GDP-L-galactose Phosphorylase Research Articles

Characterization of the GGP gene family in potato (Solanum tuberosum L.) and Pepper (Capsicum annuum L.) and its expression analysis under hormonal and abiotic stresses

GDP-l-galactose phosphorylase (GGP) is a key rate-limiting enzyme in plant ascorbic acid synthesis, which plays an important role in plant growth and development as well as stress response. However, the presence of GGP and its function in potato and pepper are not known. In this study, we first identified two GGP genes in each potato and pepper genomes using a genome-wide search approach. We then analyzed their physicochemical properties, conserved domains, protein structures and phylogenetic relationships. Phylogenetic tree analysis revealed that members of the potato and pepper GGP gene families are related to eggplant (Solanum melongena L.), Arabidopsis (Arabidopsis thaliana L.), tobacco (Nicotiana tabacum L.) and tomato (Solanum lycopersicum L.), with tomato being the most closely related. The promoter sequences mainly contain homeopathic elements such as light-responsive, hormone-responsive and stress-responsive, with light-responsive elements being the most abundant. By analyzing the structure of the genes, it was found that there is no transmembrane structure or signal peptide in the GGP gene family of potatoes and peppers, and that all of its members are hydrophilic proteins. The expression profiles of different tissues show that StGGP1 has the highest expression levels in leaves, StGGP2 has the highest expression levels in stamens, and CaGGPs have the highest expression levels in the early stages of fruit development (Dev1). It was found that StGGPs and CaGGPs genes showed different response to phytohormones and abiotic stresses. Abscisic acid (ABA) treatment induced the most significant change in the expression of StGGPs, while the expression of CaGGPs showed the most pronounced change under methyl jasmonate (MeJA) treatment. StGGPs responded mainly to dark treatment, whereas CaGGPs responded mainly to NaCl stress. These results provide an important basis for a detailed study about the functions of GGP homologous genes in potato and pepper in response to abiotic stresses.

Deciphering the Genomic Characterization of the GGP Gene Family and Expression Verification of CmGGP1 Modulating Ascorbic Acid Biosynthesis in Melon Plants.

Ascorbic acid (AsA), also known as vitamin C, is a well-known antioxidant found in living entities that plays an essential role in growth and development, as well as in defensive mechanisms. GDP-L-galactose phosphorylase (GGP) is a candidate gene regulating AsA biosynthesis at the translational and transcriptional levels in plants. In the current study, we conducted genome-wide bioinformatic analysis and pinpointed a single AsA synthesis rate-limiting enzyme gene in melon (CmGGP1). The protein prediction analysis depicted that the CmGGP1 protein does not have a signaling peptide or transmembrane structure and mainly functions in the chloroplast or nucleus. The constructed phylogenetic tree analysis in multispecies showed that the CmGGP1 protein has a highly conserved motif in cucurbit crops. The structural variation analysis of the CmGGP1 gene in different domesticated melon germplasms showed a single non-synonymous type-base mutation and indicated that this gene was selected by domestication during evolution. Wild-type (WT) and landrace (LDR) germplasms of melon depicted close relationships to each other, and improved-type (IMP) varieties showed modern domestication selection. The endogenous quantification of AsA content in both the young and old leaves of nine melon varieties exhibited the major differentiations for AsA synthesis and metabolism. The real-time quantitative polymerase chain reaction (qRT-PCR) analysis of gene co-expression showed that AsA biosynthesis in leaves was greater than AsA metabolic consumption, and four putative interactive genes (MELO3C025552.2, MELO3C007440.2, MELO3C023324.2, and MELO3C018576.2) associated with the CmGGP1 gene were revealed. Meanwhile, the CmGGP1 gene expression pattern was noticed to be up-regulated to varying degrees in different acclimated melons. We believe that the obtained results would provide useful insights for an in-depth genetic understanding of the AsA biosynthesis mechanism, aimed at the development of improving crop plants for melon.

Multi-regulated GDP-l-galactose phosphorylase calls the tune in ascorbate biosynthesis.

Ascorbate is involved in numerous vital processes, in particular in response to abiotic but also biotic stresses whose frequency and amplitude increase with climate change. Ascorbate levels vary greatly depending on species, tissues, or stages of development, but also in response to stress. Since its discovery, the ascorbate biosynthetic pathway has been intensely studied and it appears that GDP-l-galactose phosphorylase (GGP) is the enzyme with the greatest role in the control of ascorbate biosynthesis. Like other enzymes of this pathway, its expression is induced by various environmental and also developmental factors. Although mRNAs encoding it are among the most abundant in the transcriptome, the protein is only present in very small quantities. In fact, GGP translation is repressed by a negative feedback mechanism involving a small open reading frame located upstream of the coding sequence (uORF). Moreover, its activity is inhibited by a PAS/LOV type photoreceptor, the action of which is counteracted by blue light. Consequently, this multi-level regulation of GGP would allow fine control of ascorbate synthesis. Indeed, experiments varying the expression of GGP have shown that it plays a central role in response to stress. This new understanding will be useful for developing varieties adapted to future environmental conditions.

The ascorbate biosynthesis pathway in plants is known, but there is a way to go with understanding control and functions.

Ascorbate (vitamin C) is one of the most abundant primary metabolites in plants. Its complex chemistry enables it to function as an antioxidant, as a free radical scavenger, and as a reductant for iron and copper. Ascorbate biosynthesis occurs via the mannose/l-galactose pathway in green plants, and the evidence for this pathway being the major route is reviewed. Ascorbate accumulation is leaves is responsive to light, reflecting various roles in photoprotection. GDP-l-galactose phosphorylase (GGP) is the first dedicated step in the pathway and is important in controlling ascorbate synthesis. Its expression is determined by a combination of transcription and translation. Translation is controlled by an upstream open reading frame (uORF) which blocks translation of the main GGP-coding sequence, possibly in an ascorbate-dependent manner. GGP associates with a PAS-LOV protein, inhibiting its activity, and dissociation is induced by blue light. While low ascorbate mutants are susceptible to oxidative stress, they grow nearly normally. In contrast, mutants lacking ascorbate do not grow unless rescued by supplementation. Further research should investigate possible basal functions of ascorbate in severely deficient plants involving prevention of iron overoxidation in 2-oxoglutarate-dependent dioxygenases and iron mobilization during seed development and germination.

Blue light promotes ascorbate synthesis by deactivating the PAS/LOV photoreceptor that inhibits GDP-L-galactose phosphorylase.

Ascorbate (vitamin C) is an essential antioxidant in fresh fruits and vegetables. To gain insight into the regulation of ascorbate metabolism in plants, we studied mutant tomato plants (Solanum lycopersicum) that produce ascorbate-enriched fruits. The causal mutation, identified by a mapping-by-sequencing strategy, corresponded to a knock-out recessive mutation in a class of photoreceptor named PAS/LOV protein (PLP), which acts as a negative regulator of ascorbate biosynthesis. This trait was confirmed by CRISPR/Cas9 gene editing and further found in all plant organs, including fruit that accumulated 2 to 3 times more ascorbate than in the WT. The functional characterization revealed that PLP interacted with the 2 isoforms of GDP-L-galactose phosphorylase (GGP), known as the controlling step of the L-galactose pathway of ascorbate synthesis. The interaction with GGP occurred in the cytoplasm and the nucleus, but was abolished when PLP was truncated. These results were confirmed by a synthetic approach using an animal cell system, which additionally demonstrated that blue light modulated the PLP-GGP interaction. Assays performed in vitro with heterologously expressed GGP and PLP showed that PLP is a noncompetitive inhibitor of GGP that is inactivated after blue light exposure. This discovery provides a greater understanding of the light-dependent regulation of ascorbate metabolism in plants.

Overexpression of banana GDP-L-galactose phosphorylase (GGP) modulates the biosynthesis of ascorbic acid in Arabidopsis thaliana

l-Ascorbic acid (AsA) is a potent antioxidant and essential micronutrient for the growth and development of plants and animals. AsA is predominantly synthesized by the Smirnoff-Wheeler (SW) pathway in plants where the GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting step. In the present study, AsA was estimated in twelve banana cultivars, where Nendran carried the highest (17.2 mg/100 g) amount of AsA in ripe fruit pulp. Five GGP genes were identified from the banana genome database, and they were located at chromosome 6 (4 MaGGPs) and chromosome 10 (1 MaGGP). Based on in-silico analysis, three potential MaGGP genes were isolated from the cultivar Nendran and subsequently overexpressed in Arabidopsis thaliana. Significant enhancement in AsA (1.52 to 2.20 fold) level was noted in the leaves of all three MaGGPs overexpressing lines as compared to non-transformed control plants. Among all, MaGGP2 emerged as a potential candidate for AsA biofortification in plants. Further, the complementation assay of Arabidopsis thaliana vtc-5-1 and vtc-5-2 mutants with MaGGP genes overcome the AsA deficiency that showed improved plant growth as compared to non-transformed control plants. This study lends strong affirmation towards development of AsA biofortified plants, particularly the staples that sustain the personages in developing countries.

Over-expression of GGP1 and GPP genes enhances ascorbate content and nutritional quality of tomato

L-Ascorbic acid (AsA), a strong antioxidant, serves as an enzyme cofactor and redox status marker, modulating a plethora of biological processes. As tomato commercial varieties and hybrids possess relatively low amounts of AsA, the improvement of fruit AsA represents a strategic goal for enhanced human health. Previously, we have suggested that GDP-L-Galactose phosphorylase (GGP) and L-galactose-1-phosphate phosphatase (GPP) can serve as possible targets for AsA manipulation in tomato (Solanum lycopersicon L.) fruit. To this end, we produced and evaluated T3 transgenic tomato plants carrying these two genes under the control of CaMV-35S and two fruit specific promoters, PPC2 and PG-GGPI. The transgenic lines had elevated levels of AsA, with the PG-GGP1 line containing 3-fold more AsA than WT, without affecting fruit characteristics. Following RNA-Seq analysis, 164 and 13 DEGs were up- or down-regulated, respectively, between PG-GGP1 and WT pink fruits. PG-GGP1 fruit had a distinct number of up-regulated transcripts associated with cell wall modification, ethylene biosynthesis and signaling, pollen fertility and carotenoid metabolism. The elevated AsA accumulation resulted in the up regulation of AsA associated transcripts and alternative biosynthetic pathways suggesting that the entire metabolic pathway was influenced, probably via master regulation. We show here that AsA-fortification of tomato ripe fruit via GGP1 overexpression under the action of a fruit specific promoter PG affects fruit development and ripening, reduces ethylene production, and increased the levels of sugars, and carotenoids, supporting a robust database to further explore the role of AsA induced genes for agronomically important traits, breeding programs and precision gene editing approaches.

How does light facilitate vitamin C biosynthesis in leaves?

Plants store ascorbate in high concentrations, particularly in their leaves. Ascorbate is an excellent antioxidant that acts as an indispensable photoprotectant. The d-mannose/l-galactose pathway is responsible for ascorbate biosynthesis in plants. Light facilitates ascorbate biosynthesis in a light intensity-dependent manner to enhance ascorbate pool size in leaves, and photosynthesis is required for this process. Light- and photosynthesis-dependent activation of the rate-limiting enzyme GDP-l-galactose phosphorylase (GGP) plays a critical role in ascorbate pool size regulation. In addition, the tight regulation of ascorbate biosynthesis by ascorbate itself has been proposed. Ascorbate represses GGP translation in a dose-dependent manner through the upstream open reading frame in the 5'-untranslated regions of the gene, which may compete with the light-dependent activation of ascorbate biosynthesis. This review focuses on ascorbate biosynthesis based on past and latest findings and critically discusses how light activates this process.

Kiwifruit MYBS1-like and GBF3 transcription factors influence l-ascorbic acid biosynthesis by activating transcription of GDP-L-galactose phosphorylase 3.

Summary Plant‐derived Vitamin C (l‐ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP‐l‐galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP.AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2‐hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R‐subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene‐editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G‐box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co‐expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA.This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.

One-Step Biosynthesis of Vitamin C in Saccharomyces cerevisiae.

Vitamin C (VC) is comprehensively applied in foods, cosmetics, pharmaceuticals, and especially clinical medicine. Nowadays, the industrial production of VC mainly relies on the classic two-step fermentation route, and researchers have explored the way for one-step fermentation of VC in recent years. In this study, a VC biosynthesis pathway that directly produced VC from glucose was reconstructed in Saccharomyces cerevisiae, and the protein engineering and metabolic engineering strategies were adopted to improve it. First, five exogenous modules from Arabidopsis were introduced into the chassis cells by synthetic biology approaches to obtain the strain YLAA harboring VC biosynthesis. In addition, L-galactose dehydrogenase (L-GalDH) and L-galactono-1,4-lactone dehydrogenase (L-GLDH) were fused and expressed in S. cerevisiae cells for the first time, which increased the intracellular VC accumulation by 2.78-fold, reaching 9.97 ± 0.09 mg/L. Through copy number engineering, it was further confirmed that the last step catalyzed by L-GLDH is the rate-limiting step. GDP-L-galactose phosphorylase (GPP) encoded by vtc2 is another rate-limiting enzyme confirmed by GAL1p overexpression results. Finally, by balancing gene expression and cell growth, the highest production strain with overexpressing vtc2 by multicopy plasmids was constructed. The VC accumulation reached 24.94 ± 1.16 mg/L, which was currently the highest production from glucose in S. cerevisiae. The production of the recombinant strain reached nearly 44 mg/L with the exogenous addition of L-galactose or glutathione. The results further emphasized the importance of the step catalyzed by GPP. The investigation provided experience for the efficient biosynthesis of VC and the determination of rate-limiting steps.

Overproduction of ascorbic acid impairs pollen fertility in tomato.

Ascorbate is a major antioxidant buffer in plants. Several approaches have been used to increase the ascorbate content of fruits and vegetables. Here, we combined forward genetics with mapping-by-sequencing approaches using an ethyl methanesulfonate (EMS)-mutagenized Micro-Tom population to identify putative regulators underlying a high-ascorbate phenotype in tomato fruits. Among the ascorbate-enriched mutants, the family with the highest fruit ascorbate level (P17C5, up to 5-fold wild-type level) had strongly impaired flower development and produced seedless fruit. Genetic characterization was performed by outcrossing P17C5 with cv. M82. We identified the mutation responsible for the ascorbate-enriched trait in a cis-acting upstream open reading frame (uORF) involved in the downstream regulation of GDP-l-galactose phosphorylase (GGP). Using a specific CRISPR strategy, we generated uORF-GGP1 mutants and confirmed the ascorbate-enriched phenotype. We further investigated the impact of the ascorbate-enriched trait in tomato plants by phenotyping the original P17C5 EMS mutant, the population of outcrossed P17C5 × M82 plants, and the CRISPR-mutated line. These studies revealed that high ascorbate content is linked to impaired floral organ architecture, particularly anther and pollen development, leading to male sterility. RNA-seq analysis suggested that uORF-GGP1 acts as a regulator of ascorbate synthesis that maintains redox homeostasis to allow appropriate plant development.

Molecular evolution of GDP-L-galactose phosphorylase, a key regulatory gene in plant ascorbate biosynthesis.

Ascorbic acid (AsA) is a widespread antioxidant in living organisms, and plays essential roles in the growth and development of animals and plants as well as in the response to abiotic stress tolerance. The GDP-L-galactose phosphorylase (GGP) is a key regulatory gene in plant AsA biosynthesis that can regulate the concentration of AsA at the transcriptional and translational levels. The function and regulation mechanisms of GGP have been well understood; however, the molecular evolutionary patterns of the gene remain unclear. In this study, a total of 149 homologous sequences of GGP were sampled from 71 plant species covering the major groups of Viridiplantae, and the phylogenetic relationships, gene duplication and molecular evolution analyses of the genes were systematically investigated. Results showed that GGP genes are present throughout the plant kingdom and five shared whole-genome duplications and several lineage-specific whole-genome duplications were found, which led to the rapid expansion of GGPs in seed plants, especially in angiosperms. The structure of GGP genes was more conserved in land plants, but varied greatly in green algae, indicating that GGP may have undergone great differentiation in the early stages of plant evolution. Most GGP proteins had a conserved motif arrangement and composition, suggesting that plant GGPs have similar catalytic functions. Molecular evolutionary analyses showed that GGP genes were predominated by purifying selection, indicating that the gene is functionally conserved due to its vital importance in AsA biosynthesis. Most of the branches under positive selection identified by the branch-site model were mainly in the chlorophytes lineage, indicating episodic diversifying selection may contribute to the evolution of GGPs, especially in the chlorophyte lineage. The conserved function of GGP and its rapid expansion in angiosperms maybe one of the reasons for the increase of AsA content in angiosperms, enabling angiosperms to adapt to changing environments.

The effect of low ascorbic acid content on tomato fruit ripening.

The oxidant/antioxidant balance affects the ripening time of tomato fruit. Ripening of tomato fruit is associated with several modifications such as loss of cell wall firmness and transformation of chloroplasts to chromoplasts. Besides a peak in H2O2, reactive oxygen species (ROS) are observed at the transition stage. However, the role of different components of oxidative stress metabolism in fruit ripening has been scarcely addressed. Two GDP-L-galactose phosphorylase (GGP) Solanum lycopersicum L. cv Micro-Tom mutants which have fruit with low ascorbic acid content (30% of wild type) were used in this work to unravel the participation of ascorbic acid and H2O2 in fruit maturation. Both GGP mutants show delayed fruit maturation with no peak of H2O2; treatment with ascorbic acid increases its own concentration and accelerates ripening only in mutants to become like wild type plants. Unexpectedly, the treatment with ascorbic acid increases H2O2 synthesis in both mutants resembling what is observed in wild type fruit. Exogenous supplementation with H2O2 decreases its own synthesis delaying fruit maturation in plants with low ascorbic acid content. The site of ROS production is localized in the chloroplasts of fruit of all genotypes as determined by confocal microscopy analysis. The results presented here demonstrate that both ascorbic acid and H2O2 actively participate in tomato fruit ripening.

Deficiency of GDP-L-galactose phosphorylase, an enzyme required for ascorbic acid synthesis, reduces tomato fruit yield.

Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. Reduced GDP-L-galactose phosphorylase expression and deficiency of ascorbic acid content lead to decreased fruit set and yield in tomato plants. GDP-L-galactose phosphorylase (GGP) catalyzes the first step committed to ascorbic acid synthesis. The participation of GDP-L-galactose phosphorylase and ascorbate in tomato fruit production and quality was studied in this work using two SlGGP1 deficient EMS Micro-Tom mutants. The SlGGP1 mutants display decreased concentrations of ascorbate in roots, leaves, flowers, and fruit. The initiation of anthesis is delayed in ggp1 plants but the number of flowers is similar to wild type. The number of fruits is reduced in ggp1 mutants with an increased individual weight. However, the whole fruit biomass accumulation is reduced in both mutant lines. Fruits of the ggp1 plants produce more ethylene and show higher firmness and soluble solids content than the wild type after the breaker stage. Leaf CO2 uptake decreases about 50% in both ggp1 mutants at saturating light conditions; however, O2 production in an enriched CO2 atmosphere is only 19% higher in wild type leaves. Leaf conductance that is largely reduced in both mutants may be the main limitation for photosynthesis. Sink-source assays and hormone concentration were measured to determine restrictions to fruit yield. Manipulation of leaf area/fruit number relationship demonstrates that the number of fruits and not the provision of photoassimilates from the source restricts biomass accumulation in the ggp1 lines. The lower gibberellins concentration measured in the flowers would contribute to the lower fruit set, thus impacting in tomato yield. Taken as a whole these results demonstrate that ascorbate biosynthetic pathway critically participates in tomato development and fruit production.

Genome-wide identification and characterization of the GDP-L-galactose phosphorylase gene family in bread wheat

BackgroundAscorbate is a powerful antioxidant in plants and an essential micronutrient for humans. The GDP-L-galactose phosphorylase (GGP) gene encodes the rate-limiting enzyme of the L-galactose pathway—the dominant ascorbate biosynthetic pathway in plants—and is a promising gene candidate for increasing ascorbate in crops. In addition to transcriptional regulation, GGP production is regulated at the translational level through an upstream open reading frame (uORF) in the long 5′-untranslated region (5’UTR). The GGP genes have yet to be identified in bread wheat (Triticum aestivum L.), one of the most important food grain sources for humans.ResultsBread wheat chromosomal groups 4 and 5 were found to each contain three homoeologous TaGGP genes on the A, B, and D subgenomes (TaGGP2-A/B/D and TaGGP1-A/B/D, respectively) and a highly conserved uORF was present in the long 5’UTR of all six genes. Phylogenetic analyses demonstrated that the TaGGP genes separate into two distinct groups and identified a duplication event of the GGP gene in the ancestor of the Brachypodium/Triticeae lineage. A microsynteny analysis revealed that the TaGGP1 and TaGGP2 subchromosomal regions have no shared synteny suggesting that TaGGP2 may have been duplicated via a transposable element. The two groups of TaGGP genes have distinct expression patterns with the TaGGP1 homoeologs broadly expressed across different tissues and developmental stages and the TaGGP2 homoeologs highly expressed in anthers. Transient transformation of the TaGGP coding sequences in Nicotiana benthamiana leaf tissue increased ascorbate concentrations more than five-fold, confirming their functional role in ascorbate biosynthesis in planta.ConclusionsWe have identified six TaGGP genes in the bread wheat genome, each with a highly conserved uORF. Phylogenetic and microsynteny analyses highlight that a transposable element may have been responsible for the duplication and specialized expression of GGP2 in anthers in the Brachypodium/Triticeae lineage. Transient transformation of the TaGGP coding sequences in N. benthamiana demonstrated their activity in planta. The six TaGGP genes and uORFs identified in this study provide a valuable genetic resource for increasing ascorbate concentrations in bread wheat.

Isolation and Characterization of GDP-L-Galactose Phosphorylase Gene of Vitamin C Biosynthesis Pathway from Kiwi

Introduction: Vitamin C is a major antioxidant in plants and plays an important role in reducing the activity of reactive oxygen species. In humans, the main role of this molecule is the elimination of activity of active oxygen species along with being cofactor for many enzymes. Human is one of the few mammalian species that can not synthesize this vitamin and needs to get it through food sources. The GDP-L-galactose phosphorylase (GGP) gene is one of the most important genes in the biosynthetic pathway of vitamin C, which codes for the GDPL-galactose phosphorylase enzyme. Isolation of GGP gene is an important step in transferring it to elevate vitamin C biosynthesis in plants. Materials and Methods: In current study, the isolation of this gene from kiwi plant was carried out and then was cloned in the pTG19-T plasmid via T/A cloning and subsequently sequenced to confirm it. Results: Sequencing analysis of the GGP gene showed that this fragment contains 1383 bp and the start and stop codons were ATG and TGA, respectively. The bioinformatics analysis of this gene can provide important information on gene and protein structure. The alignment of cloned sequence was done with other Actinidia DNA sequences. The results based on neighbor-joining alignment showed that some of the mutations in nucleotides were related to the third nucleotide in a specific codon. Also, the minimum distance of protein sequences was observed between isolated GGP and Actinidia chinensis. Conclusions: Based on analyses, isolated gene (GGP) can be used for increase vitamin C content in other plants such as cucumber and for resistance to environmental stresses in different plants.

L-ascorbic acid metabolism in an ascorbate-rich kiwifruit (Actinidia. Eriantha Benth.) cv. ‘White’ during postharvest

Kiwifruit (Actinidia eriantha Benth.) ‘White’, a novel cultivar with higher L-ascorbic acid (AsA) level, is registered in China. Changes in AsA, related metabolites, enzymatic activity, and gene expression associated with AsA biosynthesis and recycling process were investigated in this paper. The results indicated that AsA biosynthesis through L-galactose pathway supplemented by D-galacturonic acid pathway and AsA recycling collectively contributed to accumulating and remaining higher AsA level in kiwifruit cv. ‘White’ during postharvest. Moreover, L-galactose dehydrogenase (GalDH) activity and relative expressions of the genes encoding GDP-D-mannose pyrophosphorylase (GMP), L-galactose-1-P phosphatase (GPP), GDP-L-galactose phosphorylase (GGP), GalDH and D-galacturonate reductase (GalUR) were important for regulation of AsA biosynthesis, and the activity and expression of dehydroascorbate reductase (DHAR) were primarily responsible for regulation of AsA recycling in kiwifruit ‘White’ during postharvest.

Gene expression and enzyme activities of the D-mannose/L-galactose pathway influence L-ascorbic acid content in Myrciaria dubia

The aim of this work was to elucidate the molecular and biochemical mechanisms that control L-ascorbic acid (AsA) content variation in Myrciaria dubia. The AsA was quantified by high-performance liquid chromatography, gene expression by real-time quantitative PCR, and enzyme activities by spectrophotometric methods from leaves and immature fruits of two genotypes (Md-60,06 and Md-02,04) with pronounced (about 2 times) differences in the AsA content. In either genotype, the fruit peel had ∼ 1.5 times more AsA than the fruit pulp and ∼ 15.0 times more than the leaf. All tissues examined demonstrated the capability for AsA biosynthesis through the D-mannose/L-galactose pathway because mRNAs of the six key genes [GDP-D-mannose pyrophosphorylase (GMP), GDP-D-mannose-3′,5′-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1-4-lactone dehydrogenase (GLDH)] and catalytic activities of the corresponding enzymes (GMP, GDH, and GLDH) were detected. The differential expressions of genes and enzyme activities mostly correlated with the respective AsA content. Thus, the expression of several genes of the D-mannose/L-galactose pathway determined the AsA content variation in tissues of M. dubia.

English

Moringa (Moringa oleifera), which is a semi-tropical plant, is used as food and for the production of medicines and oil products, because of a large amount of various nutrients including ascorbic acid (AsA). Although Moringa leaf has a high AsA content, the molecular mechanisms of AsA accumulation in Moringa have received little attention. In this study, we isolated Moringa cDNAs for enzymes, belonging to the major AsA biosynthesis pathway (Smirnoff-Wheeler pathway) in higher plants. The predicted amino acid sequences showed 70% or more similarity to those of Arabidopsis. Quantitative RT-PCR indicated that Moringa GDP-L-galactose phosphorylase (GGP) is most highly expressed in Moringa during leaf development and light exposure. A significant high promoter activity of the Moringa GGP gene was detected by promoter assay in Arabidopsis protoplast. Key words: Moringa oleifera, ascorbic acid, biosynthesis enzymes, gene expression.

Manipulation of the rice L-galactose pathway: evaluation of the effects of transgene overexpression on ascorbate accumulation and abiotic stress tolerance.

Ascorbic acid (AsA) is the most abundant water-soluble antioxidant in plants, and it plays a crucial role in plant growth, development and abiotic stress tolerance. In the present study, six key Arabidopsis or rapeseed genes involved in AsA biosynthesis were constitutively overexpressed in an elite Japonica rice cultivar. These genes encoded the GDP-mannose pyrophosphorylase (GMP), GDP-mannose-3',5'-epimerase (GME), GDP-L-galactose phosphorylase (GGP), L-galactose-1-phosphate phosphatase (GPP), L-galactose dehydrogenase (GDH), and L-galactono-1,4-lactone dehydrogenase (GalLDH). The effects of transgene expression on rice leaf AsA accumulation were carefully evaluated. In homozygous transgenic seedlings, AtGGP transgenic lines had the highest AsA contents (2.55-fold greater than the empty vector transgenic control), followed by the AtGME and AtGDH transgenic lines. Moreover, with the exception of the AtGPP lines, the increased AsA content also provoked an increase in the redox state (AsA/DHA ratio). To evaluate salt tolerance, AtGGP and AtGME transgenic seedlings were exposed to salt stress for one week. The relative plant height, root length and fresh weight growth rates were significantly higher for the transgenic lines compared with the control plants. Altogether, our results suggest that GGP may be a key rate-limiting step in rice AsA biosynthesis, and the plants with elevated AsA contents demonstrated enhanced tolerance for salt stress.