Metabolic Engineering of Plant L-Ascorbic Acid Biosynthesis: Recent Trends and Applications

ABSTRACT

Vitamin C (L-ascorbic acid; AsA) is the major soluble antioxidant found in plants and is also an essential component of human nutrition. Although numerous biotechnological methods have been exploited to increase its yield, pressures such as commercial competition and environmental concerns make it urgent to find a new way for industrial production of plant-derived AsA. Engineering plant AsA has now become feasible because of our increased understanding of its biosynthetic pathway. Several possible strategies could be followed to increase AsA production, such as overcoming the rate limiting steps in the biosynthetic pathway, promoting recycling, and reducing catabolism. For these purposes, genes of plant, microbial and animal origins have been successfully used. Several examples will be given to illustrate these various approaches. The existing and potential achievements in increasing AsA production would provide the opportunity for enhancing nutritional quality and stress tolerance of crop plants.     

Biosynthesis and Catabolism of L-Ascorbic Acid in Plants

L-Ascorbic acid (AsA) is a vital antioxidant compound that plays a critical role in the cellular metabolism of plants and animals. Research on plant AsA metabolism experienced a significant resurgence after 1998 following the identification of AsA-deficient Arabidopsis mutants and the elucidation of a biosynthetic pathway accepted by the overwhelming majority of the plant science community. The identification and cloning of novel biosynthetic genes and the ensuing metabolic engineering of plant AsA content has however revealed a more complex picture. Additional biosynthetic routes have been identified and unexpected biochemical phenotypes were observed upon expression of animal AsA biosynthetic genes. The isolation of novel AsA conjugates from plant tissues and the evidence for long distance transport of AsA in plants have provided additional facets to its functionality. Although some progress has been made regarding the impact of AsA recycling on pool size, we still do not have a clear picture of the biochemistry of AsA degradation. This communication comprehensively reviews new developments in the AsA metabolic system and prompts directions for future research.     

Metal/metalloid stress tolerance in plants: role of ascorbate,its redox couple,and associated enzymes

The enhanced generation of reactive oxygen species (ROS) under metal/metalloid stress is most common in plants, and the elevated ROS must be successfully metabolized in order to maintain plant growth, development, and productivity. Ascorbate (AsA) is a highly abundant metabolite and a water-soluble antioxidant, which besides positively influencing various aspects in plants acts also as an enigmatic component of plant defense armory. As a significant component of the ascorbate-glutathione (AsA-GSH) pathway, it performs multiple vital functions in plants including growth and development by either directly or indirectly metabolizing ROS and its products. Enzymes such as monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) and dehydroascorbate reductase (DHAR, EC 1.8.5.1) maintain the reduced form of AsA pool besides metabolically controlling the ratio of AsA with its oxidized form (dehydroascorbate, DHA). Ascorbate peroxidase (APX, EC 1.11.1.11) utilizes the reduced AsA pool as the specific electron donor during ROS metabolism. Thus, AsA, its redox couple (AsA/DHA), and related enzymes (MDHAR, DHAR, and APX) cumulatively form an AsA redox system to efficiently protect plants particularly against potential anomalies caused by ROS and its products. Here we present a critical assessment of the recent research reports available on metal/metalloid-accrued modulation of reduced AsA pool, AsA/DHA redox couple and AsA-related major enzymes, and the cumulative significance of these antioxidant system components in plant metal/metalloid stress tolerance.     

Analysis of GDP-D-mannose pyrophosphorylase gene promoter from acerola (Malpighia glabra) and increase in ascorbate content of transgenic tobacco expressing the acerola gene

GDP-D-mannose pyrophosphorylase (GMP) is an important enzyme in the Smirnoff-Wheeler's pathway for the biosynthesis of ascorbic acid (AsA) in plants. We have reported recently that the expression of the acerola (Malpighia glabra) GMP gene, designated MgGMP, correlates with the AsA content of the plant. The acerola plant has very high levels of AsA relative to better studied model plants such as Arabidopsis. Here we found that the GMP mRNA levels in acerola are higher than those from Arabidopsis and tomato. Also, the transient expression of the uidA reporter gene in the protoplasts of Nicotiana tabacum cultures showed the MgGMP gene promoter to have higher activity than the cauliflower mosaic virus 35S and Arabidopsis GMP promoters. The AsA content of transgenic tobacco plants expressing the MgGMP gene including its promoter was about 2-fold higher than that of the wild type.     

Recent advances in the role and biosynthesis of ascorbic acid in plants

The past few years have provided many advances in the role and biosynthesis of L -ascorbic acid (AsA) in plants. There is an increasing body of evidence confirming that AsA plays an important role in the detoxification of reactive oxygen species. The role of AsA in photoprotection has been confirmed in vivo with the use of Arabidopsis mutants. A player in the defence against reactive oxygen species, AsA peroxidase, has been extensively studied at the molecular level, and regulation of this key enzymatic activity appears to occur at several levels. As a cofactor in the hydroxylation of prolyl and lysl-residues by peptidyl-prolyl and -lysyl hydroxylases, AsA plays a part in cell wall synthesis, defence, and possibly cell division. The maintenance of reduced levels of AsA appears to be highly regulated, involving the interplay of both monodehydroascorbate and dehydroascorbate reductases and possibly auxin. A major breakthrough in plant AsA biosynthesis has been made recently, and strong biochemical and genetic evidence suggest that GDP-mannose and L -galactose are key substrates. In addition, evidence for an alternative AsA biosynthetic pathway(s) exists and awaits additional scrutiny. Finally, newly described Arabidopsis mutants deficient in AsA will further increase our understanding of AsA biosynthesis     

Lignin: genetic engineering and impact on pulping

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.     

Lignin: Genetic Engineering and Impact on Pulping

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.     

myo-inositol oxygenase offers a possible entry point into plant ascorbate biosynthesis

Two biosynthetic pathways for ascorbate (l-ascorbic acid [AsA]; vitamin C) in plants are presently known, the mannose/l-galactose pathway and an l-GalUA pathway. Here, we present molecular and biochemical evidence for a possible biosynthetic route using myo-inositol (MI) as the initial substrate. A MI oxygenase (MIOX) gene was identified in chromosome 4 (miox4) of Arabidopsis ecotype Columbia, and its enzymatic activity was confirmed in bacterially expressed recombinant protein. Miox4 was primarily expressed in flowers and leaves of wild-type Arabidopsis plants, tissues with a high concentration of AsA. Ascorbate levels increased 2- to 3-fold in homozygous Arabidopsis lines overexpressing the miox4 open reading frame, thus suggesting the role of MI in AsA biosynthesis and the potential for using this gene for the agronomic and nutritional enhancement of crops.     

Metabolic engineering in food crops to enhance ascorbic acid production: crop biofortification perspectives for human health

Ascorbic acid (AsA) also known as vitamin C is considered as an essential micronutrient in the diet of humans. The human body is unable to synthesize AsA, thus solely dependent on exogenous sources to accomplish the nutritional requirement. AsA plays a crucial role in different physiological aspects of human health like bone formation, iron absorption, maintenance and development of connective tissues, conversion of cholesterol to bile acid and production of serotonin. It carries antioxidant properties and is involved in curing various clinical disorders such as scurvy, viral infection, neurodegenerative diseases, cardiovascular diseases, anemia, and diabetes. It also plays a significant role in COVID-19 prevention and recovery by improving the oxygen index and enhancing the production of natural killer cells and T-lymphocytes. In plants, AsA plays important role in floral induction, seed germination, senescence, ROS regulation and photosynthesis. AsA is an essential counterpart of the antioxidant system and helps to defend the plants against abiotic and biotic stresses. Surprisingly, the deficiencies of AsA are spreading in both developed and developing countries. The amount of AsA in the major food crops such as wheat, rice, maize, and other raw natural plant foods is inadequate to fulfill its dietary requirements. Hence, the biofortification of AsA in staple crops would be feasible and cost-effective means of delivering AsA to populations that may have limited access to diverse diets and other interventions. In this review, we endeavor to provide information on the role of AsA in plants and human health, and also perused various biotechnological and agronomical approaches for elevating AsA content in food crops.     

Gene expression of ascorbic acid biosynthesis related enzymes of the Smirnoff-Wheeler pathway in acerola (Malpighia glabra)

The Smirnoff-Wheeler (SW) pathway has been proven to be the only significant source of l-ascorbic acid (AsA; vitamin C) in the seedlings of the model plant Arabidopsis thaliana. It is yet uncertain whether the same pathway holds for all other plants and their various organs as AsA may also be synthesized through alternative pathways. In this study, we have cloned some of the genes involved in the SW-pathway from acerola (Malpighia glabra), a plant containing enormous amount of AsA, and examined the expression patterns of these genes in the plant. The AsA contents of acerola leaves were about 8-fold more than that of Arabidopsis with 5-700-fold higher mRNA abundance in AsA-biosynthesizing genes. The unripe fruits have the highest AsA content but the accumulation was substantially repressed as the fruit transitions to maturation. The mRNAs encoding these genes showed correlation in their expression with the AsA contents of the fruits. Although very little AsA was recorded in the seeds the mRNAs encoding all the genes, with the exception of the mitochondrially located L-galactono-1,4-lactone dehydrogenase, were clearly detected in the seeds of the unripe fruits. In young leaves of acerola, the expression of most genes were repressed by the dark and induced by light. However, the expression of GDP-D-mannose pyrophosphorylase similar to that encoded by A. thaliana VTC1 was induced in the dark. The expressions of all the genes surged after 24h following wounding stress on the young leaves. These findings will advance the investigation into the molecular factors regulating the biosynthesis of abundant AsA in acerola.     

L-Ascorbic acid is accumulated in source leaf phloem and transported to sink tissues in plants

L-Ascorbic acid (AsA) was found to be loaded into phloem of source leaves and transported to sink tissues. When L-[(14)C]AsA was applied to leaves of intact plants of three different species, autoradiographs and HPLC analysis demonstrated that AsA was accumulated into phloem and transported to root tips, shoots, and floral organs, but not to mature leaves. AsA was also directly detected in Arabidopsis sieve tube sap collected from an English green aphid (Sitobion avenae) stylet. Feeding a single leaf of intact Arabidopsis or Medicago sativa with 10 or 20 mM L-galactono-1,4-lactone (GAL-L), the immediate precursor of AsA, lead to a 7- to 8-fold increase in AsA in the treated leaf and a 2- to 3-fold increase of AsA in untreated sink tissues of the same plant. The amount of AsA produced in treated leaves and accumulated in sink tissues was proportional to the amount of GAL-L applied. Studies of the ability of organs to produce AsA from GAL-L showed mature leaves have a 3- to 10-fold higher biosynthetic capacity and much lower AsA turnover rate than sink tissues. The results indicate AsA transporters reside in the phloem, and that AsA translocation is likely required to meet AsA demands of rapidly growing non-photosynthetic tissues. This study also demonstrates that source leaf AsA biosynthesis is limited by substrate availability rather than biosynthetic capacity, and sink AsA levels may be limited to some extent by source production. Phloem translocation of AsA may be one factor regulating sink development because AsA is critical to cell division/growth.     

Feedback inhibition of spinach L-galactose dehydrogenase by L-ascorbate

We have studied the enzymological properties of L-galactose dehydrogenase (l-GalDH), a key enzyme in the biosynthetic pathway of l-ascorbate (AsA) in plants. L-GalDH was purified approximately 560-fold from spinach leaves. The enzyme was a homodimer with a subunit mass of 36 kDa. We also cloned the full-length cDNA of spinach L-GalDH, which contained an open reading frame encoding 322 amino acid residues with a calculated molecular mass of 35,261 Da. The deduced amino acid sequence of the cDNA showed 82, 79 and 75% homology to L-GalDH from kiwifruit, apple and Arabidopsis, respectively. Recombinant enzyme expressed from the cDNA in Escherichia coli showed L-GalDH activity. Southern blot analysis revealed that the spinach L-GalDH gene occurs in a single copy. Northern blot analysis suggests that L-GalDH is expressed in different organs of spinach. The purified native L-GalDH showed high specificity for L-galactose with a Km of 116.2+/-3.2 microM. Interestingly, spinach L-GalDH exhibited reversible inhibition by AsA, the end-product of the biosynthetic pathway. The inhibition kinetics indicated a linear-competitive inhibition with a Ki of 133.2+/-7.2 microM, suggesting feedback regulation in AsA synthesis in the plant.     

L-Ascorbic acid and L-galactose are sources for oxalic acid and calcium oxalate in Pistia stratiotes

Axenic Pistia stratiotes L. plants were pulse-chase labeled with [14C]oxalic acid, L[1-14C]ascorbic acid, L-6-14C]ascorbic acid, D-[1-14C]erythorbic acid, L-[1-14C]galactose, or [1-14C]glycolate. Specific radioactivities of L-ascorbic acid (AsA), free oxalic acid (OxA) and calcium oxalate (CaOx) in labeled plants were compared. Samples of leaf tissue were fixed for microautoradiography and examined by confocal microscopy. Results demonstrate a biosynthetic role for AsA as precursor of OxA and its crystalline deposition product, CaOx, in idioblast cells of P. stratiotes and support the recent discovery of Wheeler, Jones and Smirnoff (Wheeler, G.L., Jones M.A., & Smirnoff, N. (1998). The biosynthetic pathway of vitamin C in higher plants. Nature, 393, 365-369) that L-galactose is a key intermediate in the conversion of D-glucose to AsA in plants. D-[1-14C]erythorbic acid (a diastereomeric analog of AsA) is utilized also by P. stratiotes as a precursor of OxA and its calcium salt deposition product in idioblasts. Labeled OxA is rapidly incorporated into CaOx in idioblasts, but microautoradiography shows there is also significant incorporation of carbon from OxA into other components of growing cells, contrary to the dogma that OxA is a relatively stable end product of metabolism. Glycolate is a poor substrate for synthesis of OxA and CaOx formation, further establishing AsA as th immediate precursor in the synthesis of OxA used for calcium precipitation in crystal idioblasts.     

Suppression of tomato <Emphasis Type="Italic">SlGGP</Emphasis> aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.     

Metabolic engineering of plants for alkaloid production.

Alkaloids purified from plants provide many pharmacologically active compounds, including leading chemotherapy drugs. As is generally true of secondary metabolites, overall productivity is low, making commercial production expensive. Alternative production methods remain impractical, leaving the plant as the best source for these valuable chemicals. Recently, significant progress in characterizing the biosynthetic pathways leading to various alkaloids has been made, and a number of relevant genes have been cloned. Metabolic engineering employing such genes provides a promising technology for improved productivity in plant cell cultures, plant tissue cultures, or intact plants. In exploring solutions though, metabolic engineers must be careful to recognize the limitations inherent in designing plant systems.     

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.     

Deciphering Ascorbic Acid Regulatory Pathways in Ripening Tomato Fruit Using a Weighted Gene Correlation Network Analysis Approach

Genotype is generally determined by the co-expression of diverse genes and multiple regulatory pathways in plants. Gene co-expression analysis combining with physiological trait data provides very important information about the gene function and regulatory mechanism. L-Ascorbic acid （AsA）, which is an essential nutrient component for human health and plant metabolism, plays key roles in diverse biological processes such as cell cycle, cell expansion, stress resistance, hormone synthesis, and signaling. Here, we applied a weighted gene correlation network analysis approach based on gene expression values and AsA content data in ripening tomato （Solanum lycopersicum L.） fruit with different AsA content levels, which leads to identification of AsA relevant modules and vital genes in AsA regulatory pathways. Twenty- four modules were compartmentalized according to gene expression profiling. Among these modules, one negatively related module containing genes involved in redox processes and one positively related module enriched with genes involved in AsA biosynthetic and recycling pathways were further analyzed. The present work herein indicates that redox pathways as well as hormone-signal pathways are closely correlated with AsA accumulation in ripening tomato fruit, and allowed us to prioritize candidate genes for follow-up studies to dissect this interplay at the biochemical and molecular level.     

An Inhibitor of Tryptophan-Dependent Biosynthesis of Indole-3-Acetic Acid Alters Seedling Development in Arabidopsis

Although polar transport and the TIR1-dependent signaling pathway of the plant hormone auxin/indole-3-acetic acid (IAA) are well characterized, understanding of the biosynthetic pathway(s) leading to the production of IAA is still limited. Genetic dissection of IAA biosynthetic pathways has been complicated by the metabolic redundancy caused by the apparent existence of several parallel biosynthetic routes leading to IAA production. Valuable complementary tools for genetic as well as biochemical analysis of auxin biosynthesis would be molecular inhibitors capable of acting in vivo on specific or general components of the pathway(s), which unfortunately have been lacking. Several indole derivatives have been previously identified to inhibit tryptophan-dependent IAA biosynthesis in an in vitro system from maize endosperm. We examined the effect of one of them, 6-fluoroindole, on seedling development of Arabidopsis thaliana and tested its ability to inhibit IAA biosynthesis in feeding experiments in vivo. We demonstrated a correlation of severe developmental defects or growth retardation caused by 6-fluoroindole with significant downregulation of de novo synthesized IAA levels, derived from the stable isotope-labeled tryptophan pool, upon treatment. Hence, 6-fluoroindole shows important features of an inhibitor of tryptophan-dependent IAA biosynthesis both in vitro and in vivo and thus may find use as a promising molecular tool for the identification of novel components of the auxin biosynthetic pathway(s).     

The Arabidopsis YUCCA1 flavin monooxygenase functions in the indole-3-pyruvic acid branch of auxin biosynthesis

The effects of auxins on plant growth and development have been known for more than 100 years, yet our understanding of how plants synthesize this essential plant hormone is still fragmentary at best. Gene loss- and gain-of-function studies have conclusively implicated three gene families, CYTOCHROME P450 79B2/B3 (CYP79B2/B3), YUCCA (YUC), and TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1/TRYPTOPHAN AMINOTRANSFERASE-RELATED (TAA1/TAR), in the production of this hormone in the reference plant Arabidopsis thaliana. Each of these three gene families is believed to represent independent routes of auxin biosynthesis. Using a combination of pharmacological, genetic, and biochemical approaches, we examined the possible relationships between the auxin biosynthetic pathways defined by these three gene families. Our findings clearly indicate that TAA1/TARs and YUCs function in a common linear biosynthetic pathway that is genetically distinct from the CYP79B2/B3 route. In the redefined TAA1-YUC auxin biosynthetic pathway, TAA1/TARs are required for the production of indole-3-pyruvic acid (IPyA) from Trp, whereas YUCs are likely to function downstream. These results, together with the extensive genetic analysis of four pyruvate decarboxylases, the putative downstream components of the TAA1 pathway, strongly suggest that the enzymatic reactions involved in indole-3-acetic acid (IAA) production via IPyA are different than those previously postulated, and a new and testable model for how IAA is produced in plants is needed.