Intra-canopy LED lighting outperformed top LED lighting in improving tomato yield and expression of the genes responsible for lycopene, phytoene and vitamin C synthesis.

Greenhouses located at high latitudes and in cloudy areas often experience a low quality and quantity of light, especially during autumn and winter. This low daily light integral (DLI) reduces production rate, quality, and nutritional value of many crops. This study was conducted on Sakhiya RZ F1 tomato plants to evaluate the impact of LED lights on the growth and nutritional value of tomatoes in a greenhouse with low daily light due to cloudy weather. The treatments included LED growth lights in three modes: top lighting, intra-canopy lighting, and combined top and intra-canopy lighting. The results showed that although the combined top and intra-canopy lighting reached the maximum increase in tomato yield, exposure to intra-canopy LED lighting alone outperformed in tomato fruit yield increase (28.46%) than exposure to top LED lighting alone (12.12%) when compared to no supplemental lighting during the entire production year. Intra-canopy exposure demonstrated the highest increase in tomato lycopene (31.3%), while top and intra-canopy lighting exhibited the highest increase in vitamin C content (123.4%) compared to the control. The LED light treatment also had a very positive effect on the expression of genes responsible for metabolic cycles, including Psy1, LCY-ß, and VTC2 genes, which had collinearity with the increase in tomato fruit production.

Effect of light on ascorbic acid biosynthesis and bioinformatics analysis of related genes in Chinese chives.

Ascorbic acid (AsA) is an essential nutritional component and powerful antioxidant in vegetables, and in plants, AsA levels are regulated by light. AsA levels in the leaves of Chinese chive (Allium tuberosum Rottler ex Spr), a popular vegetable, are poorly understood. Thus, this study was performed to assess the influence of light on AsA biosynthesis in chive and select related genes (AtuGGP1 and AtuGME1); in addition, bioinformatic analyses and gene expression level assays were performed. The biological information obtained for AtuGGP1 and AtuGME1 was analysed with several tools, including NCBI, DNAMAN, and MEGA11. After different light treatments were performed, the Chive AsA content and AtuGGP1 and AtuGME1 expression levels were determined. These results suggest that 1) compared with natural light, continuous darkness inhibited AsA synthesis in chives. 2) The amino acid sequences of AtuGGP1 and AtuGME1 are very similar to those of other plants. 3) The trends observed for the expression levels of AtuGGP1 and AtuGME1 were consistent with the AsA content observed in chives. Hence, we speculated that light controls AsA biosynthesis in chives by regulating AtuGGP1 and AtuGME1 expression. This study provided impactful and informative evidence regarding the functions of GGP and GME in chives.

[Biosynthesis of water-soluble vitamins].

Vitamins are a class of organic substances essential for maintaining the normal physiological function of organisms. Most vitamins cannot be synthesized by the human body, and a small number of vitamins can only be synthesized in a limited manner, which cannot meet the body needs. Therefore, people need to take food or drugs containing vitamins to meet the body needs. Nowadays, vitamins are widely used in medicine, food or feed additives, cosmetics and other industries, and the demand for vitamins is growing. Vitamins are mainly produced by chemical synthesis and biosynthesis. Compared with chemical synthesis, biosynthesis of vitamins is praised for the environmental friendliness, high safety, and low costs. Therefore, it is of great practical significance to study the biosynthesis methods of vitamins. This paper reviews the research progress in the methods and summarizes the research results in the biosynthesis of water-soluble vitamins (B vitamins and vitamin C) in recent years and then makes an outlook on the future development in this field.

Combining transcriptomics and HPLC to uncover variations in quality formation between 'Benihoppe' and 'Fenyu No.1' strawberries.

'Benihoppe' and 'Fenyu No.1' are representative varieties of red and pink strawberries in China, possess distinct hue and flavor profiles. This study analyzed the underlying biochemical and molecular differences of two varieties utilizing transcriptomics and high-performance liquid chromatography (HPLC). Ripening 'Benihoppe' fruits accumulated more sucrose and pelargonin-3-glucoside (P3G) with a little cyanidin and higher firmness. Whereas ripening 'Fenyu No.1' fruits contained more fructose, glucose, malic acid and ascorbic acid (AsA), but less P3G and citric acid. Moreover, genotype significantly influenced phenolic compounds contents in strawberries. Transcriptome analysis revealed that pectin degradation (PL, PG, PE), sucrose synthesis (CWINV, SUS, TPS) and citric acid metabolism (α-OGDH, ICDH, GAD, GS, GDH, PEPCK, AST) were weakened in 'Benihoppe' fruit. In contrast, the synthesis of sucrose (CWINH, SPS), citric acid (CS, PEPC), anthocyanin (F3H, F3'H, F3'5'H, DFR, UFGT and ANS), and citric acid transport (V-ATPase) was enhanced. In 'Fenyu No.1' fruit, the degradation of sucrose, citric acid, and pectin was enhanced, along with the synthesis of malic acid (ME) and ascorbic acid (PMM, MDHAR and GaLUR). However, anthocyanins synthesis, glucose metabolism (HK, G6PI, PFK, G6PDH, PGK, PGM, ENO, PK), fructose metabolism (FK), citric acid synthesis and transport, and AsA degradation (AO, APX) were relatively weak. RT-qPCR results corroborated the transcriptome data. In conclusion, this study revealed the distinctions and characteristics of strawberries with different fruit colors regarding texture, flavor and color formation processes. These findings offer valuable insights for regulating metabolic pathways and identifying key candidate genes to improve strawberry quality.

Genome-wide identification of Rosa roxburghii CML family genes identifies an RrCML13-RrGGP2 interaction involved in calcium-mediated regulation of ascorbate biosynthesis.

Calmodulin-like proteins (CMLs) are an essential family of calcium sensors involved in multiple Ca2+-mediated cellular processes in plants. Rosa roxburghii Tratt, known for the abundance of L-ascorbic acid (AsA) in its fruits, is widely distributed in calcium-rich soil of the karst region in southwestern China. The aim of this study was to identify key CMLs that respond to exogenous Ca2+ levels and regulate AsA biosynthesis in R. roxburghii. A genome-wide scan revealed the presence of 41 RrCML genes with 1-4 EF-hand motif (s) unevenly distributed across the 7 chromosomes of R. roxburghii. qRT-PCR analysis revealed that RrCML13, RrCML10, and RrCML36 responded significantly to exogenous Ca2+ treatment, and RrCML13 was positively correlated with GDP-L-galactose phosphorylase encoding gene (RrGGP2) expression and AsA content in the developing fruit. Overexpression of RrCML13 in fruits and roots significantly promoted the transcription of RrGGP2 and the accumulation of AsA, while virus-induced silencing of RrCML13 reduced the transcription of RrGGP2 and the content of AsA. Furthermore, Moreover, the yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analysis confirmed the interaction between RrCML13 and RrGGP2 proteins, indicating that RrCML13 plays a regulatory role in calcium-mediated AsA biosynthesis. This study enhances our understanding of R. roxburghii CMLs and sheds light on the calcium-mediated regulation of AsA biosynthesis.

Inferences on the evolution of the ascorbic acid synthesis pathway in insects using Phylogenetic Tree Collapser (PTC), a tool for the automated collapsing of phylogenetic trees using taxonomic information.

When inferring the evolution of a gene/gene family, it is advisable to use all available coding sequences (CDS) from as many species genomes as possible in order to infer and date all gene duplications and losses. Nowadays, this means using hundreds or even thousands of CDSs, which makes the inferred phylogenetic trees difficult to visualize and interpret. Therefore, it is useful to have an automated way of collapsing large phylogenetic trees according to a taxonomic term decided by the user (family, class, or order, for instance), in order to highlight the minimal set of sequences that should be used to recapitulate the full history of the gene/gene family being studied at that taxonomic level, that can be refined using additional software. Here we present the Phylogenetic Tree Collapser (PTC) program (https://github.com/pegi3s/phylogenetic-tree-collapser), a flexible tool for automated tree collapsing using taxonomic information, that can be easily used by researchers without a background in informatics, since it only requires the installation of Docker, Podman or Singularity. The utility of PTC is demonstrated by addressing the evolution of the ascorbic acid synthesis pathway in insects. A Docker image is available at Docker Hub (https://hub.docker.com/r/pegi3s/phylogenetic-tree-collapser) with PTC installed and ready-to-run.

GmHXK2 promotes the salt tolerance of soybean seedlings by mediating AsA synthesis, and auxin synthesis and distribution.

BACKGROUND: Salt is an important factor that affects crop productivity. Plant hexokinases (HXKs) are key enzymes in the glycolytic pathway and sugar signaling transduction pathways of plants. In previous studies, we identified and confirmed the roles of GmHXK2 in salt tolerance. RESULTS: In this study, we analyzed the tissue-specific expression of GmHXK2 at different growth stages throughout the plant's life cycle. The results showed that GmHXK2 was expressed significantly in all tissues at vegetative stages, including germination and seedling. However, no expression was detected in the pods, and there was little expression in flowers during the later mature period. Arabidopsis plants overexpressing the GmHXK2 (OE) had more lateral roots. The OE seedlings also produced higher levels of auxin and ascorbic acid (AsA). Additionally, the expression levels of genes PMM, YUC4/YUC6/YUC8, and PIN/LAX1,LAX3, which are involved respectively in the synthesis of AsA and auxin, as well as polar auxin transport, were upregulated in OE plants. This upregulation occurred specifically under exogenous glucose treatment. AtHKT1, AtSOS1, and AtNHX1 were up-regulated in OE plants under salt stress, suggesting that GmHXK2 may modulate salt tolerance by maintaining ion balance within the cells and alleviating damage caused by salt stress. Additionally, we further confirmed the interaction between GmHXK2 and the protein GmPMM through yeast two-hybridization and bimolecular fluorescence complementation assays, respectively. CONCLUSION: The expression of GmHXK2 gene in plants is organ-specific and developmental stage specific. GmHXK2 not only regulates the synthesis of AsA and the synthesis and distribution of auxin, but also promotes root elongation and induces lateral root formation, potentially enhancing soil water absorption. This study reveals the crosstalk between sugar signaling and hormone signaling in plants, where GmHXK2 acts as a glucose sensor through its interaction with GmPMM, and sheds light on the molecular mechanism by which GmHXK2 gene is involved in salt tolerance in plants.

The chromosome-level genome and functional database accelerate research about biosynthesis of secondary metabolites in Rosa roxburghii.

Rosa roxburghii Tratt, a valuable plant in China with long history, is famous for its fruit. It possesses various secondary metabolites, such as L-ascorbic acid (vitamin C), alkaloids and poly saccharides, which make it a high nutritional and medicinal value. Here we characterized the chromosome-level genome sequence of R. roxburghii, comprising seven pseudo-chromosomes with a total size of 531 Mb and a heterozygosity of 0.25%. We also annotated 45,226 coding gene loci after masking repeat elements. Orthologs for 90.1% of the Complete Single-Copy BUSCOs were found in the R. roxburghii annotation. By aligning with protein sequences from public platform, we annotated 85.89% genes from R. roxburghii. Comparative genomic analysis revealed that R. roxburghii diverged from Rosa chinensis approximately 5.58 to 13.17 million years ago, and no whole-genome duplication event occurred after the divergence from eudicots. To fully utilize this genomic resource, we constructed a genomic database RroFGD with various analysis tools. Otherwise, 69 enzyme genes involved in L-ascorbate biosynthesis were identified and a key enzyme in the biosynthesis of vitamin C, GDH (L-Gal-1-dehydrogenase), is used as an example to introduce the functions of the database. This genome and database will facilitate the future investigations into gene function and molecular breeding in R. roxburghii.

Structure, mechanism, and evolution of the last step in vitamin C biosynthesis.

Photosynthetic organisms, fungi, and animals comprise distinct pathways for vitamin C biosynthesis. Besides this diversity, the final biosynthetic step consistently involves an oxidation reaction carried out by the aldonolactone oxidoreductases. Here, we study the origin and evolution of the diversified activities and substrate preferences featured by these flavoenzymes using molecular phylogeny, kinetics, mutagenesis, and crystallographic experiments. We find clear evidence that they share a common ancestor. A flavin-interacting amino acid modulates the reactivity with the electron acceptors, including oxygen, and determines whether an enzyme functions as an oxidase or a dehydrogenase. We show that a few side chains in the catalytic cavity impart the reaction stereoselectivity. Ancestral sequence reconstruction outlines how these critical positions were affixed to specific amino acids along the evolution of the major eukaryotic clades. During Eukarya evolution, the aldonolactone oxidoreductases adapted to the varying metabolic demands while retaining their overarching vitamin C-generating function.

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event.

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

[Verification of the peanut vitamin C synthesis-related gene AhPMM and its role in stress resistance].

Vitamin C plays an important role in plant antioxidation, photosynthesis, growth and development, and metabolism. In this study, a gene AhPMM, which is involved in vitamin C synthesis and responds significantly to low temperature, NaCl, polyethylene glycol (PEG) and abscisic acid (ABA) treatments, was cloned from peanut. An AhPMM overexpression vector was constructed, and transferred to a peanut variety Junanxiaohong using the pollen tube injection method. PCR test on the T3 generation transgenic peanut plants showed a transgenics positive rate of 42.3%. HPLC was used to determine the content of reducing vitamin C (AsA) and total vitamin C in the leaves of transgenic plants. The results showed that the content of AsA in some lines increased significantly, up to 1.90 times higher than that of the control, and the total vitamin content increased by up to 1.63 times compared to that of the control. NaCl and ABA tolerance tests were carried out on transgenic seeds. The results showed that the salt tolerance of transgenic seeds was significantly enhanced and the sensitivity to ABA was weakened compared to that of the non-transgenic control. Moreover, the salt tolerance of the transgenic plants was also significantly enhanced compared to that of the non-transgenic control. The above results showed that AhPMM gene not only increased the vitamin C content of peanut, but also increased the salt tolerance of transgenic peanut seeds and plants. This study may provide a genetic source for the molecular breeding of peanut for enhanced salt tolerance.

Alternative pathways leading to ascorbate biosynthesis in plants: lessons from the last 25 years.

l-Ascorbic acid (AsA) is an antioxidant with important roles in plant stress physiology, growth, and development. AsA also plays an essential role in human health, preventing scurvy. Humans do not synthesize AsA, which needs to be supplied via a diet rich in fresh produce. Research efforts have provided progress in the elucidation of a complex metabolic network with at least four routes leading to AsA formation in plants. In this review, three alternative pathways, namely the d-galacturonate, the l-gulose, and the myo-inositol pathways, are presented with the supporting evidence of their operation in multiple plant species. We critically discuss feeding studies using precursors and their conversion to AsA in plant organs, and research where the expression of key genes encoding enzymes involved in the alternative pathways showed >100% AsA content increase in the transgenics and in many cases accompanied by enhanced tolerance to multiple stresses. We propose that the alternative pathways are vital in AsA production in response to stressful conditions and to compensate in cases where the flux through the d-mannose/l-galactose pathway is reduced. The genes and enzymes that have been characterized so far in these alternative pathways represent important tools that are being used to develop more climate-tolerant crops.

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.

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.

Structural insights into the Smirnoff-Wheeler pathway for vitamin C production in the Amazon fruit camu-camu.

l-Ascorbic acid (AsA, vitamin C) is a pivotal dietary nutrient with multifaceted importance in living organisms. In plants, the Smirnoff-Wheeler pathway is the primary route for AsA biosynthesis, and understanding the mechanistic details behind its component enzymes has implications for plant biology, nutritional science, and biotechnology. As part of an initiative to determine the structures of all six core enzymes of the pathway, the present study focuses on three of them in the model species Myrciaria dubia (camu-camu): GDP-d-mannose 3',5'-epimerase (GME), l-galactose dehydrogenase (l-GalDH), and l-galactono-1,4-lactone dehydrogenase (l-GalLDH). We provide insights into substrate and cofactor binding and the conformational changes they induce. The MdGME structure reveals a distorted substrate in the active site, pertinent to the catalytic mechanism. Mdl-GalDH shows that the way in which NAD+ association affects loop structure over the active site is not conserved when compared with its homologue in spinach. Finally, the structure of Mdl-GalLDH is described for the first time. This allows for the rationalization of previously identified residues which play important roles in the active site or in the formation of the covalent bond with FAD. In conclusion, this study enhances our understanding of AsA biosynthesis in plants, and the information provided should prove useful for biotechnological applications.

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.

F-box protein MdAMR1L1 regulates ascorbate biosynthesis in apple by modulating GDP-mannose pyrophosphorylase.

Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.

Supplementary Far-Red and Blue Lights Influence the Biomass and Phytochemical Profiles of Two Lettuce Cultivars in Plant Factory.

Three different LED spectra (W: White light; WFR: W + far-red light; WB: W + blue light) with similar photosynthetic photon flux density (PPFD) were designed to explore the effects of supplementary far-red and blue lights on leaf color, biomass and phytochemicals of two cultivars of red-leaf lettuce ("Yanzhi" and "Red Butter") in an artificial lighting plant factory. Lettuce plants under WB had redder leaf color and significantly higher contents of pigments, such as chlorophyll a, chlorophyll b, chlorophyll (a + b) and anthocyanins. The accumulation of health-promoting compounds, such as vitamin C, vitamin A, total phenolic compounds, total flavonoids and anthocyanins in the two lettuce cultivars were obviously enhanced by WB. Lettuce under WFR showed remarkable increase in fresh weight and dry weight; meanwhile, significant decreases of pigments, total phenolic compounds, total flavonoids and vitamin C were found. Thus, in the plant factory system, the application of WB can improve the coloration and quality of red leaf lettuce while WFR was encouraged for the purpose of elevating the yield of lettuce.

Comparative Transcriptome Analysis Revealed the Key Genes Regulating Ascorbic Acid Synthesis in Actinidia.

Actinidia (kiwifruit) is known as 'the king of vitamin C' due to its rich ascorbic acid (AsA) concentration, which makes it an important model for studying the regulation of AsA metabolism. Herein, transcriptomic analysis was employed to identify candidate genes that regulate AsA synthesis in Actinidia species with 100-fold variations in fruit AsA content (A. latifolia and A. rufa). Approximately 1.16 billion high-quality reads were generated, and an average of 66.68% of the data was uniquely aligned against the reference genome. AsA-associated DEGs that predominately respond to abiotic signals, and secondary metabolic pathways were identified. The key candidate genes, for instance, GDP-L-galactose phosphorylase-3 (GGP3), were explored according to integrated analysis of the weighted gene co-expression network and L-galactose pathway. Transgenic kiwifruit plants were generated, and the leaves of GGP3 (OE-GGP3) overexpressing lines had AsA contents 2.0- to 6.4-fold higher than those of the wild type. Transcriptomic analysis of transgenic kiwifruit lines was further implemented to identify 20 potential downstream target genes and understand GGP3-regulated cellular processes. As a result, two transcription factors (AcESE3 and AcMYBR) were selected to carry out yeast two-hybrid and BiFC assays, which verified that there were obvious AcESE3-AcMYBR and AcESE3-AcGGP3 protein-protein interactions. This study provides insight into the mechanism of AsA synthesis and provides candidate factors and genes involved in AsA accumulation in kiwifruit.

Enzymatic Production of Ascorbic Acid-2-Phosphate by Engineered Pseudomonas aeruginosa Acid Phosphatase.

l-Ascorbic acid-2-phosphate (AsA-2P) is stable in aqueous solution and at high temperatures and is widely used in foods, pharmaceuticals, cosmetics, and fodders; however, practical application of enzymatic synthesis methods to promote industrial-scale production of AsA-2P remains a major challenge. In this study, we enhanced the phosphorylation efficiency of Pseudomonas aeruginosa acid phosphatase (APase; EC 3.1.3.2) for AsA-2P production via protein engineering. Among the mutants obtained, we selected the most efficient mutant (Var5; G125E/D135T/S136N), which exhibited an increased kcat of 18.6 s-1 and a Km for AsA of 223.9 mM. In addition, Var5 exhibited a maximum enzyme activity of 2080.4 U/L after 10 h of fermentation, which was 80% higher than the wild-type enzyme. Furthermore, under optimal conditions, Var5 showed a maximal conversion of 48.6% and achieved a final AsA-2P titer of 61.5 g/L at 8 h, which is considerably higher than that reported for other similar biocatalytic approaches. These findings demonstrate the potential of this method for the large-scale production of AsA-2P.