The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis.

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.

Deciphering the molecular basis of tissue-specific gene expression in plants: Can synthetic biology help?

Gene expression differences between distinct cell types are orchestrated by specific sets of transcription factors and epigenetic regulators acting upon the genome. In plants, the mechanisms underlying tissue-specific gene activity remain largely unexplored. Although transcriptional and epigenetic profiling of individual organs, tissues, and more recently, of single cells can easily detect the molecular signatures of different biological samples, how these unique cell identities are established at the mechanistic level is only beginning to be decoded. Computational methods, including machine learning, used in combination with experimental approaches, enable the identification and validation of candidate cis-regulatory elements driving cell-specific expression. Synthetic biology shows great promise not only as a means of testing candidate DNA motifs but also for establishing the general rules of nature driving promoter architecture and for the rational design of genetic circuits in research and agriculture to confer tissue-specific expression to genes or molecular pathways of interest.

Vitamin C Content in Fruits: Biosynthesis and Regulation.

Throughout evolution, a number of animals including humans have lost the ability to synthesize ascorbic acid (ascorbate, vitamin C), an essential molecule in the physiology of animals and plants. In addition to its main role as an antioxidant and cofactor in redox reactions, recent reports have shown an important role of ascorbate in the activation of epigenetic mechanisms controlling cell differentiation, dysregulation of which can lead to the development of certain types of cancer. Although fruits and vegetables constitute the main source of ascorbate in the human diet, rising its content has not been a major breeding goal, despite the large inter- and intraspecific variation in ascorbate content in fruit crops. Nowadays, there is an increasing interest to boost ascorbate content, not only to improve fruit quality but also to generate crops with elevated stress tolerance. Several attempts to increase ascorbate in fruits have achieved fairly good results but, in some cases, detrimental effects in fruit development also occur, likely due to the interaction between the biosynthesis of ascorbate and components of the cell wall. Plants synthesize ascorbate de novo mainly through the Smirnoff-Wheeler pathway, the dominant pathway in photosynthetic tissues. Two intermediates of the Smirnoff-Wheeler pathway, GDP-D-mannose and GDP-L-galactose, are also precursors of the non-cellulosic components of the plant cell wall. Therefore, a better understanding of ascorbate biosynthesis and regulation is essential for generation of improved fruits without developmental side effects. This is likely to involve a yet unknown tight regulation enabling plant growth and development, without impairing the cell redox state modulated by ascorbate pool. In certain fruits and developmental conditions, an alternative pathway from D-galacturonate might be also relevant. We here review the regulation of ascorbate synthesis, its close connection with the cell wall, as well as different strategies to increase its content in plants, with a special focus on fruits.