AsA Content Research Articles

The roles of ROS production-scavenging system in Lasiodiplodia theobromae (Pat.) Griff. & Maubl.-induced pericarp browning and disease development of harvested longan fruit

Effects of Lasiodiplodia theobromae on reactive oxygen species (ROS) production-scavenging system during L. theobromae-induced pericarp browning and disease development of harvested “Fuyan” longans were investigated. Compared with control longans, L. theobromae-inoculated longans exhibited higher pericarp browning index and fruit disease index, higher pericarp O2− generation rate and MDA content. Moreover, L. theobromae infection also resulted in lower contents of pericarp AsA and GSH, lower levels of pericarp DPPH radical scavenging ability and reducing power. Additionally, L. theobromae infection decreased the activities of pericarp SOD, CAT and APX from day 2 to day 5. These findings suggested that L. theobromae-induced pericarp browning and disease development of harvested longans might be due to reduction of ROS scavenging ability and increase in ROS production, which might stimulate membrane lipid peroxidation, disrupt cellular membrane structure, and cause the loss of cellular compartmentalization and disease resistance, in turn, resulting in pericarp browning and disease development.

Jasmonic acid-induced NO activates MEK1/2 in regulating the metabolism of ascorbate and glutathione in maize leaves.

This study investigated the relationship between MEK1/2 and nitric oxide (NO) in jasmonic acid (JA)-regulated metabolism of ascorbate and glutathione in maize leaves. The results showed that JA increased the activities of APX, GR, MDHAR, DHAR, GalLDH, and γ-ECS; the contents of AsA and GSH; and the production of NO. Above increases except for γ-ECS activity and NO production were all suppressed by pre-treatments with MEK1/2 inhibitors PD98059 and U0126. Above increases were all suppressed by pre-treatments with nitric oxide synthase (NOS) inhibitor L-NAME and NO scavenger cPTIO. The results of western blot showed that JA enhanced the phosphorylation level of MEK1/2. Pre-treatments with L-NAME and cPTIO suppressed JA-induced phosphorylation level of MEK1/2. Our results suggested that JA-induced NO activated MEK1/2 by increasing the phosphorylation level, which, in turn, resulted in the upregulation of ascorbate and glutathione metabolism in maize leaves.

Pectic enzymes as potential enhancers of ascorbic acid production through the D-galacturonate pathway in Solanaceae

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family.

Sound waves increases the ascorbic acid content of alfalfa sprouts by affecting the expression of ascorbic acid biosynthesis-related genes

Previous studies have shown that sound wave treatment can affect the expression of plant genes and improve the growth. So, we investigated the ability of sound waves to increase AsA (l-ascorbic acid) content in alfalfa (Medicago sativa) sprouts in this study. Sprouts were exposed to a range of sound wave frequencies for two 1-h periods per day for various numbers of days. Most sound wave treated sprouts had a higher AsA content than untreated sprouts. In addition, the activity level of superoxide dismutase, an enzyme with potent antioxidative properties, was increased in sound wave-treated sprouts. The AsA content varied in response to sound wave treatment. Most processing conditions, including 500 and 1000 Hz, increased AsA content by 24–50%; however, some treatment conditions caused reduced AsA content during sprout growth. Furthermore, AsA content during sprout storage was increased by most sound wave treatment conditions, with 13–36% increases observed following 800 and 1000 Hz sound wave treatments compared to untreated sprouts. To investigate the mechanisms underlying changes in AsA content, we analyzed the expression levels of AsA biosynthesis-related genes. We found that several genes, including VTC1, VTC2, VTC4, GME, L-GalDH, GLDH, MDHAR, and DHAR1, displayed differential expression in response to sound wave treatment. Therefore, sound wave treatment may be a viable method for increasing the nutritional contents of sprouted vegetables.

Overexpression of E3 Ubiquitin Ligase Gene AdBiL Contributes to Resistance against Chilling Stress and Leaf Mold Disease in Tomato.

Ubiquitination is a common regulatory mechanism, playing a critical role in diverse cellular and developmental processes in eukaryotes. However, a few reports on the functional correlation between E3 ubiquitin ligases and reactive oxygen species (ROS) or reactive nitrogen species (RNS) metabolism in response to stress are currently available in plants. In the present study, the E3 ubiquitin ligase gene AdBiL (Adi3 Binding E3 Ligase) was introduced into tomato line Ailsa Craig via Agrobacterium-mediated method. Transgenic lines were confirmed for integration into the tomato genome using PCR. Transcription of AdBiL in various transgenic lines was determined using real-time PCR. Evaluation of stress tolerance showed that T1 generation of transgenic tomato lines showed only mild symptoms of chilling injury as evident by higher biomass accumulation and chlorophyll content than those of non-transformed plants. Compared with wild-type plants, the contents of AsA, AsA/DHA, GSH and the activity of GaILDH, γ-GCS and GSNOR were increased, while H2O2, , MDA, NO, SNOs, and GSNO accumulations were significantly decreased in AdBiL overexpressing plants in response to chilling stress. Furthermore, transgenic tomato plants overexpressing AdBiL showed higher activities of enzymes such as G6PDH, 6PGDH, NADP-ICDH, and NADP-ME involved in pentose phosphate pathway (PPP). The transgenic tomato plants also exhibited an enhanced tolerance against the necrotrophic fungus Cladosporium fulvum. Tyrosine nitration protein was activated in the plants infected with leaf mold disease, while the inhibition could be recovered in AdBiL gene overexpressing lines. Taken together, our results revealed a possible physiological role of AdBiL in the activation of the key enzymes of AsA–GSH cycle, PPP and down-regulation of GSNO reductase, thereby reducing oxidative and nitrosative stress in plants. This study demonstrates an optimized transgenic strategy using AdBiL gene for crop improvement against biotic and abiotic stress factors.

Beneficial effects of melatonin in overcoming drought stress in wheat seedlings

Melatonin plays an important role in abiotic stress in plant, but its role in wheat drought tolerance is less known. To verify its role, wheat seedlings (Triticum aestivum L. ‘Yan 995’) at 60% and 40% of field capacity were treated with 500 μM melatonin in this study. Melatonin treatment significantly enhanced the drought tolerance of wheat seedlings, as demonstrated by decreased membrane damage, more intact grana lamella of chloroplast, higher photosynthetic rate, and maximum efficiency of photosystem II, as well as higher cell turgor and water holding capacity in melatonin-treated seedlings. Besides, melatonin markedly decreased the content of hydrogen peroxide and superoxide anion in melatonin-treated seedlings, which is attributed to the increased total antioxidant capacity, GSH and AsA contents, as well as enzyme activity including ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX), and glutathione transferase (GST). The GSH-AsA related genes including APX, MDHAR, and DHAR were commonly upregulated by melatonin and correlated to the antioxidant enzyme activity as well as the content of GSH and AsA, indicating that the increase of GSH and AsA was attributed to the expression of these genes. Our result confirmed the mitigation potential of melatonin in drought stress and certain mechanisms of melatonin-induced GSH and AsA accumulation, which could deepen our understanding of melatonin-induced drought tolerance in wheat.

Anatomy of a subtropical intrathermocline eddy

An interdisciplinary survey of a subtropical intrathermocline eddy was conducted within the Canary Eddy Corridor in September 2014. The anatomy of the eddy is investigated using near submesoscale fine resolution two-dimensional data and coarser resolution three-dimensional data. The eddy was four months old, with a vertical extension of 500m and 46km radius. It may be viewed as a propagating negative anomaly of potential vorticity (PV), 95% below ambient PV. We observed two cores of low PV, one in the upper layers centered at 85m, and another broader anomaly located between 175m and the maximum sampled depth in the three-dimensional dataset (325m). The upper core was where the maximum absolute values of normalized relative vorticity (or Rossby number), |Ro| =0.6, and azimuthal velocity, U=0.5ms−1, were reached and was defined as the eddy dynamical core. The typical biconvex isopleth shape for intrathermocline eddies induces a decrease of static stability, which causes the low PV of the upper core. The deeper low PV core was related to the occurrence of a pycnostad layer of subtropical mode water that was embedded within the eddy. The eddy core, of 30km radius, was in near solid body rotation with period of ~4 days. It was encircled by a thin outer ring that was rotating more slowly. The kinetic energy (KE) content exceeded that of available potential energy (APE), KE/APE=1.58; this was associated with a low aspect ratio and a relatively intense rate of spin as indicated by the relatively high value of Ro. Inferred available heat and salt content anomalies were AHA=2.9×1018 J and ASA=14.3×1010kg, respectively. The eddy AHA and ASA contents per unit volume largely exceed those corresponding to Pacific Ocean intrathermocline eddies. This suggests that intrathermocline eddies may play a significant role in the zonal conduit of heat and salt along the Canary Eddy Corridor.

Tomato SlGGP-LIKE gene participates in plant responses to chilling stress and pathogenic infection

Plants are always exposed to abiotic and biotic stresses which can adversely affect their growth and development. As an important antioxidant, AsA plays a vital role in plant defence against damage caused by stresses. In this study, we cloned a tomato GDP-L-galactose phosphorylase-like (SlGGP-LIKE) gene and investigated its role in resistance to abiotic and biotic stresses by using antisense transgenic (AS) tomato lines. The AsA content in AS plants was lower than that in WT plants. Under chilling stress, the growth of AS plants was inhibited significantly, and they yielded higher levels of ROS, REC and MDA but demonstrated weaker APX activity than that shown by WT plants. Additionally, the declined values of Pn, Fv/Fm, oxidisable P700, and D1 protein content of PSII in AS lines were significant. Furthermore, the effect on xanthophyll cycle of AS plants was more severe than that on WT plants, and the ratio of zeaxanthin (Z)/(V + A + Z) and (Z + 0.5 A)/(V + A + Z) in AS lines was lower than that in WT plants. In spite of chilling stress, under Pseudomonas syringae pv.tomato (Pst) DC3000 strain infection, AS plants showed lesser bacterial cell growth and dead cells than those shown by WT plants. This finding indicated that AS plants demonstrated stronger resistance against pathogenic infection. Results suggest that SlGGP-LIKE gene played an important role in plant defence against chilling stress and pathogenic infection.

Expression profiles of the genes involved in l-ascorbic acid biosynthesis and recycling in Rosa roxburghii leaves of various ages

Vitamin C (l-ascorbic acid, AsA) serves as an antioxidant and enzyme cofactor in plants. Rosa roxburghii Tratt is a rose species that is recognized for its high AsA content. However, leaf AsA metabolism has not been explored in detail, especially at the gene expression level. In the present study, AsA accumulation and the relative expression levels of genes putatively involved in AsA biosynthesis and recycling, as well as enzyme activities in leaves of different ages were investigated. Leaf AsA content remained at its highest level during the first 50 days of development, and then markedly decreased, which correlated with the expression and activity of monodehydroascorbate reductase (MDHAR) (p < 0.05). The dehydroascorbate (DHA) and T-AsA (AsA + DHA) contents increased with leaf development. Both of them reached the peak in 50-day-old leaves and then decreased rapidly. The highest expression levels of three genes, i.e. l-galactono-1,4-lactone dehydrogenase (GalLDH), d-galacturonic acid reductase (GalUR), and myo-inositol oxygenase (MIOX) involved in AsA synthesis were observed in 50-day-old leaves, although only GalUR strongly correlated with the rate of T-AsA accumulation (p < 0.05). Furthermore, incubating leaf discs in vitro with its precursor candidates showed that d-galacturonic acid (d-GalUA) was the most effective precursor of AsA biosynthesis. These results suggest that Rosa roxburghii leaves accumulated high levels of AsA (9 μmol g−1 FW at most), especially at young to mature ages. AsA and AsA pool size accumulation in the leaves were regulated by both biosynthesis and recycling, and GalUR in the GalUA pathway and MDHAR in the recycling pathway could play important roles in this process.

Characterization of Two Arabidopsis L-Gulono-1,4-lactone Oxidases, AtGulLO3 and AtGulLO5, Involved in Ascorbate Biosynthesis.

L-Ascorbic acid (AsA, vitamin C) is an essential antioxidant for plants and animals. There are four known ascorbate biosynthetic pathways in plants: the L-galactose, L-gulose, D-galacturonate, and myo-inositol routes. These pathways converge into two AsA precursors: L-galactono-1,4-lactone and L-gulono-1,4-lactone (L-GulL). This work focuses on the study of L-gulono-1,4-lactone oxidase (GulLO), the enzyme that works at the intersect of the gulose and inositol pathways. Previous studies have shown that feeding L-gulono-1,4-lactone to multiple plants leads to increased AsA. There are also reports showing GulLO activity in plants. We describe the first detailed characterization of a plant enzyme specific to oxidize L-GulL to AsA. We successfully purified a recombinant Arabidopsis GulLO enzyme (called AtGulLO5) in a transient expression system. The biochemical properties of this enzyme are similar to the ones of bacterial isozymes in terms of substrate specificity, subcellular localization, use of flavin adenine dinucleotide (FAD) as electron acceptor, and specific activity. AtGulLO5 is an exclusive dehydrogenase with an absolute specificity for L-GulL as substrate thus differing from the existing plant L-galactono-1,4-lactone dehydrogenases and mammalian GulLOs. Feeding L-GulL to N. benthamiana leaves expressing AtGulLO5 constructs led to increased foliar AsA content, but it was not different from that of controls, most likely due to the observed low catalytic efficiency of AtGulLO5. Similar results were also obtained with another member of the AtGulLO family (AtGulLO3) that appears to have a rapid protein turnover. We propose that AsA synthesis through L-GulL in plants is regulated at the post-transcriptional level by limiting GulLO enzyme availability.

The effects of cadmium on the biochemical and physiological parameters of Eruca sativa.

In this study, Eruca sativa (Rocket) seedlings were treated with different cadmium (Cd) concentrations (0, 150, 300 and 450 μg ∙ g-1). The effects of Cd on lipid peroxidation, enzymatic (APx, CAT, GPX, SOD) and non-enzymatic antioxidants (total ascorbate, dehydroascorbate, ascorbate, non-protein thiol), fresh and dry masses, water content were determined. Also, Cd content of the leaves and the roots were analysed. The highest cadmium accumulation of leaves was at 450 μg ∙ g-1 Cd treatment and the accumulation was 2.62 times greater than those in the roots. The translocation factor was 3.89 at 300 μg ∙ g-1 Cd treatment. Cd treatments caused decreases of fresh, dry mass and water content of leaves and roots. Malondialdehyde content, which is an index of lipid peroxidation, was increased in proportion with the increase in Cd. While there was not change in the activity of GPX according to control, a decrease in activities of SOD, CAT and APX were observed with the increase of cadmium concentration. Although a significant increase in the amounts of non-protein thiol groups and proline were observed in 450 μg ∙ g-1 Cd treated plants, Cd did not lead to a significant change in AsA, DHA and total AsA contents. According to the results of the research, E. sativa may be a Cd hyperaccumulator plant and we suggest that the plant may be a candidate plant for remediation of Cd-contaminated soil.

Glutathione improves survival of cryopreserved embryogenic calli of Agapanthus praecox subsp. orientalis

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H2O2, OH·, and O2·−) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in OH· generation activity and the contents of H2O2 and MDA, as well as enhancement in the inhibition of O2·− generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.

GDP-D-mannose pyrophosphorylase from Pogonatherum paniceum enhances salinity and drought tolerance of transgenic tobacco.

Pogonatherum paniceum is a highly drought- and salt-tolerant plant species that is typically used for ecological restoration and the conservation of soil and water in many countries. Understanding the molecular mechanisms underlying plant abiotic stress responses, especially to salinity and drought stresses, in species such as P. paniceum could be important to broader crop improvement efforts. GDP-D-mannose pyrophosphorylase (GMPase) is the limiting enzyme in the synthesis of L-ascorbic acid (AsA), which plays a crucial role in the detoxification of reactive oxygen species (ROS). We have cloned and characterized the cDNA of the PpGMP gene of P. paniceum encoding a GMPase. The full-length cDNA sequence contains 1411 nucleotides encoding a putative protein with 361 amino acid residues and an approximate molecular mass of 39.68 kDa. The GMPase transcript was up-regulated in P. paniceum plants subjected to salinity and drought stress, respectively. Transgenic tobacco expressing PpGMPase exhibited enhanced salinity and drought resistance, a higher seed germination rate, better growth performance, a higher AsA content, a more stable redox state, higher superoxide dismutase (SOD) activity, and lower levels of malonaldehyde (MDA) and H2O2 under drought and salinity stress. Taken together, expression of PpGMPase in tobacco conferred salinity and drought stress tolerance by increasing the content of AsA, thereby enhancing ROS-detoxifying functions. Thus, PpGMP is a potential candidate gene for crop improvement.

Autotoxicity in cucumber (Cucumis sativus L.) seedlings is alleviated by silicon through an increase in the activity of antioxidant enzymes and by mitigating lipid peroxidation

Autotoxicity in plants limits their growth and that of nearby plants of the same species, which has obvious implications in crop yield and quality. Silicon (Si) has been shown to increase plant tolerance to autotoxic stress. However, the physiological mechanisms underlying the effects of Si in alleviating autotoxicity during germination in cucumber (Cucumis sativus L.) are unknown. Cinnamic acid derivatives, such as 3-phenylpropionic acid (PA), are a class of autotoxins present in cucumber root exudates. Our objective was to investigate Si-induced autotoxic stress tolerance in cucumber seedlings by focusing on the effects of Si on the induction of antioxidant defense pathways. We found that PA treatment significantly reduced seed germination, radicle length, lateral root number, fresh weight, AsA and GSH contents, and the activities of SOD, CAT, and APX in cucumber seedlings, while it increased membrane permeability and levels of MDA, proline, O 2 - , and H2O2. Application of Si enhanced growth of PA-treated plants and significantly increased germination rate, radicle length, lateral root number, fresh weight, AsA and GSH levels, and SOD, CAT, POD, and APX activities. These results suggest that exogenous Si alleviates autotoxicity caused by PA during seed germination by increasing antioxidant enzyme activities and mitigating lipid peroxidation.

Involvement of ABA- and H2O2-dependent cytosolic glucose-6-phosphate dehydrogenase in maintaining redox homeostasis in soybean roots under drought stress

The roles of abscisic acid (ABA) and hydrogen peroxide (H2O2) in inducing glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and the possible roles of G6PDH in regulating ascorbate-glutathione (AsA-GSH) cycle were investigated in soybean (Glycine max L.) roots under drought stress. Drought caused a marked increase of the total and cytosolic G6PDH activities and triggered a rapid ABA and H2O2 accumulation in soybean roots. Exogenous ABA or H2O2 treatment elevated the total and cytosolic G6PDH activities, whereas suppressing ABA or H2O2 production inhibited the drought-induced increase in total and cytosolic G6PDH activities, suggesting that ABA and H2O2 are required for drought-induced increase of total G6PDH activity, namely cytosolic G6PDH activity. Furthermore, ABA induced H2O2 production by stimulating NADPH oxidase activity under drought stress. Moreover, drought significantly increased the contents of AsA and GSH and the activities of key enzymes in AsA-GSH cycle, while application of G6PDH inhibitor to seedlings significantly reduced the above effect induced by drought. Taken together, these results indicate that H2O2 acting as a downstream signaling molecule of ABA mediates drought-induced increase in cytosolic G6PDH activity, and that enhanced cytosolic G6PDH activity maintains cellular redox homeostasis by regulating AsA-GSH cycle in soybean roots.

Exogenous calcium alleviates cadmium-induced oxidative stress in rice (Oryza sativa L.) seedlings by regulating the antioxidant defense and glyoxalase systems

The present study was undertaken to investigate the regulatory role of exogenous application of calcium (Ca) in enhancing the antioxidant defense and glyoxalase systems in mitigating cadmium (Cd) stress in rice. Hydroponically grown 14-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.25 and 0.5 mM CdCl2 alone and in combination with 2.5 mM CaCl2 for 3 days. Exposure to Cd caused chlorosis, leaf rolling symptoms, and growth inhibition. A higher concentration of Cd in the growth medium resulted in higher Cd accumulation, which induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense system. Cadmium treatment increased the methylglyoxal (MG) level. Calcium supplementation in the Cd-treated growth medium reduced Cd uptake. Application of Ca also significantly increased the (ascorbate) AsA content, increased the activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), monodehydroascorbate reductase (MDHAR), and dehydroascorbate reductase (DHAR) in the antioxidant system, and increased the glyoxalase I (Gly I) and glyoxalase II (Gly II) activities in the glyoxalase system in rice seedlings exposed to both levels of Cd. Exogenous Ca application regulated the antioxidant defense and glyoxalase systems, which reversed overproduced ROS and detoxified MG, which in turn reduced Cd toxicity.

De novo assembly and functional annotation of Myrciaria dubia fruit transcriptome reveals multiple metabolic pathways for L-ascorbic acid biosynthesis.

BackgroundMyrciaria dubia is an Amazonian fruit shrub that produces numerous bioactive phytochemicals, but is best known by its high L-ascorbic acid (AsA) content in fruits. Pronounced variation in AsA content has been observed both within and among individuals, but the genetic factors responsible for this variation are largely unknown. The goals of this research, therefore, were to assemble, characterize, and annotate the fruit transcriptome of M. dubia in order to reconstruct metabolic pathways and determine if multiple pathways contribute to AsA biosynthesis.ResultsIn total 24,551,882 high-quality sequence reads were de novo assembled into 70,048 unigenes (mean length = 1150 bp, N50 = 1775 bp). Assembled sequences were annotated using BLASTX against public databases such as TAIR, GR-protein, FB, MGI, RGD, ZFIN, SGN, WB, TIGR_CMR, and JCVI-CMR with 75.2 % of unigenes having annotations. Of the three core GO annotation categories, biological processes comprised 53.6 % of the total assigned annotations, whereas cellular components and molecular functions comprised 23.3 and 23.1 %, respectively. Based on the KEGG pathway assignment of the functionally annotated transcripts, five metabolic pathways for AsA biosynthesis were identified: animal-like pathway, myo-inositol pathway, L-gulose pathway, D-mannose/L-galactose pathway, and uronic acid pathway. All transcripts coding enzymes involved in the ascorbate-glutathione cycle were also identified. Finally, we used the assembly to identified 6314 genic microsatellites and 23,481 high quality SNPs.ConclusionsThis study describes the first next-generation sequencing effort and transcriptome annotation of a non-model Amazonian plant that is relevant for AsA production and other bioactive phytochemicals. Genes encoding key enzymes were successfully identified and metabolic pathways involved in biosynthesis of AsA, anthocyanins, and other metabolic pathways have been reconstructed. The identification of these genes and pathways is in agreement with the empirically observed capability of M. dubia to synthesize and accumulate AsA and other important molecules, and adds to our current knowledge of the molecular biology and biochemistry of their production in plants. By providing insights into the mechanisms underpinning these metabolic processes, these results can be used to direct efforts to genetically manipulate this organism in order to enhance the production of these bioactive phytochemicals.The accumulation of AsA precursor and discovery of genes associated with their biosynthesis and metabolism in M. dubia is intriguing and worthy of further investigation. The sequences and pathways produced here present the genetic framework required for further studies. Quantitative transcriptomics in concert with studies of the genome, proteome, and metabolome under conditions that stimulate production and accumulation of AsA and their precursors are needed to provide a more comprehensive view of how these pathways for AsA metabolism are regulated and linked in this species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2225-6) contains supplementary material, which is available to authorized users.

Implication of the antioxidant system in chilling injury tolerance in the red peel of grapefruit

Previous observations have indicated that the red peel areas of grapefruit with high lycopene concentrations were more tolerant to CI than yellow peel areas (Lado et al., 2015a). Because lycopene is a carotene with powerful antioxidant capacity, this study investigated whether the CI tolerance of the lycopene-accumulating rind of grapefruit may be due to an enhancement of the enzymatic and/or non-enzymatic antioxidant systems. Total antioxidant capacity, antioxidant metabolite (GSH and AsA) contents, and antioxidant enzyme (GR [glutathione reductase], ascorbate peroxidase [APX], catalase [CAT] and superoxide dismutase [SOD]) activity and gene expression were measured in the peel of Star Ruby grapefruit with contrasting CI tolerance during storage at 2°C for up to 58d. The peel of CI-tolerant fruit exhibited a lower lipid peroxidation level (MDA content). The hydrogen peroxide concentration was similar after 3 weeks of storage, when the differences in chilling damage between sensitive and tolerant fruit were noticeable, suggesting that the increase in H2O2 is a response of flavedo cells to cold stress that is not necessarily related to the development of CI. Moreover, CI tolerance was not associated with enhancement of either total antioxidant capacity or glutathione and AsA contents, indicating that such antioxidant responses may be cold-mediated and not directly linked to chilling tolerance. Analysis of singlet oxygen scavenging capacity by the SOAC assay revealed considerably higher activity in the lycopene-accumulating peel than in the yellow peel at harvest time and throughout the entire cold storage and shelf-life period. Enzymatic activity and gene expression analyses of GR, APX and SOD did not reveal the involvement of these antioxidant enzymes in the protection against CI. However, high CAT activity was detected in the peel of CI-tolerant fruit, although this difference did not correlate with changes in the expression levels of the CAT1 and CAT2 genes. Therefore, the boost in singlet oxygen scavenging capacity is likely the primary factor responsible for CI tolerance in the lycopene-accumulating peel of grapefruit.

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.

Metabolic engineering of vitamin C production in Arabidopsis

Vitamin C (L-ascorbic acid, AsA) is a compound which provides major nutritional value, both for plants and humans. In this study, three distinct metabolic engineering strategies, including overexpression of biosynthesis enzyme, suppression of catabolism enzyme and switching the sub-cellular localization of compartment enzyme, were employed to enhance the production of AsA in Arabidopsis thaliana. The results showed that (1) overexpression of L-Galactose-1-P Phosphatase (GalPPase) enhanced AsA content to 3.96 µmol/g FW, which was 1.6-fold more than that in their wild-type (WT) counterparts; (2) RNAi suppression of ascorbate oxidase (AO) resulted in a significant increase of AsA accumulation (0.86 µmol/g FW) in apoplast; (3) both mitochondrion-target and none-target overexpression of L-Galactose dehydrogenase (GalDH) did not significantly promote AsA production compared with WT (1.96 µmol/g), however a dramatic enhancement was observed following infiltration with L-galactono-1, 4-lactone (L-GalL), both in transgenic and WT plants. The best line produced AsA with the content of 3.90 µmol/g FW, which was about 2-fold of that in the untreated control (1.99 µmol/g FW). This study provides new strategies including GalPPase overexpression, AO suppression as well as L-GalL feeding for modern breeding aimed at stimulating the AsA content in plants.