title: HPLC-MS/MS analyses show that the near-Starchless aps1 and pgm leaves accumulate wild type levels of ADPglucose: further evidence for the occurrence of important ADPglucose biosynthetic pathway(s) alternative to the pPGI-pPGM-AGP pathway.
abstract: In leaves, it is widely assumed that starch is the end-product of a metabolic pathway exclusively taking place in the chloroplast that (a) involves plastidic phosphoglucomutase (pPGM), ADPglucose (ADPG) pyrophosphorylase (AGP) and starch synthase (SS), and (b) is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase (pPGI). This view also implies that AGP is the sole enzyme producing the starch precursor molecule, ADPG. However, mounting evidence has been compiled pointing to the occurrence of important sources, other than the pPGI-pPGM-AGP pathway, of ADPG. To further explore this possibility, in this work two independent laboratories have carried out HPLC-MS/MS analyses of ADPG content in leaves of the near-starchless pgm and aps1 mutants impaired in pPGM and AGP, respectively, and in leaves of double aps1/pgm mutants grown under two different culture conditions. We also measured the ADPG content in wild type (WT) and aps1 leaves expressing in the plastid two different ADPG cleaving enzymes, and in aps1 leaves expressing in the plastid GlgC, a bacterial AGP. Furthermore, we measured the ADPG content in ss3/ss4/aps1 mutants impaired in starch granule initiation and chloroplastic ADPG synthesis. We found that, irrespective of their starch contents, pgm and aps1 leaves, WT and aps1 leaves expressing in the plastid ADPG cleaving enzymes, and aps1 leaves expressing in the plastid GlgC accumulate WT ADPG content. In clear contrast, ss3/ss4/aps1 leaves accumulated ca. 300 fold-more ADPG than WT leaves. The overall data showed that, in Arabidopsis leaves, (a) there are important ADPG biosynthetic pathways, other than the pPGI-pPGM-AGP pathway, (b) pPGM and AGP are not major determinants of intracellular ADPG content, and (c) the contribution of the chloroplastic ADPG pool to the total ADPG pool is low.
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Abdellatif
authors.initials: A
authors.lastname: Bahaji
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Edurne
authors.initials: E
authors.lastname: Baroja-Fernández
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Angela María
authors.initials: AM
authors.lastname: Sánchez-López
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Francisco José
authors.initials: FJ
authors.lastname: Muñoz
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Goizeder
authors.initials: G
authors.lastname: Almagro
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Montero
authors.affiliation: Servicio de Apoyo a la Investigación, Universidad Pública de Navarra, Campus de Arrosadia, Iruña, Nafarroa, Spain.
authors.forename: Pablo
authors.initials: P
authors.lastname: Pujol
authors.affiliation: Servicio de Apoyo a la Investigación, Universidad Pública de Navarra, Campus de Arrosadia, Iruña, Nafarroa, Spain.
authors.forename: Regina
authors.initials: R
authors.lastname: Galarza
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Kaneko
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata, Japan.
authors.forename: Kazusato
authors.initials: K
authors.lastname: Oikawa
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata, Japan.
authors.forename: Kaede
authors.initials: K
authors.lastname: Wada
authors.affiliation: Department of Applied Biological Chemistry, Niigata University, Niigata, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Mitsui
authors.affiliation: Instituto de Agrobiotecnología, Universidad Pública de Navarra/Consejo Superior de Investigaciones Científicas/Gobierno de Navarra, Mutiloabeti, Nafarroa, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Pozueta-Romero
mesh_terms: D000245:Adenosine Diphosphate Glucose / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D051599:Glucose-1-Phosphate Adenylyltransferase / Q000235:genetics / Q000378:metabolism*; D005956:Glucose-6-Phosphate Isomerase; D018515:Plant Leaves / Q000201:enzymology* / Q000378:metabolism*; D018087:Plastids / Q000201:enzymology / Q000378:metabolism; D013214:Starch Synthase / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D029681:Arabidopsis Proteins; D000245:Adenosine Diphosphate Glucose; D013214:Starch Synthase; D051599:Glucose-1-Phosphate Adenylyltransferase; D005956:Glucose-6-Phosphate Isomerase