Coordinated Effect of Ascorbate Biosynthesis and Recycling in Maize Seed Germination and Seedling Establishment under Low Temperature

The impacts of low temperature occasionally encountered at higher latitude regions on maize seed germination present significant threats to yield and cultivation. Exploring the association of antioxidant system with low temperature (LT) germination could support the breeding strategies for better responding to LT disturbance. In this study, we have examined the germination rate and growth potential of a set of elite maize inbred accessions under LT and normal temperature (NT) conditions in the field. These accessions were found to have variable germination rate and growth potential when grown at LT, whereas the difference is not significant under NT. Physiological study revealed lower hydrogen peroxide content in LT tolerant accessions when compared with sensitive ones. LT-tolerant and LT-sensitive lines maintained similar content of ascorbate (AsA) and glutathione (GSH), whereas the reduced substrate content of which were significantly higher in LT-tolerant accessions. Consistently, activities of ascorbate peroxidase and dehydroascorbate reductase, the enzyme components that responsible for the AsA-GSH recycling, were much higher in LT-tolerant lines. Transcription profile revealed the increased expression of ZmVTC2 gene in LT-tolerant inbred line, which was rate limited step in AsA biosynthesis. These data indicates that the coordinated improvement of AsA biosynthesis and AsA-GSH recycling increase the pool size of the total antioxidants, which ameliorate LT-induced oxidative stress during maize seed germination.

Increased Ascorbate Biosynthesis Does Not Improve Nitrogen Fixation Nor Alleviate the Effect of Drought Stress in Nodulated Medicago truncatula Plants

Legume plants are able to establish nitrogen-fixing symbiotic relations with Rhizobium bacteria. This symbiosis is, however, affected by a number of abiotic constraints, particularly drought. One of the consequences of drought stress is the overproduction of reactive oxygen (ROS) and nitrogen species (RNS), leading to cellular damage and, ultimately, cell death. Ascorbic acid (AsA), also known as vitamin C, is one of the antioxidant compounds that plants synthesize to counteract this oxidative damage. One promising strategy for the improvement of plant growth and symbiotic performance under drought stress is the overproduction of AsA via the overexpression of enzymes in the Smirnoff-Wheeler biosynthesis pathway. In the current work, we generated Medicago truncatula plants with increased AsA biosynthesis by overexpressing MtVTC2, a gene coding for GDP-L-galactose phosphorylase. We characterized the growth and physiological responses of symbiotic plants both under well-watered conditions and during a progressive water deficit. Results show that increased AsA availability did not provide an advantage in terms of plant growth or symbiotic performance either under well-watered conditions or in response to drought.

VARIABILITY OF GMP GENES-HOMOLOGOUS IN WILD TOMATO SPECIES FROM THE LYCOPERSICON SECTION

Domestication led to a regression of ascorbate levels in cultivated tomato fruits. In this work, we for the first time sequenced and characterized 11 GMP homologous genes from wild tomato species that play a central role in the metabolic pathway of ascorbate biosynthesis in plants

L-Ascorbate Biosynthesis Involves Carbon Skeleton Rearrangement in the Nematode Caenorhabditis elegans

Ascorbate (AsA) is required as a cofactor and is widely distributed in plants and animals. Recently, it has been suggested that the nematode Caenorhabditis elegans also synthesizes AsA. However, its biosynthetic pathway is still unknown. To further understand AsA biosynthesis in C. elegans, we analyzed the incorporation of the 13C atom into AsA using gas chromatography-mass spectrometry (GC-MS) in worms fed with D-Glc (1-13C)-labeled Escherichia coli. GC-MS analysis revealed that AsA biosynthesis in C. elegans, similarly to that in mammalian systems, involves carbon skeleton rearrangement. The addition of L-gulono-1,4-lactone, an AsA precursor in the mammalian pathway, significantly increased AsA level in C. elegans, whereas the addition of L-galactono-1,4-lactone, an AsA precursor in the plant and Euglena pathway, did not affect AsA level. The suppression of E03H4.3 (an ortholog of gluconolactonase) or the deficiency of F54D5.12 (an ortholog of L-gulono-1,4-lactone oxidase) significantly decreased AsA level in C. elegans. Although N2- and AsA-deficient F54D5.12 knockout mutant worm (tm6671) morphologies and the ratio of collagen to non-collagen protein did not show any significant differences, the mutant worms exhibited increased malondialdehyde levels and reduced lifespan compared with the N2 worms. In conclusion, our findings indicate that the AsA biosynthetic pathway is similar in C. elegans and mammals.

Organization and control of the ascorbate biosynthesis pathway in plants

ABSTRACTThe enzymatic steps involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler, SW pathway) has been well established and here we comprehensively analyze the subcellular localization, potential physical interactions of SW pathway enzymes and assess their role in control of ascorbate synthesis. Transient expression of GFP-fusions in Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) mutants complemented with genomic constructs showed that while GME is cytosolic, VTC1, VTC2, VTC4, and l-GalDH have cytosolic and nuclear localization. While transgenic lines GME-GFP, VTC4-GFP and l-GalDH-GFP driven by their endogenous promoters accumulated the fusion proteins, the functional VTC2-GFP protein is detected at low level using immunoblot in a complemented vtc2 null mutant. This low amount of VTC2 protein and the extensive analyses using multiple combinations of SW enzymes in N. benthamiana supported the role of VTC2 as the main control point of the pathway on ascorbate biosynthesis. Interaction analysis of SW enzymes using yeast two hybrid did not detect the formation of heterodimers, although VTC1, GME and VTC4 formed homodimers. Further coimmunoprecipitation (CoIP) analysis indicted that consecutive SW enzymes, as well as the first and last enzymes (VTC1 and l-GalDH), associate thereby adding a new layer of complexity to ascorbate biosynthesis. Finally, metabolic control analysis incorporating known kinetic characteristics, showed that previously reported feedback repression at the VTC2 step confers a high flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.One sentence summaryMetabolic engineering, genetic analysis and functional mutant complementation identify GDP-l-galactose phosphorylase as the main control point in ascorbate biosynthesis in green tissues.

Biosynthetic Gene Pyramiding Leads to Ascorbate Accumulation with Enhanced Oxidative Stress Tolerance in Tomato

Ascorbic acid (AsA) has high antioxidant activities, and its biosynthesis has been well studied by engineering of a single structural gene (SG) in staple crops, such as tomato (Solanum lycopersicum). However, engineering the AsA metabolic pathway by multi-SG for biofortification remains unclear. In this study, pyramiding transgenic lines including GDP-Mannose 3′,5′-epimerase (GME) × GDP-d-mannose pyrophosphorylase (GMP), GDP-l-Gal phosphorylase (GGP) × l-Gal-1-P phosphatase (GPP) and GME × GMP × GGP × GPP, were obtained by hybridization of four key genes to get over-expression transgenic plants (GME, GMP, GGP, and GPP) in tomato. Pyramiding lines exhibited a significant increase in total ascorbate in leaves and red fruits except for GGP × GPP. Expression analysis indicated that increased accumulation of AsA in pyramiding transgenic lines is due to multigene regulation in AsA biosynthesis. Substrate feeding in leaf and fruit suggested that AsA biosynthesis was mainly contributed by the d-Man/l-Gal pathway in leaves, while alternative pathways may contribute to AsA accumulation in tomato fruit. Pyramiding lines showed an enhanced light response, stress tolerance, and AsA transport capacity. Also, fruit shape, fruit size, and soluble solids were slightly affected by pyramiding. This study provides the first comprehensive analysis of gene pyramiding for ascorbate biosynthesis in tomato. SGs pyramiding promotes AsA biosynthesis, which in turn enhances light response and oxidative stress tolerance. Also, the data revealed an alternative ascorbate biosynthesis pathway between leaves and fruit of tomato.