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

Abstract

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.

Keywords: ascorbate biosynthesis; genetic control; metabolic pathways; regulation; vitamin C.

Figure 1

Proposed pathways for AsA biosynthesis in plants. The photosynthetic process provides the pool of hexose monophosphates as precursors for some AsA biosynthetic pathways. From left to right: D-galacturonic acid pathway (violet), L-galactose pathway (blue), L-gulose pathway (red), and myo-inositol pathway (black). The last enzymatic reaction for AsA biosynthesis occurs in the mitochondria.

Figure 2

L-galactose pathway for AsA biosynthesis in plants. The eight enzymes involved in AsA biosynthesis are phosphomannose isomerase (PMI: EC 5.3.1.8), phosphomannomutase (PMM: EC 5.4.2.8), GDP-D-mannose pyrophosphorylase (GMP: EC 2.7.7.13), GDP-D-mannose 3’,5’-epimerase (GME: EC 5.1.3.18), GDP-L-galactose phosphorylase (GGP: EC 2.7.7.69), L-galactose-1-phosphate phosphatase (GPP: EC 3.1.3.25), L-galactose dehydrogenase (GDH: EC 1.1.1.117), and L-galactono-1,4-lactone dehydrogenase (GLDH: EC 1.3.2.3).

Figure 3

Experimental and predicted tridimensional structures of enzymes from the L-galactose pathway for AsA biosynthesis. Experimentally determined tridimensional structures by X-ray diffraction for the following enzymes: GMP from Arabidopsis thaliana “thale cress” (PDB code: 7X8K), GME from Arabidopsis thaliana “thale cress” (PDB code: 2C54), GDH from Spinacia oleracea “spinach” (PDB code: 7SMI), and GLDH from Myrciaria dubia “camu-camu” (PDB code: 7SML). Predicted tridimensional structures determined by using SWISS-MODEL for the following enzyme: PMI from Arabidopsis thaliana using as a template the three-dimensional structure of PMI from Candida albicans (PDB code 1PMI). Predicted tridimensional structures determined by using AlphaFold for the following enzymes: GGP from Arabidopsis thaliana (AlphaFoldDB: Q8RWE8), and GPP from Arabidopsis thaliana (AlphaFoldDB: Q9M8S8). Tridimensional structures were drawn using RasMol v2.7.5.

Figure 4

Genetic strategies for regulation of AsA biosynthesis in plants. When plants are under biotic and abiotic stress, they quickly up-regulate and/or down-regulate the expression of several genes involved directly or indirectly in AsA biosynthesis. Thus, several stressors induce the biosynthesis of jasmonic acid and other jasmonates, which starts intracellular signaling that finally induces the expression of AsA biosynthetic genes, translation of the corresponding enzymes, and increases the intracellular levels of AsA. The light, on its part, also starts an intracellular signaling activating transcription factors that bind with light-responsive promoter elements (LRE) and induces the transcription of gene-encoding enzymes of the L-galactose pathway. Until now, also were identified several transcription factors that activate (e.g., SlICE1, AtERF98, SlHZ24, BZR1, etc.) or inactivate (i.e., NF-Y, ABI4, SINFYA10, and ZmbHLH55) the transcription of the genes. In addition, alternative splicing is a probable mechanism involved in the regulation of AsA biosynthesis. Finally, the GGP gene is controlled via post-transcriptional repression by a conserved cis-acting upstream open reading frame (5’-uORF) present in the 5’-untranslated region of its mRNAs.

Figure 5

Biochemical strategies for regulation of AsA biosynthesis in plants. In plants, several biochemical mechanisms have been described, including control of the enzyme levels, control of the enzyme catalytic activity, feedback inhibition of regulatory enzymes, and other processes.