Plant Ascorbic Acid Biosynthesis Research Articles

Auxin and abscisic acid antagonistically regulate ascorbic acid production via the SlMAPK8-SlARF4-SlMYB11 module in tomato.

Ascorbic acid (AsA) is a multifunctional phytonutrient that is essential for the human diet as well as plant development. While much is known about AsA biosynthesis in plants, how this process is regulated in tomato (Solanum lycopersicum) fruits remains unclear. Here, we found that auxin treatment inhibited AsA accumulation in the leaves and pericarps of tomato. The auxin response factor gene SlARF4 is induced by auxin to mediate auxin-induced inhibition of AsA accumulation. Specifically, SlARF4 transcriptionally inhibits the transcription factor gene SlMYB11, thereby modulating AsA accumulation by regulating the transcription of the AsA biosynthesis genes l-galactose-1-phosphate phosphatase, l-galactono-1,4-lactone dehydrogenase, and dehydroascorbate. By contrast, abscisic acid (ABA) treatment increased AsA accumulation in tomato under drought stress. ABA induced the expression of the mitogen-activated protein kinase gene SlMAPK8. We demonstrate that SlMAPK8 phosphorylates SlARF4 and inhibits its transcriptional activity, whereas SlMAPK8 phosphorylates SlMYB11 and activates its transcriptional activity. SlMAPK8 functions in ABA-induced AsA accumulation and drought stress tolerance. Moreover, ABA antagonizes the effects of auxin on AsA biosynthesis. Therefore, auxin- and ABA-induced regulation of AsA accumulation is mediated by the SlMAPK8-SlARF4-SlMYB11 module in tomato during fruit development and drought stress responses, shedding light on the roles of phytohormones in regulating AsA accumulation to mediate stress tolerance.

Maize bHLH55 functions positively in salt tolerance through modulation of AsA biosynthesis by directly regulating GDP-mannose pathway genes

Ascorbic acid (AsA) is an antioxidant and enzyme co-factor that is vital to plant development and abiotic stress tolerance. However, the regulation mechanisms of AsA biosynthesis in plants remain poorly understood. Here, we report a basic helix-loop-helix 55 (ZmbHLH55) transcription factor that regulates AsA biosynthesis in maize. Analysis of publicly available transcriptomic data revealed that ZmbHLH55 is co-expressed with several genes of the GDP-mannose pathway. Experimental data showed that ZmbHLH55 forms homodimers localized to the cell nuclei, and it exhibits DNA binding and transactivation activity in yeast. Under salt stress conditions, knock down mutant (zmbhlh55) in maize accumulated lower levels of AsA compared with wild type, accompanied by lower antioxidant enzymes activity, shorter root length, and higher malondialdehyde (MDA) level. Gene expression data from the WT and zmbhlh55 mutant, showed that ZmbHLH55 positively regulates the expression of ZmPGI2, ZmGME1, and ZmGLDH, but negatively regulates ZmGMP1 and ZmGGP. Furthermore, ZmbHLH55-overexpressing Arabidopsis, under salt conditions, showed higher AsA levels, increased rates of germination, and elevated antioxidant enzyme activities. In conclusion, these results have identified previously unknown regulation mechanisms for AsA biosynthesis, indicating that ZmbHLH55 may be a potential candidate to enhance plant salt stress tolerance in the future.

Molecular Evolution of GDP-D-Mannose Epimerase (GME), a Key Gene in Plant Ascorbic Acid Biosynthesis.

The widespread ascorbic acid (AsA) plays a vital role in plant development and abiotic stress tolerance, but AsA concentration varies greatly among different plants. GDP-D-mannose epimerase (GME), which catalyzes GDP-D-mannose to GDP-L-galactose or GDP-L-gulose, is a key enzyme in plant AsA biosynthesis pathway. Functions and expression patterns of GME have been well studied in previous works, however, little information is known about the evolutionary patterns of the gene. In this study, GME gene structure, corresponding conserved protein motifs and evolutionary relationships were systematically analyzed. A total of 111 GME gene sequences were retrieved from 59 plant genomes, which representing almost all the major lineages of Viridiplantae: dicotyledons, monocotyledons, gymnosperms, pteridophytes, bryophytes, and chlorophytes. Results showed that homologs of GME were widely present in Viridiplantae. GME gene structures were conservative in higher plants, while varied greatly in the basal subgroups of the phylogeny including lycophytes, bryophytes, and chlorophytes, suggesting GME gene structure might have undergone severe differentiation at lower plant and then gradually fixed as plant evolution. The basic motifs of GME were strongly conserved throughout Viridiplantae, suggesting the conserved function of the protein. Molecular evolution analysis showed that strong purifying selection was the predominant force in the evolution of GME. A few branches and sites under episodic diversifying selection were identified and most of the branches located in the subgroup of chlorphytes, indicating episodic diversifying selection at a few branches and sites may play a role in the evolution of GME and diversifying selection may have occurred at the early stage of Viridiplantae. Our results provide novel insights into functional conservation and the evolution of GME.

Ascorbic Acid-A Potential Oxidant Scavenger and Its Role in Plant Development and Abiotic Stress Tolerance.

Over-production of reactive oxygen species (ROS) in plants under stress conditions is a common phenomenon. Plants tend to counter this problem through their ability to synthesize ROS neutralizing substances including non-enzymatic and enzymatic antioxidants. In this context, ascorbic acid (AsA) is one of the universal non-enzymatic antioxidants having substantial potential of not only scavenging ROS, but also modulating a number of fundamental functions in plants both under stress and non-stress conditions. In the present review, the role of AsA, its biosynthesis, and cross-talk with different hormones have been discussed comprehensively. Furthermore, the possible involvement of AsA-hormone crosstalk in the regulation of several key physiological and biochemical processes like seed germination, photosynthesis, floral induction, fruit expansion, ROS regulation and senescence has also been described. A simplified and schematic AsA biosynthetic pathway has been drawn, which reflects key intermediates involved therein. This could pave the way for future research to elucidate the modulation of plant AsA biosynthesis and subsequent responses to environmental stresses. Apart from discussing the role of different ascorbate peroxidase isoforms, the comparative role of two key enzymes, ascorbate peroxidase (APX) and ascorbate oxidase (AO) involved in AsA metabolism in plant cell apoplast is also discussed particularly focusing on oxidative stress perception and amplification. Limited progress has been made so far in terms of developing transgenics which could over-produce AsA. The prospects of generation of transgenics overexpressing AsA related genes and exogenous application of AsA have been discussed at length in the review.

Ascorbic Acid Accumulation is Transcriptionally Modulated in High-Pigment-1 Tomato Fruit

Ascorbic acid (AsA) is one of the most important antioxidants as well as being a growth regulator and enzymatic cofactor in plants. The biosynthesis of AsA in plants is well characterized while the regulation of its accumulation remains largely unexplored. In the present work, the AsA concentration throughout tomato fruit development and ripening was lower in High-Pigment-1 (hp1) with differential accumulation pattern as compared to wild-type (WT) plants. The transcriptional abundance of genes involved in AsA synthesis was also investigated during tomato fruit development. The result showed that transcriptional profiles of 14 genes involved in AsA biosynthesis were differentially regulated in hp1 fruits. The expression of genes encoding GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3′,5′-epimerase (GME1 and GME2) and l-galactose-1-phosphate phosphatase in hp1 was generally higher than that in WT, while the expression of gene encoding monodehydroascorbate reductase was lower than in WT. The expression abundance of GMP (P < 0.01) and GME1 (P < 0.05) were negatively correlated with AsA accumulation in WT tomato fruits. In the hp1 fruits, however, the expression level of gene encoding the last step enzyme, l-galactono-1,4-lactone dehydrogenase (GLDH), was negatively correlated with AsA accumulation (P < 0.05). Thus the reduced AsA accumulation in hp1 tomato fruits is associated with negative transcriptional regulation of GLDH, which offsets the increased expression of GMP and GME1. The probable mechanism of AsA down-regulation in hp1 fruit is discussed.

Engineering ascorbic acid biosynthetic pathway in Arabidopsis leaves by single and double gene transformation

Six genes, which encode enzymes involved in ascorbic acid (AsA) biosynthesis, including guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-galactose guanylyltransferase (GGT), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH) and L-galactono-1,4-lactone dehydrogenase (GLDH) were transformed into Arabidopsis thaliana, to evaluate the contribution of each gene to AsA accumulation. Additionally, two combinations, GGT-GPP and GGT-GLDH, were co-transformed into Arabidopsis with a reliable double-gene transformation system. AsA content of GGT transgenic lines was 2.9-fold higher as compared to the control, and co-transformation led up to 4.1-fold AsA enhancement. These results provided further evidence that GGT is the key enzyme in plant AsA biosynthesis.

A Temperature-sensitive Mutation in the Arabidopsis thaliana Phosphomannomutase Gene Disrupts Protein Glycosylation and Triggers Cell Death

Eukaryotic phosphomannomutases (PMMs) catalyze the interconversion of mannose 6-phosphate to mannose 1-phosphate and are essential to the biosynthesis of GDP-mannose. As such, plant PMMs are involved in ascorbic acid (AsA) biosynthesis and N-glycosylation. We report on the conditional phenotype of the temperature-sensitive Arabidopsis thaliana pmm-12 mutant. Mutant seedlings were phenotypically similar to wild type seedlings when grown at 16-18 degrees C but died within several days after transfer to 28 degrees C. This phenotype was observed throughout both vegetative and reproductive development. Protein extracts derived from pmm-12 plants had lower PMM protein and enzyme activity levels. In vitro biochemical analysis of recombinant proteins showed that the mutant PMM protein was compromised in its catalytic efficiency (K cat/K m). Despite significantly decreased AsA levels in pmm-12 plants, AsA deficiency could not account for the observed phenotype. Since, at restrictive temperature, total glycoprotein patterns were altered and glycosylation of protein-disulfide isomerase was perturbed, we propose that a deficiency in protein glycosylation is responsible for the observed cell death phenotype.

NAD +-specific d-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from d-arabinose by d-arabinose dehydrogenase (ARA) and d-arabinono-γ-lactone oxidase. We found an NAD +-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD +-specific ARA activity with K m = 0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP +-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD +-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.

Cloning, Expression, and Mapping of GDP-D-mannose Pyrophosphorylase cDNA from Tomato ( Lycopersicon esculentum)

GDP-D-mannose pyrophosphorylase (GMP, EC 2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant ascorbic acid biosynthesis. Using potato GMP cDNA sequence as a querying probe, 65 highly homologous tomato ESTs were obtained from dbEST of GenBank and the putative cDNA sequence of tomato GMP was assembled. The full-length GMP cDNA of tomato was cloned by RACE-PCR with primers designed according to the assembled cDNA sequence. The full-length cDNA sequence contained a complete open reading frame (ORF) of 1 086 bp, which encoded 361 amino acid residues. This gene was designated as LeGMP (GenBank accession No. AY605668). Homology analysis of LeGMP showed a 96% identity with potato GMP and the deduced amino acid showed 99%, 97%, 91% and 89% homology with GMP from potato, tobacco, alfalfa and Arabidopsis thaliana, respectively. Northern blot analysis showed that LeGMP was constitutively expressed in roots, stems, leaves, flowers and fruits of tomato; but the expression levels varied. LeGMP was mapped to 3-D using 75 tomato introgression lines (ILs), each containing a single homozygous RFLP-defined chromosome segment from the green-fruited species Lycopersicon pennellii.

Ascorbic acid biosynthesis: a precursor study on plants

Since the first isolation of ascorbic acid (AsA) in 1928, few papers have been published regarding the biosynthesis of AsA in plants, especially in fruits. It took as long as 1998, before Wheeler, Jones and Smirnoff, based on a study with Arabidopsis leaves, proposed what can be considered the main pathway of biosynthesis of AsA, in which L-galactose (L-GAL) is a key precursor. This paper reports the effectiveness of some precursors (cold or radiolabeled) in the biosynthesis of AsA in different plants: green sweet pepper, white-pulp guava, red-pulp guava, papaya and strawberry at two ripening stages (mature green and ripe for papaya and mature green and half red for strawberry) and broccoli. The 'Smirnoff-Wheeler' pathway was functioning and active in all sources studied, as demonstrated by the increase in AsA contents and incorporation of labeled precursors into AsA. In papaya, the AsA content in the ripe fruit was higher than in the mature green, indicating the synthesis of AsA during ripening. On the other hand, the AsA content in the mature green strawberry was similar to that of the half red fruits. Our data demonstrate that L-GAL and L-Galactono-1,4-lactone (L-GL) are effective precursors for the biosynthesis of AsA in fruits and also provided additional evidence for the participation of D-mannose (D-MAN) and D-glucose-1P in the biosynthesis of AsA in plants.

BIOSYNTHESIS OF ASCORBIC ACID IN PLANTS: A Renaissance.

The structure of the familiar antioxidant L-ascorbic acid (vitamin C) was described in 1933 yet remarkably, its biosynthesis in plants remained elusive until only recently. It became clear from radioisotopic labeling studies in the 1950s that plant ascorbic acid biosynthesis does not proceed in toto via a route similar to that in mammals. The description in 1996 of an Arabidopsis thaliana mutant deficient in ascorbic acid prompted renewed research effort in this area, and subsequently in 1998 a new pathway was discovered that is backed by strong biochemical and molecular genetic evidence. This pathway proceeds through the intermediates GDP-D-mannose, L-galactose, and L-galactono-1,4-lactone. Much research has focused on the properties of the terminal enzyme responsible for conversion of the aldonolactone to ascorbate, and on related enzymes in both mammals and fungi. Two of the plant biosynthetic genes have been studied at the molecular level and additional ascorbate-deficient A. thaliana mutants may hold the key to other proteins involved in plant ascorbate metabolism. An analysis of the biosynthesis of ascorbate and its analogues in algae and fungi as well as the study of alternative proposed pathways should broaden our understanding of ascorbate metabolism in plants. With a biosynthetic pathway in hand, research on areas such as the control of ascorbate biosynthesis and the physiological roles of ascorbate should progress rapidly.

Metabolic engineering of an alternative pathway for ascorbic acid biosynthesis in plants

Plants and most animals can synthesize their own L-ascorbic acid (vitamin C), but a mutation in the L-gulono-γ-lactone oxidase gene in the primate lineage makes it necessary for humans to acquire this vital compound from their diet. Despite the fact that plants and animals synthesize ascorbic acid via different pathways, transgenic tobacco and lettuce plants expressing a rat cDNA encoding L-gulono-γ-lactone oxidase accumulated up to seven times more ascorbic acid than untransformed plants. These results demonstrate that basal levels of ascorbic acid in plants can be significantly increased by expressing a single gene from the animal pathway.