accumulation of coproducts in bioenergy crops may represent a valuable option for enhancing the commercial value of biomass and attaining sustainable lignocellulosic biorefineries.
... Plant molecular pharming (PMP) is based on the ability of plants to express different genes for preventive and therapeutic purposes and it holds promise [11]. The production and commercialization of recombinant proteins and pharmaceutical compounds from plants have been optimized and relatively successful over the past few years [11][12][13][14][15]. The concept of PMP as a biofactory was demonstrated earlier on [16] and presents many advantages over conventional systems that rely on bacteria, yeast, insect and animal cell cultures for pharmaceutical products [13]. ...
Millions of people around the world suffer from heavy social and
health burdens related to HIV/AIDS and its associated
opportunistic infections. To reduce these burdens, preventive
and therapeutic vaccines are required. Effective HIV vaccines
have been under investigation for several decades using
different animal models. Potential plant-made HIV vaccine
candidates have also gained attention in the past few years. In
addition to this, broadly neutralizing antibodies produced in
plants which can target conserved viral epitopes and neutralize
mutating HIV strains have been identified. Numerous epitopes
of envelope glycoproteins and capsid proteins of HIV-1 are a
part of HIV therapy. Here, we discuss some recent findings
aiming to produce anti-HIV-1 recombinant proteins in
engineered plants for AIDS prophylactics and therapeutic
treatments.
... In recent years, plant based natural products are extensively employed in pharmaceutical, nutraceutical and dietary supplements. Nogueira et al. (2018) proposed molecular factories of plants for nutritional and industrial production of isoprenoids including tocopherols and carotenoids which is a major constituent of human diet. The plant-based natural products are widely used as medicines for the ailment of deadly diseases, including HIV and new diseases that are spreading in recent times (Vora et al. 2018). ...
Medicinal plants are known to contain substances which could be useful for the treatment of diseases or for the production of drugs. These substances occur naturally in plants (leaves, stems, barks, and root) and are referred to as secondary metabolites because they are synthesized to protect the plant other than for growth just like the primary metabolites. Fortunately for humans, most of these secondary metabolites possess medicinal potentials which are active against many diseases. Before the advent of modern analytical techniques for the screening of plant actives, the traditional plants have been used primitively to alleviate symptoms of illnesses such as stomach ache, toothache, body pain and inflammation, diarrhea, malaria, typhoid, diabetes etc. This chapter presents an overview of the usefulness of medicinal plants as sources of drugs, the drug discovery process, the efficacy and safety of phytochemicals, current uses, advances in screening tools, problems and the way forward.
... Phenolic compounds may release during an extraction course that might be detrimental to the downstream processing by expression of proteins from leafy crops and this could be unstable green leaf host system is easy to engineer, it can lead to problems if the protein interferes, with plant development and also purification is difficult due to the presence of pigments, alkaloids, and polyphenols [58][59][60] high protease activity host system has been used for production of biopharmaceuticals, yeast is also a biotechnological host for the production of biopharmaceuticals [43][44][45] large-scale production of recombinant proteins depends upon microorganisms used to be the best suitable host, proteins expressed in mammalian cells [46] have similar properties as the natural origin, but still it is the less suitable for expression of the therapeutic proteins because of the inability of large-scale production and highly expensive culture cost [47]. Microalgae are being used as a production system for the production of vaccines and biopharmaceuticals [48,49] other new approaches are raised by advancing and rewriting the DNA of host [50] CRISPR has 9 technology can be used for creation off GMOs without the introduction of foreign gene, no doubt gene editing is a powerful technology [51][52][53]. Plant biotechnology is a joint venture of tissue culture and genetic engineering genetically modified organisms are products with altered genetic material to get desired protein utilizing recombinant technology plant part which is Utilize should be well studied and allow effective risk assessment of the transgene of interest. selection of host system is important for efficient protein production. ...
... In fact, any synthetic biology approach will be limited by natural genetic variation, as described for carotenoids (Harjes et al., 2008;Lee et al., 2012;Schulz et al., 2016;Sulli et al., 2017;Price et al., 2018;Teixeira da Silva et al., 2018). Nonetheless, metabolic engineering of the pathway has been demonstrated for many crops (Giuliano, 2017;Majer et al., 2017;Nogueira et al., 2017Nogueira et al., , 2018Camagna et al., 2018;McQuinn et al., 2018;Sankari et al., 2018). These strategies have also included the successful application of gene editing technology (Dahan-Meir et al., 2018) which in the future could be used to incorporate favorable allelic variation from wild species. ...
... Over the last few years, systems biology helps to understand the molecular, cellular, and physiological response induced by plants under stress conditions which provides preliminary information for genetic manipulation of the plant towards the generation of stress tolerance [121]. Currently, scientists are also trying to involve a modern approach like genome editing by using CRISPR/Cas, metabolic pathway modeling, enzyme engineering, and nanotechnology in the field of plant science to improve the genetic machinery of plants against stress [122]. There are very few reports which are using these technologies, but there are some limitations like complex nature of plant genomes, the high cost of reagents used in systems biology, etc., which needs to overcome and require more efforts for amicable options to understand such mechanisms. ...
Plants are continually facing biotic and abiotic stresses, and hence, they need to respond and adapt to survive. Plant response during multiple and combined biotic and abiotic stresses is highly complex and varied than the individual stress. These stresses resulted alteration of plant behavior through regulating the levels of microRNA, heat shock proteins, epigenetic variations. These variations can cause many adverse effects on the growth and development of the plant. Further, in natural conditions, several abiotic stresses causing factors make the plant more susceptible to pathogens infections and vice-versa. A very intricate and multifaceted interactions of various biomolecules are involved in metabolic pathways that can direct towards a cross-tolerance and improvement of plant’s defence system. Systems biology approach plays a significant role in the investigation of these molecular interactions. The valuable information obtained by systems biology will help to develop stress-resistant plant varieties against multiple stresses. Thus, this review aims to decipher various multilevel interactions at the molecular level under combinatorial biotic and abiotic stresses and the role of systems biology to understand these molecular interactions.
There is a remarkable acceleration in the production of nutraceuticals which is increasingly associated with escalated commercial demands. Several techniques are being applied for the production of nutraceuticals. The in vitro plant culture system is regarded as a fundamental technique for the production of fast multiplication of extraordinary plant genotypes, disease-free plants, and plant genome transformation. However, each technique possesses its limitations. Fortunately, most of the obstacles of in vitro plant techniques can now be overcome through the promising new technology CRISPR/Cas9, which allows change of specific regions of the genome with an increased precision of the insertion and offering excellent reproducibility. This chapter discusses the production of nutraceuticals and briefly summarizes its challenges and opportunities.
Carotenoid biosynthesis has now been subjected to metabolic engineering for over two decades. The outputs clearly show that carotenoid formation is an integral component of metabolism. Perturbations can affect intermediary metabolism and other isoprenoids. The advances in omic technologies have enabled the quantitative assessment of changes in the transcriptome, proteome and metabolome in response to altered carotenoid biosynthesis. In the present article, the approaches and procedures relating to the capture of the metabolome in response to modulation of the carotenoid biosynthetic pathway are described. These data will contribute to the fundamental understanding of metabolic biology, underpinning future rationale design of New Plant Breeding Techniques (NPBTs) and associated regulatory affairs.
An overview of the field of plant tissue culture is presented in this chapter, which includes a general history, successive developments, and key contributions by several leading scientists. The development of cell and tissue culture of plant organs has contributed to promoting research in several areas of plant biotechnology. It has made great progress and significant contributions to the advancement of modern agriculture, production of secondary substances used in pharmaceutical and biochemical industries, food security, and conservation of plant genetic resources. This chapter briefly presents the current status of plant tissue culture applications in Vietnam (as a developing country) and the world as well. Low-cost tissue culture technology will be one of the top priorities in the development of agriculture, horticulture, and forestry in many developing countries in order to produce affordable high-quality crop materials without affecting the quality of products created. Last but not least, some future developments in this field, including the great potential of plant micropropagation and the increasingly important role of new biological techniques, are covered in this chapter.
Synthetic biology programs for increased production of bioactive plant-derived terpenes initially focused on linear aspects of their biosynthetic pathways. Yet, the spatial organization of terpene pathways, typically across multiple cellular compartments, seriously encumbers engineering success. Here, we discuss the recent advances in endoplasmic reticulum, peroxisome and other organellar engineering and illustrate how this is being applied to increase terpene pathway performances in plants and yeasts. We also discuss how specialized transporters could present potent novel tools to connect cellular compartments. Altogether, these new perspectives demonstrate how synthetic biology can offer real-world solutions for efficient and sustainable production of high-value terpenes and eventually address the shortcomings of extraction from natural resources.
Plants play an essential role in many aspects of human health, including nutrition and medicine. Advancements in biology and engineering have hastened research regarding plant‐based medicines. More specifically, biotechnology has played a significant role in identifying new ways that plants can be utilised. There are three major directions that plants are being developed to improve our health. The first concerns the use of plants as production platforms for modern pharmaceuticals. The second deals with plant biofortification strategies to prevent malnutrition and defeat chronic diseases. The third describes the identification and analysis of bioactive compounds derived from plants in modern medicine. All three sections bring to light mankind's continuous reliance on plants for our current and future health needs.
Key Concepts
• Plants play an essential role in many aspects of human health, including nutrition and medicine.
• Plants can be used as production platforms for biopharmaceuticals.
• Biofortified crops such as Golden Rice and Banana21 can prevent Vitamin A deficiency.
• Plant secondary metabolites can be used as a source of bioactive molecules to improve human health.
• However, significant opposition to biotechnology has slowed the research and development of plant-based health products.
Covering: up to 2019 As abundant natural products, isoprenoids have many useful industrial applications in the manufacturing of drugs, fragrances, food additives, colorants, rubber and advanced biofuels. The microbial production of isoprenoids has received much attention in recent years. Metabolic engineering approaches and synthetic biology have been utilized to reconstruct and optimize the metabolic pathways for isoprenoid production in cell factories. In this review, the recent advances in isoprenoid production using microbes are summarized, with a focus on MEP and MVA pathway engineering, downstream isoprenoid pathway engineering and microbial host engineering, which mainly includes central carbon pathway engineering. Finally, future perspectives for the improvement of isoprenoid production are discussed.
Prenylation of protein is a post‐translational modification (PTM) that involves the covalent thioether attachment of farnesyl or geranylgeranyl group to Cys residues present in the C‐terminal domain of proteins and is situated in the common consensus sequence of CAAX motif. Both farnesyl and geranylgeranyl groups are isoprenoids and hence the modification is called prenylation. Farnesyl is a 15‐carbon isoprenoid, whereas geranylgeranyl is a 20‐carbon isoprenoid. Following prenylation, the terminal three amino acid residues, i.e. AAX residues are removed by CAAX motif‐specific proteases followed by methylation of the carboxyl group of prenylated Cys residue that is a newly generated terminal amino acid residue. More than 120 human proteins have been identified as prenylated with either farnesyl or geranylgeranyl group. Among the human proteins that are prenylated are G‐proteins, Ras family of GTPases, laminins, protein kinases, and protein phosphatases. Prenylation is an enzymatic reaction mediated by either farnesyltransferases or geranylgeranyltransferases. Farnesyltransferase mediates the transfer of farnesyl group from farnesylpyrophosphate, whereas gernylgeranyltransferase mediates the transfer of geranylgeranyl group from geranylgeranylpyrophosphate. These transferases are heterodimeric proteins consisting of a common α‐subunit and a distinct β‐subunit. Prenylation is involved in controlling the protein membrane trafficking and modulation of immune responses. In addition, prenylation is also involved in tethering the G‐protein to inner surface of the plasma membrane in living cells that help the protein interactions with its receptors. So far, no prenylation has been observed in recombinant therapeutic proteins (RTPs) including monoclonal antibodies (mAbs). Enzymatic and gas‐chromatography in conjunction with mass spectrometry methods have been used to detect and analyze prenylation of proteins.
This chapter contains sections titled: Introduction Taxol: History and Clinical Impact Endophytes The Plausibility of Horizontal Gene Transfer (HGT) Hypothesis Endophytes as Biological Factories of Functional Metabolites Taxol Producing Endophytic Fungi Molecular Basis of Taxol Production by Taxus Plants (Taxol Biosynthetic Pathway) Metabolic Engineering for Synthesis of Taxol: Next Generation Tool Future Perspectives Acknowledgements
Plants play an essential role in many aspects of human health, including nutrition and medicine. Advancements in biology and engineering have hastened research regarding plant-based medicines. The following chapter outlines three major directions that plants are being developed to improve our health. The first concerns the use of plants as production platforms for modern pharmaceuticals. The second deals with plant biofortification strategies to prevent malnutrition and defeat chronic diseases. The third describes the identification and analysis of bioactive compounds derived from plants in modern medicine. All three sections bring to light mankind’s continuous reliance on plants for our current and future health needs.
Plants have a remarkable capacity for the production of a wide range of metabolites. Much has been reported and reviewed on the diversity of these metabolites and how it is achieved, for example through the evolution of enzyme families. In comparison, relatively little is known on the extraordinary metabolic productivity of dedicated organs where many of these metabolites are synthesized and accumulate. Plant glandular trichomes are such specialized metabolite factories, for which recent omics analyses have shed new light on the adaptive metabolic strategies that support high metabolic fluxes. In photosynthetic trichomes such as those of the Solanaceae, these include CO2 refixation and possibly C4-like metabolism which contribute to the high productivity of these sink organs.
The profile of secondary metabolites in plants reflects the balance of biosynthesis, degradation and storage, including the availability of precursors and products that affect the metabolic equilibrium. We investigated the impact of the precursor-product balance on the carotenoid pathway in the endosperm of intact rice plants because this tissue does not normally accumulate carotenoids, allowing us to control each component of the pathway. We generated transgenic plants expressing the maize phytoene synthase gene (ZmPSY1) and the bacterial phytoene desaturase gene (PaCRTI), which are sufficient to produce b-carotene in the presence of endogenous lycopene b-cyclase. We combined this mini-pathway with the Arabidopsis thaliana genes AtDXS (encoding 1-deoxy-D-xylulose 5-phosphate synthase, which supplies metabolic precursors) or AtOR (the ORANGE gene, which promotes the formation of a metabolic sink). Analysis of the resulting transgenic plants suggested that the supply of isoprenoid precursors from the MEP pathway is one of the key factors limiting carotenoid accumulation in the endosperm and that the overexpression of AtOR increased the accumulation of carotenoids in part by up-regulating a series of endogenous carotenogenic genes. The identification of metabolic bottlenecks in the pathway will help to refine strategies for the creation of engineered plants with specific carotenoid profiles.
ELife digest
Malaria is by far the most devastating tropical disease in the world. It affects hundreds of millions of people – mainly in Africa and Asia – with almost half a million deaths every year. The most effective therapies against malaria all include the drug artemisinin, which is naturally found in an Asian plant called Artemisia annua. Unfortunately, the artemisinin content of A. annua plants is relatively low and the demand for this drug outstrips the supply of the plant. The costly production process makes artemisinin-based treatments inaccessible to many of the people in the most badly affected regions, and so researchers have been trying to find new ways to produce this drug.
Genetically modifying crop plants, such as tobacco, to produce artemisinin or related compounds could potentially provide a more sustainable and cheaper source of the drug. Inside plant cells, a structure called the nucleus contains DNA that encodes most of a plant’s genes, but compartments called mitochondria and chloroplasts also contain some DNA. Existing methods to genetically modify plants are able to insert a few genes into either the nucleus or the chloroplasts at a time. However, the production of artemisinin in A. annua involves many different genes that act at different stages of the process, and the precise roles played by many of them remain unclear.
Fuentes et al. developed a new approach to insert many of the A. annua genes involved in artemisinin production into tobacco plants at the same time, instead of one-by-one. The new method, referred to as COSTREL, takes advantage of the researchers’ ability to insert new genes into both the nucleus and the chloroplast of the tobacco plants. In the first step, Fuentes et al. inserted a core set of genes that are essential to make artemisinin into the chloroplast. This enabled the plants to produce a molecule called artemisinic acid, which the researchers can extract from the plants and convert into artemisinin by simple chemical reactions.
After testing different arrangements of the genes in the chloroplast, the plant line that had the highest levels of artemisinic acid was used to introduce a set of “accessory” genes into the nuclear DNA. These accessory genes are not strictly required to make the drug, but they help to regulate the process in a largely unknown manner. The experiments generated hundreds of genetically modified plant lines that each have different combinations of the accessory genes. Fuentes et al. examined these lines and were able to identify plants that could produce large amounts of artemisinic acid. Therefore, these findings lay the foundations for a cheap way to produce this life-saving drug in tobacco. In the future, the COSTREL method developed by Fuentes et al. could also be used to genetically engineer other complex biochemical processes into plants.
DOI: http://dx.doi.org/10.7554/eLife.13664.002
Genome editing has emerged as a technology with a potential to revolutionize plant breeding. In this study, we report on generating, in less than ten months, Tomelo, a non- transgenic tomato variety resistant to the powdery mildew fungal pathogen using the CRISPR/ Cas9 technology. We used wholegenome sequencing to show that Tomelo does not carry any foreign DNA sequences but only carries a deletion that is indistinguishable from naturally occurring mutations. We also present evidence for CRISPR/ Cas9 being a highly precise tool, as we did not detect off- target mutations in Tomelo. Using our pipeline, mutations can be readily introduced into elite or locally adapted tomato varieties in less than a year with relatively minimal effort and investment.
Beta-carotene adds nutritious value and determines the color of many fruits including melon. In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) 'golden' SNP through a yet to be discovered mechanism. In Arabidopsis, OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here we identified a CmOr nonsense mutation (Cmor-lowβ), which lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the 'golden' SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblotting analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in 'golden' SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including crtiso ('yofi'), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The 'yofi' F2 segregants accumulated prolycopene independently of the 'golden' SNP. Moreover, Cmor-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 days after anthesis until ripening, suggesting that CmOr regulates chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels for maintaining carotenoid biosynthesis metabolic flux, but that the mechanism of the CmOr 'golden' SNP involves an inhibited metabolism downstream to β-carotene to dramatically affect both carotenoid content and plastid fate.
Soybean (Glycine max L.) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization (CGH) analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO) which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.
Significance
Studies on the importance of vitamin A for human health continue to draw significant worldwide attention. However, the instability of provitamin A in crops resulted in a significant reduction of the potential nutrition values of these food crops. Our work demonstrates that provitamin A can be stabilized in sorghum by the coexpression of vitamin E through ectopic expression of homogentisate geranylgeranyltransferase ( HGGT ) and that vitamin E can enhance the stability of provitamin A in planta . This research has the potential to impact directly the lives of the millions of people who suffer from vitamin A deficiency, and we believe that these results will be applicable to enhancing provitamin A stability in many food crops.
Catharanthus roseus produces bioactive terpenoid indole alkaloids ( TIA s), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood.
The jasmonic acid ( JA )‐responsive AP 2/ ERF transcription factor ( TF ), ORCA 3, and its regulator, Cr MYC 2, play key roles in TIA biosynthesis. ORCA 3 forms a physical cluster with two uncharacterized AP 2/ ERF s, ORCA 4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA 4, while overlapping functionally with ORCA 3, modulates an additional set of TIA genes. Unlike ORCA 3, ORCA 4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, Cr MYC 2 is capable of activating ORCA 3 and co‐regulating TIA pathway genes concomitantly with ORCA 3.
The ORCA gene cluster and Cr MYC 2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, Cr MAPKK 1. Overexpression of Cr MAPKK 1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation.
This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post‐translational regulatory mechanisms that govern TIA biosynthesis in C. roseus .
Controlling the rate of softening to extend shelf life was a key target for researchers engineering genetically modified (GM) tomatoes in the 1990s, but only modest improvements were achieved. Hybrids grown nowadays contain 'non-ripening mutations' that slow ripening and improve shelf life, but adversely affect flavor and color. We report substantial, targeted control of tomato softening, without affecting other aspects of ripening, by silencing a gene encoding a pectate lyase.
The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by site-specific nucleases to initiate DNA repair reactions that are either based on non-homologous end joining (NHEJ) or homologous recombination (HR). Recently, the CRISPR/Cas system emerged as the most important tool for genome engineering due to its simple structure and its applicability to a wide range of organisms. Here, we review the current status of its various applications in plants, where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. Furthermore, tremendous progress in plant genome engineering by HR was obtained by the setup of replicon mediated and in planta gene targeting techniques. Finally, other complex approaches that rely on the induction of more than one DNA lesion at a time such as paired nickases to avoid off-site effects or controlled genomic deletions are beginning to be applied routinely.
Significance
Terpenoids are the largest group of plant-specialized metabolites and include many valuable bioactive compounds, such as the blockbuster anticancer drugs vincristine and vinblastine, that are monoterpenoid indole alkaloids from the medicinal plant Catharanthus roseus (Madagascar periwinkle). A master regulator was discovered that activates the biosynthesis of the iridoids, the monoterpenoid precursors of vinblastine and vincristine, and the rate-limiting branch in their biosynthetic pathway. This master regulator can be used to boost production of iridoids and monoterpenoid indole alkaloids in C. roseus cell cultures and thus represents an interesting tool for the metabolic engineering of the sustainable production of these high-value compounds in cultures of the endogenous plant species.
Bacterial blight of rice is caused by the γ-proteobacterium Xanthomonas oryzae pv. oryzae, which utilizes a group of type III TAL (transcription activator-like) effectors to induce host gene expression and condition host susceptibility. Five SWEET genes are functionally redundant to support bacterial disease, but only two were experimentally proven targets of natural TAL effectors. Here, we report the identification of the sucrose transporter gene OsSWEET13 as the disease susceptibility gene for PthXo2 and the existence of cryptic recessive resistance to PthXo2-dependent X. oryzae pv. oryzae due to promoter variations of OsSWEET13 in japonica rice. PthXo2-containing strains induce OsSWEET13 in indica rice IR24 due to the presence of an unpredicted and undescribed effector binding site not present in the alleles in japonica rice Nipponbare and Kitaake. The specificity of effector-associated gene induction and disease susceptibility is attributable to a single nucleotide polymorphism (SNP), which is also found in a polymorphic allele of OsSWEET13 known as the recessive resistance gene xa25 from the rice cultivar Minghui 63. The mutation of OsSWEET13 with CRISPR/Cas9 technology further corroborates the requirement of OsSWEET13 expression for the state of PthXo2-dependent disease susceptibility to X. oryzae pv. oryzae. Gene profiling of a collection of 104 strains revealed OsSWEET13 induction by 42 isolates of X. oryzae pv. oryzae. Heterologous expression of OsSWEET13 in Nicotiana benthamiana leaf cells elevates sucrose concentrations in the apoplasm. The results corroborate a model whereby X. oryzae pv. oryzae enhances the release of sucrose from host cells in order to exploit the host resources. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Significance
Carotenoids are indispensable to plants and humans. Despite significant achievements in carotenoid research, we still lack the fundamental knowledge of the regulatory mechanisms underlying carotenogenesis in plants. Phytoene synthase (PSY) and ORANGE (OR) are the two key proteins for carotenoid biosynthesis and accumulation in plastids. This study shows that OR family proteins interact directly with PSY and function as the major regulators of active PSY protein abundance in mediating carotenoid biosynthesis. The findings establish posttranscriptional regulation of PSY as a novel way to control carotenoid biosynthesis in plants and provide strategies for crop nutritional quality improvement.
Synthetic biology seeks to create new biological systems, including user-designed plants and plant cells. These systems can be employed for a variety of purposes, ranging from producing compounds of industrial or therapeutic value, to reducing crop losses by altering cellular responses to pathogens or climate change. To realize the full potential of plant synthetic biology, techniques are required that provide control over the genetic code - enabling targeted modifications to DNA sequences within living plant cells. Such control is now within reach owing to recent advances in the use of sequence-specific nucleases to precisely engineer genomes. We discuss here the enormous potential provided by genome engineering for plant synthetic biology.
Copyright © 2014 Elsevier Ltd. All rights reserved.
Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.
Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.
We have developed an assay based on rice embryogenic callus for the rapid functional characterization of metabolic genes. We validated the assay using a selection of well-characterized genes with known functions in the carotenoid biosynthesis pathway, allowing the rapid visual screening of callus phenotypes based on tissue color. We were then able to use the system to identify the functions of two uncharacterized genes: a chemically-synthesized β-carotene ketolase gene optimized for maize codon usage; and a wild-type Arabidopsis thaliana ortholog of the cauliflower Orange gene. In contrast to previous reports, we found that the wild-type Orange allele was sufficient to induce chromoplast differentiation. We also found that chromoplast differentiation could be induced by increasing the availability of precursors and thus driving flux through the pathway, even in the absence of Orange. Remarkably, we found that diverse endosperm-specific promoters were highly active in rice callus despite their restricted activity in mature plants. Our callus system provides a unique opportunity to predict the impact of metabolic engineering in complex pathways and provides a starting point for quantitative modeling and the rational design of engineering strategies using synthetic biology. We discuss the impact of our data on the analysis and engineering of the carotenoid biosynthesis pathway. This article is protected by copyright. All rights reserved.
Metabolic engineering of the carotenoid pathway in recent years has successfully enhanced the carotenoid contents of crop plants. It is now clear that only increasing biosynthesis is restrictive, as mechanisms to sequestrate these increased levels in the cell or organelle should be exploited. In this study, biosynthetic pathway genes were overexpressed in tomato (Solanum lycopersicum) lines and the effects on carotenoid formation and sequestration revealed. The bacterial Crt carotenogenic genes, independently or in combination, and their zygosity affect the production of carotenoids. Transcription of the pathway genes was perturbed, whereby the tissue specificity of transcripts was altered. Changes in the steady state levels of metabolites in unrelated sectors of metabolism were found. Of particular interest was a concurrent increase of the plastid-localized lipid monogalactodiacylglycerol with carotenoids along with membranous subcellular structures. The carotenoids, proteins, and lipids in the subchromoplast fractions of the transgenic tomato fruit with increased carotenoid content suggest that cellular structures can adapt to facilitate the sequestration of the newly formed products. Moreover, phytoene, the precursor of the pathway, was identified in the plastoglobule, whereas the biosynthetic enzymes were in the membranes. The implications of these findings with respect to novel pathway regulation mechanisms are discussed.
A significant number of terpenoid compounds are glycosides with the sugars linked to the active groups. Sometimes, the glycosidic residue is crucial for their activity, but in other cases glycosylation only improves pharmacokinetic parameters. Enzymatic glycosylation of terpenoids is a useful tool due to the high selectivity and the mildness of the reaction conditions, in comparison with chemical methods. Several types of biocatalysts have been used in the enzymatic glycosylation of terpenoids. These include the use of glycosyltransferases, trans-glycosidases, and whole-cell biotransformation systems capable of regenerating the cofactor, such as fungi, bacteria, plant-cell cultures, etc. Many biosynthesized terpenoid glycosides display medicinal and pharmacological properties and can be used as pro-drug substances. These terpenoid glycosides have also been employed as food additives (e.g. low-caloric sweetener compounds) and cosmetics, and even have applications as controlled-release fragrances.
The tomato (Lycopersicon esculentum) high-pigment (hp) locus was originally described as having enhanced fruit-quality characteristics and has also been shown to regulate responses
to light during growth and development. Specifically, the hp phenotype suggests that the normal HP gene-product serves as a negative regulator of light signal-transduction, as has been proposed for many of the previously
described Arabidopsis thaliana photomorphogenic mutants. Consequently, hp represents a tool for both genetic dissection of light signal-transduction and manipulation of fruit quality in tomato. As
a first step toward isolation of the HP gene, the hp locus was mapped to tomato chromosome 2, adjacent to the 45s rDNA locus, using DNA markers and an interspecific cross of L. esculentum×L. cheesmannii. We have simultaneously identified DNA markers which may be useful for gene isolation and marker-assisted selection. We have
additionally extended characterization of the hp phenotype to demonstrate increased sucrose and flavonoid accumulation in ripe hp/hp fruit. Analysis of plastid DNA copy number relative to genomic DNA content indicates that the hp locus regulates plastome DNA concentration, and possibly plastid number, in response to light.
The sweet, floral flavor typical of Muscat varieties (Muscats), due to high levels of monoterpenoids (geraniol, linalool and nerol), is highly distinct and has been greatly appreciated both in table grapes and in wine since ancient times. Muscat flavor determination in grape (Vitis vinifera L.) has up to now been studied by evaluating monoterpenoid levels through QTL analysis. These studies have revealed co-localization of 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS) with the major QTL positioned on chromosome 5.
We resequenced VvDXS in an ad hoc association population of 148 grape varieties, which included muscat-flavored, aromatic and neutral accessions as well as muscat-like aromatic mutants and non-aromatic offsprings of Muscats. Gene nucleotide diversity and intragenic linkage disequilibrium (LD) were evaluated. Structured association analysis revealed three SNPs in moderate LD to be significantly associated with muscat-flavored varieties. We identified a putative causal SNP responsible for a predicted non-neutral substitution and we discuss its possible implications for flavor metabolism. Network analysis revealed a major star-shaped cluster of reconstructed haplotypes unique to muscat-flavored varieties. Moreover, muscat-like aromatic mutants displayed unique non-synonymous mutations near the mutated site of Muscat genotypes.
This study is a crucial step forward in understanding the genetic regulation of muscat flavor in grapevine and it also sheds light on the domestication history of Muscats. VvDXS appears to be a possible human-selected locus in grapevine domestication and post-domestication. The putative causal SNP identified in Muscat varieties as well as the unique mutations identifying the muscat-like aromatic mutants under study may be immediately applied in marker-assisted breeding programs aimed at enhancing fragrance and aroma complexity respectively in table grape and wine cultivars.
Fruit-specific downregulation of the DE-ETIOLATED1 (DET1) gene product results in tomato fruits (Solanum lycopersicum) containing enhanced nutritional antioxidants, with no detrimental effects on yield. In an attempt to further our understanding of how modulation of this gene leads to improved quality traits, detailed targeted and multilevel omic characterization has been performed. Metabolite profiling revealed quantitative increases in carotenoid, tocopherol, phenylpropanoids, flavonoids, and anthocyanidins. Qualitative differences could also be identified within the phenolics, including unique formation in fruit pericarp tissues. These changes resulted in increased total antioxidant content both in the polar and nonpolar fractions. Increased transcription of key biosynthetic genes is a likely mechanism producing elevated phenolic-based metabolites. By contrast, high levels of isoprenoids do not appear to result from transcriptional regulation but are more likely related to plastid-based parameters, such as increased plastid volume per cell. Parallel metabolomic and transcriptomic analyses reveal the widespread effects of DET1 downregulation on diverse sectors of metabolism and sites of synthesis. Correlation analysis of transcripts and metabolites independently indicated strong coresponses within and between related pathways/processes. Interestingly, despite the fact that secondary metabolites were the most severely affected in ripe tomato fruit, our integrative analyses suggest that the coordinated activation of core metabolic processes in cell types amenable to plastid biogenesis is the main effect of DET1 loss of function.
Background:
As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism.
Methodology/principal findings:
In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals.
Conclusions:
The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.
The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.
The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A 'primary' intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational 'cleavage' products quantified. The processing products from our artificial polyprotein systems showed a molar excess of 'cleavage' product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and beta-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNA(Gly) ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal 'skip' from one codon to the next without the formation of a peptide bond.
Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig). Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence. Total fruit carotenoids of primary transformants (T(0)) were 2-4-fold higher than the controls, whereas phytoene, lycopene, beta-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively. The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels. The progeny (T(1) and T(2) generations) inherited both the transgene and phenotype. Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5-10-fold elevations in total phytoene synthase activity. Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway. The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.
To obtain a functioning provitamin A (beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single, combined transformation effort the cDNA coding for phytoene synthase (psy) and lycopene beta-cyclase (beta-lcy) both from Narcissus pseudonarcissus and both under the control of the endosperm-specific glutelin promoter together with a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control). This combination covers the requirements for beta-carotene synthesis and, as hoped, yellow beta-carotene-bearing rice endosperm was obtained in the T(0)-generation. Additional experiments revealed that the presence of beta-lcy was not necessary, because psy and crtI alone were able to drive beta-carotene synthesis as well as the formation of further downstream xanthophylls. Plausible explanations for this finding are that these downstream enzymes are constitutively expressed in rice endosperm or are induced by the transformation, e.g., by enzymatically formed products. Results using N. pseudonarcissus as a model system led to the development of a hypothesis, our present working model, that trans-lycopene or a trans-lycopene derivative acts as an inductor in a kind of feedback mechanism stimulating endogenous carotenogenic genes. Various institutional arrangements for disseminating Golden Rice to research institutes in developing countries also are discussed.
We applied a fusion of CRISPR-Cas9 and activation-induced cytidine deaminase (Target-AID) for point mutagenesis at genomic regions specified by single guide RNAs (sgRNAs) in two crop plants. In rice, we induced multiple herbicide-resistance point mutations by multiplexed editing using herbicide selection, while in tomato we generated marker-free plants with homozygous heritable DNA substitutions, demonstrating the feasibility of base editing for crop improvement. © 2017 Nature America, Inc., part of Springer Nature. All rights reserved.
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.
Genome editing with site-specific nucleases has become a powerful tool for functional characterization of plant genes and genetic improvement of agricultural crops. Among the various site-specific nuclease-based technologies available for genome editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) systems have shown the greatest potential for rapid and efficient editing of genomes in plant species. This article reviews the current status of application of CRISPR/Cas9 to plant genomics research, with a focus on loss-of-function and gain-of-function analysis of individual genes in the context of perennial plants and the potential application of CRISPR/Cas9 to perturbation of gene expression, and identification and analysis of gene modules as part of an accelerated domestication and synthetic biology effort.
Naturally occurring variation among wild relatives of cultivated crops is an under-exploited resource in plant breeding. Here, I argue that exotic libraries, which consist of marker-defined genomic regions taken from wild species and introgressed onto the background of elite crop lines, provide plant breeders with an important opportunity to improve the agricultural performance of modern crop varieties. These libraries can also act as reagents for the discovery and characterization of genes that underlie traits of agricultural value.
Various new plant breeding technique (NPBT) approaches have a similar aim, namely to produce improved crop varieties that are difficult to obtain through traditional breeding methods. Here we review the opportunities of products created using NPBTs. We categorize products of these NPBTs into three product classes with a different degree of genetic modification. For each product class recent examples are described to illustrate the potential for breeding new crops with improved traits. Finally, we touch upon future applications of these methods, such as cisgenic potato genotypes in which specific combinations of Phytophthora infestans resistance genes have been stacked for use in durable cultivation, or the creation of new disease resistances by knocking-out or removing S-genes using genome editing techniques.
The small bacterial-type genome of the plastid (chloroplast) can be engineered by genetic transformation, generating cells and plants with transgenic plastid genomes, also referred to as transplastomic plants. The transformation process relies on homologous recombination, thereby facilitating the site-specific alteration of endogenous plastid genes as well as the precisely targeted insertion of foreign genes into the plastid DNA. The technology has been used extensively to analyze chloroplast gene functions and study plastid gene expression at all levels in vivo. Over the years, a large toolbox has been assembled that is now nearly comparable to the techniques available for plant nuclear transformation and that has enabled new applications of transplastomic technology in basic and applied research. This review describes the state of the art in engineering the plastid genomes of algae and land plants (Embryophyta). It provides an overview of the existing tools for plastid genome engineering, discusses current technological limitations, and highlights selected applications that demonstrate the immense potential of chloroplast transformation in several key areas of plant biotechnology. Expected final online publication date for the Annual Review of Plant Biology Volume 66 is April 29, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
The advent of facile genome engineering using the bacterial RNA-guided CRISPR-Cas9 system in animals and plants is transforming biology. We review the history of CRISPR (clustered regularly interspaced palindromic repeat) biology from its initial discovery through the elucidation of the CRISPR-Cas9 enzyme mechanism, which has set the stage for remarkable developments using this technology to modify, regulate, or mark genomic loci in a wide variety of cells and organisms from all three domains of life. These results highlight a new era in which genomic manipulation is no longer a bottleneck to experiments, paving the way toward fundamental discoveries in biology, with applications in all branches of biotechnology, as well as strategies for human therapeutics.
Copyright © 2014, American Association for the Advancement of Science.
Golden rice could help to end a nutritional crisis [mdash] but only if researchers can overcome some daunting technical and political hurdles.
Allopolyploidization, the combination of the genomes from two different species, has been a major source of evolutionary innovation and a driver of speciation and environmental adaptation. In plants, it has also contributed greatly to crop domestication, as the superior properties of many modern crop plants were conferred by ancient allopolyploidization events. It is generally thought that allopolyploidization occurred through hybridization events between species, accompanied or followed by genome duplication. Although many allopolyploids arose from closely related species (congeners), there are also allopolyploid species that were formed from more distantly related progenitor species belonging to different genera or even different tribes. Here we have examined the possibility that allopolyploidization can also occur by asexual mechanisms. We show that upon grafting-a mechanism of plant-plant interaction that is widespread in nature-entire nuclear genomes can be transferred between plant cells. We provide direct evidence for this process resulting in speciation by creating a new allopolyploid plant species from a herbaceous species and a woody species in the nightshade family. The new species is fertile and produces fertile progeny. Our data highlight natural grafting as a potential asexual mechanism of speciation and also provide a method for the generation of novel allopolyploid crop species.
Xanthophylls, the pigments responsible for yellow to red coloration, are naturally occurring carotenoid compounds in many colored tissues of plants. These pigments are esterified within the chromoplast; however, little is known about the mechanisms underlying their accumulation in flower organs. In this study, we characterized two allelic tomato (Solanum lycopersicum L.) mutants, pale yellow petal (pyp) 1-1 and pyp1-2, that have reduced yellow color intensity in the petals and anthers due to loss-of-function mutations. Carotenoid analyses showed that the yellow flower organs of wild-type tomato contained high levels of xanthophylls that largely consisted of neoxanthin and violaxanthin esterified with myristic and/or palmitic acids. Functional disruption of PYP1 resulted in loss of xanthophyll esters, which was associated with a reduction in the total carotenoid content and disruption of normal chromoplast development. These findings suggest that xanthophyll esterification promotes the sequestration of carotenoids in the chromoplast and that accumulation of these esters is important for normal chromoplast development. Next-generation sequencing coupled with map-based positional cloning identified the mutant alleles responsible for the pyp1 phenotype. PYP1 most likely encodes a carotenoid modifying protein that plays a vital role in the production of xanthophyll esters in tomato anthers and petals. Our results provide insight into the molecular mechanism underlying the production of xanthophyll esters in higher plants, thereby shedding light on a longstanding mystery.This article is protected by copyright. All rights reserved.
Over 600 different carotenoids have been found in natural sources. These compounds are normal constituents in microorganisms, algae, and higher plants as well as a number of animal species. With the inherent carotenoid biosynthetic capability of natural sources, the commercial biosynthesis of these compounds by appropriate organisms is a way to produce carotenoids that cannot easily be synthesized or cannot be produced economically by chemical synthetic techniques. With the new power of biotechnology and recombinant DNA technology, the biosynthetic capability of organisms to produce carotenoids can be enhanced. This paper reviews previous and current efforts to produce carotenoids by biotechnology. In addition, the use of recombinant DNA technology to increase or modify the carotenoid biosynthetic capability of different organisms is discussed.
Measuring the extent to which individuals meet the 5 A Day dietary recommendation for fruits and vegetables can provide information on the effectiveness of public health efforts to increase consumption of these foods. However, dietary measurement is complicated by the issue of serving size. We compared two methods of measuring fruit and vegetable consumption using a random-digit-dialed telephone survey of 917 Washington state adults. The survey included two sets of questions about fruit and vegetable consumption, one providing and the other not providing standard definitions of serving size. The specific wording of questions had a large effect on the conclusions about levels of fruit and vegetable consumption. Although only 26% of respondents met the 5 A Day recommendations without serving size information, 50% met these recommendations when using a measure that included a definition of serving size.
Tomato plants transformed with a copy of the fruit-expressed phytoene synthase cDNA under control of the CaMV 35S promoter showed ectopic production of carotenoids. High expressers were reduced in stature. The dwarf character was inherited with an inverse relationship between expression of phytoene synthase and plant height. Severely affected plants also showed reduced chlorophyll content in young leaves. These dwarfs showed a 30-fold reduction in levels of gibberellin A1 (GA1) and growth was partially restored by treatment with exogenous GA3. Qualitative and quantitative changes in carotenoids were also found. It is proposed that the dwarf phenotype results from the over-production of phytoene synthase, which converts geranylgeranyl diphosphate to phytoene and thereby diverts this intermediate away from the gibberellin (GA) and phytol biosynthetic pathways.
The Or gene of cauliflower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is β-carotene, which can reach levels that are several hundred-fold higher than those in comparable wild-type tissues. The β-carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in flower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild-type tissues. Thus the unpigmented wild-type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidentified mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid-sequestering sheets.
Pollution of the aquatic environment occurs from many different sources including from oil refineries. Oil refinery effluents contain many different chemicals at different concentrations including ammonia, sulphides, phenol and hydrocarbons. The exact composition cannot however be generalised as it depends on the refinery and which units are in operation at any specific time. It is therefore difficult to predict what effects the effluent may have on the environment. Toxicity tests have shown that most refinery effluents are toxic but to varying extents. Some species are more sensitive and the toxicity may vary throughout the life cycle. Sublethal tests have found that not only can the effluents be lethal but also they can often have sublethal effects on growth and reproduction. Field studies have shown that oil refinery effluents often have an impact on the fauna, which is usually restricted to the area close to the outfall. The extent of the effect is dependent on the effluent composition, the outfall's position and the state of the recipient environment. It is possible to detect two effects that oil refinery effluent has on the environment. Firstly it has a toxic effect close to the outfall, which is seen by the absence of all or most species. Secondly there is an enrichment effect which can be distinguished as a peak in the abundance or biomass. These effects are not limited to just oil refinery effluents, which makes it difficult to distinguish the effects an oil refinery effluent has from other pollution sources. The discharge from oil refineries has reduced in quantity and toxicity over recent decades, allowing many impacted environments in estuaries and coasts to make a substantial recovery.
The genomes of DNA-containing cell organelles (mitochondria, chloroplasts) can be laterally transmitted between organisms, a process known as organelle capture. Organelle capture often occurs in the absence of detectable nuclear introgression, and the capture mechanism is unknown. Here, we have considered horizontal genome transfer across natural grafts as a mechanism underlying chloroplast capture in plants. By grafting sexually incompatible species, we show that complete chloroplast genomes can travel across the graft junction from one species into another. We demonstrate that, consistent with reported phylogenetic evidence, replacement of the resident plastid genome by the alien genome occurs in the absence of intergenomic recombination. Our results provide a plausible mechanism for organelle capture in plants and suggest natural grafting as a path for horizontal gene and genome transfer between sexually incompatible species.
Coordination of multiple transgenes is essential for metabolic engineering of biosynthetic pathways. Here, we report the utilization of two bicistronic systems involving the 2A sequence from the foot-and-mouth disease virus and the internal ribosome entry site (IRES) sequence from the crucifer-infecting tobamovirus to the biosynthesis of carotenoids in rice endosperm. Two carotenoid biosynthetic genes, phytoene synthase (Psy) from Capsicum and carotene desaturase (CrtI) from Pantoea, were linked via either the synthetic 2A sequence that was optimized for rice codons or the IRES sequence under control of the rice globulin promoter, generating PAC (Psy-2A-CrtI) and PIC (Psy-IRES-CrtI) constructs, respectively. The transgenic endosperm of PAC rice had a more intense golden color than did PIC rice, demonstrating that 2A was more efficient than IRES in coordinating gene expression. The 2A and IRES constructs were equally effective in driving transgene transcription. However, immunoblot analysis of CRTI, a protein encoded by the downstream open reading frame of the bicistronic constructs, revealed that 2A was ninefold more effective than IRES in driving translation. The PAC endosperms accumulated an average of 1.3 μg/g of total carotenoids, which was ninefold higher than was observed for PIC endosperms. In particular, accumulation of β-carotene was much higher in PAC endosperms than in PIC endosperms. Collectively, these results demonstrate that both 2A and IRES systems can coordinate the expression of two biosynthetic genes, with the 2A system exhibiting greater efficiency. Thus, the 2A expression system described herein is an effective new tool for multigene stacking in crop biotechnology.
Tissue grafting includes applications ranging from plant breeding to animal organ transplantation. Donor and recipient are
generally believed to maintain their genetic integrity, in that the grafted tissues are joined but their genetic materials
do not mix. We grafted tobacco plants from two transgenic lines carrying different marker and reporter genes in different
cellular compartments, the nucleus and the plastid. Analysis of the graft sites revealed the frequent occurrence of cells
harboring both antibiotic resistances and both fluorescent reporters. Our data demonstrate that plant grafting can result
in the exchange of genetic information via either large DNA pieces or entire plastid genomes. This observation of novel combinations
of genetic material has implications for grafting techniques and also provides a possible path for horizontal gene transfer.
