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.

Abdellatif Bahaji,Goizeder Almagro,Javier Pozueta-Romero,Manuel Montero,Ángela María Sánchez-López,Pablo Pujol,Regina Galarza,Kazusato Oikawa,Kentaro Kaneko,Jun Li,Kaede Wada,Toshiaki Mitsui,Francisco José Muñoz,Edurne Baroja-Fernández,Frederik Börnke

doi:10.1371/journal.pone.0104997

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.

Highlights

Starch is a branched homopolysaccharide of a-1,4-linked glucose subunits with a-1,6-linked glucose at the branched points
Up to 50% of the photosynthetically fixed carbon is retained within the chloroplasts of mesophyll cells during the day to synthesize starch [1,2], which is remobilized during the subsequent night to PLOS ONE | www.plosone.org aps1 and pgm Mutants Accumulate wild type (WT) ADPG Content support non-photosynthetic metabolism and growth by continued export of carbon to the rest of the plant
According to this classical view of starch biosynthesis, starch is considered the end-product of a metabolic pathway that is linked to the Calvin-Benson cycle by means of the plastidic phosphoglucose isomerase

Summary

Introduction

Starch is a branched homopolysaccharide of a-1,4-linked glucose subunits with a-1,6-linked glucose at the branched points. We measured the starch and ADPG contents in leaves of aps1 mutants expressing in the chloroplast the E. coli AGP (GlgC) [15].

Results

Conclusion