Chromosomal-scale genomes of two Rosa species provide insights into genome evolution and ascorbate accumulation.

Rosa roxburghii and Rosa sterilis, two species belonging to the Rosaceae family, are widespread in the southwest of China. These species have gained recognition for their remarkable abundance of ascorbate in their fresh fruits, making them an ideal vitamin C resource. In this study, we generated two high-quality chromosome-scale genome assemblies for R. roxburghii and R. sterilis, with genome sizes of 504 and 981.2 Mb, respectively. Notably, we present a haplotype-resolved, chromosome-scale assembly for diploid R. sterilis. Our results indicated that R. sterilis originated from the hybridization of R. roxburghii and R. longicuspis. Genome analysis revealed the absence of recent whole-genome duplications in both species and identified a series of duplicated genes that possibly contributing to the accumulation of flavonoids. We identified two genes in the ascorbate synthesis pathway, GGP and GalLDH, that show signs of positive selection, along with high expression levels of GDP-d-mannose 3', 5'-epimerase (GME) and GDP-l-galactose phosphorylase (GGP) during fruit development. Furthermore, through co-expression network analysis, we identified key hub genes (MYB5 and bZIP) that likely regulate genes in the ascorbate synthesis pathway, promoting ascorbate biosynthesis. Additionally, we observed the expansion of terpene synthase genes in these two species and tissue expression patterns, suggesting their involvement in terpenoid biosynthesis. Our research provides valuable insights into genome evolution and the molecular basis of the high concentration of ascorbate in these two Rosa species.

The D-mannose/L-galactose pathway plays a predominant role in ascorbate biosynthesis in the liverwort Marchantia polymorpha but is not regulated by light and oxidative stress.

Ascorbate plays an indispensable role in plants, functioning as both an antioxidant and a cellular redox buffer. It is widely acknowledged that the ascorbate biosynthesis in the photosynthetic tissues of land plants is governed by light-mediated regulation of the D-mannose/L-galactose (D-Man/L-Gal) pathway. At the core of this light-dependent regulation lies the VTC2 gene, encoding the rate-limiting enzyme GDP-L-Gal phosphorylase. The VTC2 expression is regulated by signals via the photosynthetic electron transport system. In this study, we directed our attention to the liverwort Marchantia polymorpha, representing one of the basal land plants, enabling us to conduct an in-depth analysis of its ascorbate biosynthesis. The M. polymorpha genome harbors a solitary gene for each enzyme involved in the D-Man/L-Gal pathway, including VTC2, along with three lactonase orthologs, which may be involved in the alternative ascorbate biosynthesis pathway. Through supplementation experiments with potential precursors, we observed that only L-Gal exhibited effectiveness in ascorbate biosynthesis. Furthermore, the generation of VTC2-deficient mutants through genome editing unveiled the inability of thallus regeneration in the absence of L-Gal supplementation, thereby revealing the importance of the D-Man/L-Gal pathway in ascorbate biosynthesis within M.  polymorpha. Interestingly, gene expression analyses unveiled a distinct characteristic of M. polymorpha, where none of the genes associated with the D-Man/L-Gal pathway, including VTC2, showed upregulation in response to light, unlike other known land plants. This study sheds light on the exceptional nature of M. polymorpha as a land plant that has evolved distinctive mechanisms concerning ascorbate biosynthesis and its regulation.

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.

Evolutionary insights into strategy shifts for the safe and effective accumulation of ascorbate in plants.

Plants accumulate high concentrations of ascorbate, commonly in their leaves, as a redox buffer. While ascorbate levels have increased during plant evolution, the mechanisms behind this phenomenon are unclear. Moreover, has the increase in ascorbate concentration been achieved without imposing any detrimental effects on the plants? In this review, we focus on potential transitions in two regulatory mechanisms related to ascorbate biosynthesis and the availability of cellular dehydroascorbate (DHA) during plant evolution. The first transition might be that the trigger for the transcriptional induction of VTC2, which encodes the rate-limiting enzyme in ascorbate biosynthesis, has shifted from oxidative stress (in green algae) to light/photosynthesis (in land plants), probably enabling the continuous accumulation of ascorbate under illumination. This could serve as a preventive system against the unpredictable occurrence of oxidative stress. The second transition might be that DHA-degrading enzymes, which protect cells from the highly reactive DHA in green algae and mosses, have been lost in ferns or flowering plants. Instead, flowering plants may have increased glutathione concentrations to reinforce the DHA reduction capacity, possibly allowing ascorbate accumulation and avoiding the toxicity of DHA. These potential transitions may have contributed to strategies for plants' safe and effective accumulation of ascorbate.

The d-mannose/l-galactose pathway is the dominant ascorbate biosynthetic route in the moss Physcomitrium patens.

Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d-mannose/l-galactose (d-Man/l-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d-galacturonate (d-GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d-Man/l-Gal pathway and an orthologous gene encoding aldonolactonase (ALase) - a key enzyme for the d-GalUA pathway - Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l-galactonic acid to the final precursor l-galactono-1,4-lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2-fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2-1 (Δvtc2-1) or PpVTC2-2 (Δvtc2-2), which encode the rate-limiting enzyme GDP-l-Gal phosphorylase in the d-Man/l-Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d-Man/l-Gal pathway, not the d-GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis.

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.

L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

Genetic and biochemical strategies for regulation of L-ascorbic acid biosynthesis in plants through the L-galactose pathway.

Vitamin C (L-ascorbic acid, AsA) is an essential compound with pleiotropic functions in many organisms. Since its isolation in the last century, AsA has attracted the attention of the scientific community, allowing the discovery of the L-galactose pathway, which is the main pathway for AsA biosynthesis in plants. Thus, the aim of this review is to analyze the genetic and biochemical strategies employed by plant cells for regulating AsA biosynthesis through the L-galactose pathway. In this pathway, participates eight enzymes encoded by the genes PMI, PMM, GMP, GME, GGP, GPP, GDH, and GLDH. All these genes and their encoded enzymes have been well characterized, demonstrating their participation in AsA biosynthesis. Also, have described some genetic and biochemical strategies that allow its regulation. The genetic strategy includes regulation at transcriptional and post-transcriptional levels. In the first one, it was demonstrated that the expression levels of the genes correlate directly with AsA content in the tissues/organs of the plants. Also, it was proved that these genes are light-induced because they have light-responsive promoter motifs (e.g., ATC, I-box, GT1 motif, etc.). In addition, were identified some transcription factors that function as activators (e.g., SlICE1, AtERF98, SlHZ24, etc.) or inactivators (e.g., SlL1L4, ABI4, SlNYYA10) regulate the transcription of these genes. In the second one, it was proved that some genes have alternative splicing events and could be a mechanism to control AsA biosynthesis. Also, it was demonstrated that a conserved cis-acting upstream open reading frame (5'-uORF) located in the 5'-untranslated region of the GGP gene induces its post-transcriptional repression. Among the biochemical strategies discovered is the control of the enzyme levels (usually by decreasing their quantities), control of the enzyme catalytic activity (by increasing or decreasing its activity), feedback inhibition of some enzymes (GME and GGP), subcellular compartmentation of AsA, the metabolon assembly of the enzymes, and control of AsA biosynthesis by electron flow. Together, the construction of this basic knowledge has been establishing the foundations for generating genetically improved varieties of fruits and vegetables enriched with AsA, commonly used in animal and human feed.

The perils of planning strategies to increase vitamin C content in plants: Beyond the hype.

Ever since the identification of vitamin C (ascorbic acid, AsA) as an essential molecule that humans cannot synthesize on their own, finding adequate dietary sources of AsA became a priority in nutrition research. Plants are the main producers of AsA for humans and other non-synthesizing animals. It was immediately clear that some plant species have more AsA than others. Further studies evidenced that AsA content varies in different plant organs, in different developmental stages/environmental conditions and even within different cell compartments. With the progressive discovery of the genes of the main (Smirnoff-Wheeler) and alternative pathways coding for the enzymes involved in AsA biosynthesis in plants, the simple overexpression of those genes appeared a suitable strategy for boosting AsA content in any plant species or organ. Unfortunately, overexpression experiments mostly resulted in limited, if any, AsA increase, apparently due to a tight regulation of the biosynthetic machinery. Attempts to identify regulatory steps in the pathways that could be manipulated to obtain unlimited AsA production were also less successful than expected, confirming the difficulties in "unleashing" AsA synthesis. A different approach to increase AsA content has been the overexpression of genes coding for enzymes catalyzing the recycling of the oxidized forms of vitamin C, namely monodehydroascorbate and dehydroascorbate reductases. Such approach proved mostly effective in making the overexpressors apparently more resistant to some forms of environmental stress, but once more did not solve the issue of producing massive AsA amounts for human diet. However, it should also be considered that a hypothetical unlimited increase in AsA content is likely to interfere with plant development, which is in many ways regulated by AsA availability itself. The present review article aims at summarizing the many attempts made so far to improve AsA production/content in plants, evidencing the most promising ones, and at providing information about the possible unexpected consequences of a pure biotechnological approach not keeping into account the peculiar features of the AsA system in plants.

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.

Chloroplast development activates the expression of ascorbate biosynthesis-associated genes in Arabidopsis roots.

Transcriptional activation of ascorbate biosynthesis-associated genes under illumination is one of the important steps in ascorbate pool size regulation in photosynthetic tissues. Several biological processes within chloroplasts such as photosynthesis are required for this activation, suggesting functional chloroplasts to play a key role. We herein found that when grown on agar plate, ascorbate content in Arabidopsis non-photosynthetic tissues, roots, are unexpectedly almost comparable to that in shoots. The high accumulation of ascorbate was particularly observed in root regions closer to the root-hypocotyl junction, in which chloroplast development occurred because of a direct exposure to light. When chloroplast development in roots were further stimulated by shoot removal, the expression of biosynthetic genes, especially VTC2 gene that encodes GDP-l-galactose phosphorylase, was activated, resulting in an increase in ascorbate pool size. These positive effects were canceled when the roots were treated with a photosynthetic inhibitor. A null mutation in the LONG HYPOCOTYL 5 (HY5) gene almost completely inhibited root greening as well as the VTC2 expression. Overall, these findings show that chloroplast development can trigger the expression of ascorbate biosynthesis-associated genes not only in leaves but also in roots.

GhVTC1, the Key Gene for Ascorbate Biosynthesis in Gossypium hirsutum, Involves in Cell Elongation Under Control of Ethylene.

L-Ascorbate (Asc) plays important roles in cell growth and plant development, and its de novo biosynthesis was catalyzed by the first rate-limiting enzyme VTC1. However, the function and regulatory mechanism of VTC1 involved in cell development is obscure in Gossypium hirsutum. Herein, the Asc content and AsA/DHA ratio were accumulated and closely linked with fiber development. The GhVTC1 encoded a typical VTC1 protein with functional conserved domains and expressed preferentially during fiber fast elongation stages. Functional complementary analysis of GhVTC1 in the loss-of-function Arabidopsis vtc1-1 mutants indicated that GhVTC1 is genetically functional to rescue the defects of mutants to normal or wild type (WT). The significant shortened primary root in vtc1-1 mutants was promoted to the regular length of WT by the ectopic expression of GhVTC1 in the mutants. Additionally, GhVTC1 expression was induced by ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), and the GhVTC1 promoter showed high activity and included two ethylene-responsive elements (ERE). Moreover, the 5'-truncted promoters containing the ERE exhibited increased activity by ACC treatment. Our results firstly report the cotton GhVTC1 function in promoting cell elongation at the cellular level, and serve as a foundation for further understanding the regulatory mechanism of Asc-mediated cell growth via the ethylene signaling pathway.

Concentration Does Matter: The Beneficial and Potentially Harmful Effects of Ascorbate in Humans and Plants.

SIGNIFICANCE: Ascorbate (Asc) is an essential compound both in animals and plants, mostly due to its reducing properties, thereby playing a role in scavenging reactive oxygen species (ROS) and acting as a cofactor in various enzymatic reactions. Recent Advances: Growing number of evidence shows that excessive Asc accumulation may have negative effects on cellular functions both in humans and plants; inter alia it may negatively affect signaling mechanisms, cellular redox status, and contribute to the production of ROS via the Fenton reaction. CRITICAL ISSUES: Both plants and humans tightly control cellular Asc levels, possibly via biosynthesis, transport, and degradation, to maintain them in an optimum concentration range, which, among other factors, is essential to minimize the potentially harmful effects of Asc. On the contrary, the Fenton reaction induced by a high-dose Asc treatment in humans enables a potential cancer-selective cell death pathway. FUTURE DIRECTIONS: The elucidation of Asc induced cancer selective cell death mechanisms may give us a tool to apply Asc in cancer therapy. On the contrary, the regulatory mechanisms controlling cellular Asc levels are also to be considered, for example, when aiming at generating crops with elevated Asc levels.

Characterization of a L-Gulono-1,4-Lactone Oxidase Like Protein in the Floral Nectar of Mucuna sempervirens, Fabaceae.

Floral nectar plays important roles in the interaction between animal-pollinated plants and pollinators. Its components include water, sugars, amino acids, vitamins, and proteins. Growing empirical evidence shows that most of the proteins secreted in nectar (nectarines) are enzymes that can tailor nectar chemistry for their animal mutualists or reduce the growth of microorganisms in nectar. However, to date, the function of many nectarines remains unknown, and very few plant species have had their nectar proteome thoroughly investigated. Mucuna sempervirens (Fabaceae) is a perennial woody vine native to China. Nectarines from this species were separated using two-dimensional gel electrophoresis, and analyzed using mass spectrometry. A L-gulonolactone oxidase like protein (MsGulLO) was detected, and the full length cDNA was cloned: it codes for a protein of 573 amino acids with a predicted signal peptide. MsGulLO has high similarity to L-gulonolactone oxidase 5 (AtGulLO5) in Arabidopsis thaliana, which was suggested to be involved in the pathway of ascorbate biosynthesis; however, both MsGulLO and AtGulLO5 are divergent from animal L-gulonolactone oxidases. MsGulLO was expressed mainly in flowers, and especially in nectary before blooming. However, cloning and gene expression analysis showed that L-galactonolactone dehydrogenase (MsGLDH), a vital enzyme in plant ascorbate biosynthesis, was expressed in all of flowers, roots, stems, and especially leaves. MsGulLO was purified to near homogeneity from raw MS nectar by gel filtration chromatography. The enzyme was determined to be a neutral monomeric protein with an apparent molecular mass of 70 kDa. MsGulLO is not a flavin-containing protein, and has neither L-galactonolactone dehydrogenase activity, nor the L-gulonolactone activity that is usual in animal GulLOs. However, it has weak oxidase activity with the following substrates: L-gulono-1,4-lactone, L -galactono-1,4-lactone, D-gluconic acid-δ-lactone, glucose, and fructose. MsGulLO is suggested to function in hydrogen peroxide generation in nectar but not in plant ascorbate biosynthesis.

Genetic Control of Ascorbic Acid Biosynthesis and Recycling in Horticultural Crops.

Ascorbic acid (AsA) is an essential compound present in almost all living organisms that has important functions in several aspects of plant growth and development, hormone signaling, as well as stress defense networks. In recent years, the genetic regulation of AsA metabolic pathways has received much attention due to its beneficial role in human diet. Despite the great variability within species, genotypes, tissues and developmental stages, AsA accumulation is considered to be controlled by the fine orchestration of net biosynthesis, recycling, degradation/oxidation, and/or intercellular and intracellular transport. To date, several structural genes from the AsA metabolic pathways and transcription factors are considered to significantly affect AsA in plant tissues, either at the level of activity, transcription or translation via feedback inhibition. Yet, all the emerging studies support the notion that the steps proceeding through GDP-L-galactose phosphorylase and to a lesser extent through GDP-D-mannose-3,5-epimerase are control points in governing AsA pool size in several species. In this mini review, we discuss the current consensus of the genetic regulation of AsA biosynthesis and recycling, with a focus on horticultural crops. The aspects of AsA degradation and transport are not discussed herein. Novel insights of how this multifaceted trait is regulated are critical to prioritize candidate genes for follow-up studies toward improving the nutritional value of fruits and vegetables.

Effect of nitrogen deficiency on ascorbic acid biosynthesis and recycling pathway in cucumber seedlings.

L-Ascorbic acid (AsA, ascorbate) is one of the most abundant natural antioxidants, and it is an important factor in the nutritional quality of cucumber. In this work, key enzymes involved in the ascorbic acid biosynthesis and recycling pathway in cucumber seedlings under nitrogen deficiency were investigated at the levels of transcription and enzyme activity. The activities of myo-inositol oxygenase (MIOX) and transcript levels of MIOXs increased dramatically, while the activities of ascorbate oxidase (AO) and glutathione reductase (GR) and transcript levels of AOs and GR2 decreased significantly in N-limited leaves, as did the ascorbate concentration, in nitrogen-deficient cucumber seedlings. The activities of other enzymes and transcript levels of other genes involved in the ascorbate recycling pathway and ascorbate synthesis pathways decreased or remained unchanged under nitrogen deficiency. These results indicate that nitrogen deficiency induced genes involved in the ascorbate-glutathione recycling and myo-inositol pathway in cucumber leaves. Thus, the AO, GR and MIOX involved in the pathways might play roles in AsA accumulation.

Transcriptomic Analysis Reveals the Metabolic Mechanism of L-Ascorbic Acid in Ziziphus jujuba Mill.

Chinese jujube (Ziziphus jujuba Mill.) is the most economically important member of the Rhamnaceae family and contains a high concentration of ascorbic acid (AsA). To explore the metabolic mechanism of AsA accumulation, we investigated the abundance of AsA in the fruit development stages, the leaf and flower of Z. jujuba cv Junzao, and the mature fruit of one type of wild jujube (Z. jujuba var. spinosa Hu, Yanchuan sour jujube). And the expression patterns of genes involved in AsA biosynthesis, degradation, and recycling were analyzed. The result showed that AsA biosynthesis during early fruit development (the enlargement stage) is the main reason for jujube high accumulation. The L-galactose pathway plays a predominant role in the biosynthesis of AsA during jujube fruit development, and the genes GMP1, GME1, GGP, and GaLDH involved in the determination of AsA concentration during fruit development and in different genotypes; the myo-inositol pathway along with the genes GME2 and GMP2 in the L-galactose pathway play a compensatory role in maintaining AsA accumulation during the ripening stage. These findings enhance our understanding of the molecular mechanism in regulating AsA accumulation for jujube.