Abstract Non-phosphorylating glyceraldehyde- 3-phosphate dehydrogenase (NP-GAPDH) is a con- served cytosolic protein found in higher plants. In photosynthetic cells, the enzyme is involved in a shuttle transfer mechanism to export NADPH from the chlo- roplast to the cytosol. To investigate the role of this enzyme in plant tissues, we characterized a mutant from Arabidopsis thaliana having an insertion at the NP-GAPDH gene locus. The homozygous mutant was determined to be null respect to NP-GAPDH, as it exhibited undetectable levels of both transcription of NP-GAPDH mRNA, protein expression and enzyme activity. Transcriptome analysis demonstrated that the insertion mutant plant shows altered expression of several enzymes involved in carbohydrate metabolism. Significantly, cytosolic phosphorylating (NAD-depen- dent) glyceraldehyde-3-phosphate dehydrogenase mRNA levels are induced in the mutant, which cor- relates with an increase in enzyme activity. mRNA levels and enzymatic activity of glucose-6-phosphate dehydrogenase were also elevated, correlating with an increase in NADPH concentration. Moreover, increased ROS levels were measured in the mutant plants. Down-regulation of several glycolytic and photosynthetic genes suggests that NP-GAPDH is important for the efficiency of both metabolic processes. The results presented demonstrate that NP-GAPDH has a relevant role in plant growth and development. Keywords Glyceraldehydes-3-P dehydrogenase Æ Plant glycolysis Æ Arabidopsis Introduction The major pathways involving carbohydrate metabo- lism, such as glycolysis, pentose phosphate pathway (PPP) and tricarboxylic acid (TCA) cycle were eluci- dated many years ago, mainly through studies per- formed in bacteria and animal cells. At the present, the occurrence and regulation of a number of critical steps remain unclear for these metabolic pathways, in par- ticular in higher plants. For instance, in plants, glycolysis exhibits unique features that suppose differences respect to compartmentalization, enzymes properties and regulation (Plaxton 1996; Fernie et al. 2004). Beyond the existence of different reports on the bio- chemical organization and characterization of plant glycolysis, the whole understanding of the metabolism is not complete. There are several recent studies describ- ing the posttranscriptional regulation and localization of glycolytic enzymes, such as phosphoglucomutase, phosphoglycerate mutase, pyruvate kinase (PK) and glyceraldehydes-3-P-dehydrogenase (GAPDH) in association with photosynthetic metabolism (Fernie S. P. Rius Æ D. F. Gomez-Casati IIB-INTECH, UNSAM-CONICET, CC 164 (7130) Chascomu ´ s, Argentina P. Casati Centro de Estudios Fotosinte ´ticos y Bioquı´micos (CEFOBI), Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina A. A. Iglesias (&) Laboratorio de Enzimologı´a Molecular, Facultad de Bioquı´mica yCiencias Biolo ´ gicas, Universidad Nacional del Litoral, Paraje ‘‘El Pozo’’, CC 242, S3000ZAA Santa Fe, Argentina e-mail: iglesias@fbcb.unl.edu.ar Plant Mol Biol (2006) 61:945–957 DOI 10.1007/s11103-006-0060-5 123 Characterization of an Arabidopsis thaliana mutant lacking a cytosolic non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase Sebastia ´n P. Rius Æ Paula Casati Æ Alberto A. Iglesias Æ Diego F. Gomez-Casati Received: 26 January 2006 / Accepted: 29 March 2006 Ó Springer Science+Business Media B.V. 2006