RT Journal Article
T1 The role of GDP-L-galactose phosphorylase in the control of ascorbate biosynthesis
A1 Fenech, Mario
A1 Amorim-Silva, Vitor
A1 Esteban del Valle, Alicia
A1 Arnoud, Dominique
A1 Ruiz-López, Noemí
A1 Castillo-Garriga, Araceli
A1 Smirnoff, Nicholas
A1 Botella-Mesa, Miguel Ángel
K1 Vitamina C-Síntesis
AB 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.
PB Oxford University Press in behalf of American Society of Plant Biologists
YR 2021
FD 2021-01-25
LK https://hdl.handle.net/10630/34529
UL https://hdl.handle.net/10630/34529
LA eng
NO Mario Fenech, Vítor Amorim-Silva, Alicia Esteban del Valle, Dominique Arnaud, Noemi Ruiz-Lopez, Araceli G Castillo, Nicholas Smirnoff, Miguel A Botella, The role of GDP-l-galactose phosphorylase in the control of ascorbate biosynthesis, Plant Physiology, Volume 185, Issue 4, April 2021, Pages 1574–1594, https://doi.org/10.1093/plphys/kiab010
NO This work was supported by a grant from the Spanish Ministerio de Educación, Cultura y Deporte para la formación del Profesorado Universitario (FPU014/01974), I Plan Propio de Investigación, Transferencia y Divulgación Científica de la Universidad de Málaga, The Ministerio de Economía, Industria y Competitividad (RYC-2013-12699), also co-financed by the European Regional Development Fund (BIO2016-81957-REDT, BIO2017-82609-R), the Spanish “Ministerio de Economía, Industria y Competitividad/FEDER” (AGL2016-75819-C2-1-R) and the Spanish Ministerio de Ciencia, Innovación y Universidades (PGC2018-098789-B-I00). N.S. was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/G021678/1 and BB/N001311/1.
DS RIUMA. Repositorio Institucional de la Universidad de Málaga
RD 19 ene 2026