GAPCp is a crucial enzyme in heterotrophic cells

Summary. A plastidial glycolytic enzyme modulates the metabolism of carbon and nitrogen in non-photosynthetic cells, and affects plant organ development. ABSTRACT 57 This study functionally characterizes the Arabidopsis thaliana plastidial glycolytic 58 isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and 59 heterotrophic cells. We expressed the enzyme in gapcp double mutants ( gapcp1gapcp2 ) 60 under the control of photosynthetic (RUBISCO small subunit RBCS2B ; RBCS ), or 61 heterotrophic (phosphate transporter PHT1.2; PHT ), cell-specific promoters. Expression 62 of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the 63 metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the 64 control of PHT promoter clearly affected Arabidopsis development, by increasing the 65 number of lateral roots and having a major effect on the AP growth and metabolite 66 profile. Our results indicate that GAPCp1 is not functionally important in 67 photosynthetic cells, but plays a fundamental role in roots and in heterotrophic cells of 68 the AP. Specifically GAPCp activity may be required in root meristems and the root cap 69 for normal primary root growth. Transcriptomic and metabolomic analyses indicate that 70 lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral 71 nutrition, and that glycerate and glutamine are the main metabolites responding to 72 GAPCp activity. Thus GAPCp could be an important metabolic connector of glycolysis 73 with other pathways such as the phosphorylated pathway of serine biosynthesis, the 74 ammonium assimilation pathway or the metabolism of GABA which in turn affect plant 75 development. 76 77 78 79 Knowledge concerning the specific roles of glycolysis in photosynthetic and heterotrophic cells is scarce. Given that gapcp1gapcp2 displays a drastic growth reduction of the AP, we explored the specific effect of GAPCp in photosynthetic cells by expressing the enzyme under the control of the RBCS promoter. Morphological and metabolomic analysis indicated that RBCS:GAPCp1 expression did not complement the reduced growth phenotype either in roots or in the AP. The metabolite profile of the AP in gapcp1gapcp2 and gapcp1gapcp2 RBCS:GAPCp1 was similar under both photosynthetic and non-photosynthetic conditions. These results indicate that plastidial glycolysis, or at least GAPCp activity, is not functionally important in photosynthetic cells. GAPCp1 expression under the control of the 35S promoter, however, restored both the growth phenotype and the metabolite profile of gapcp1gapcp2 , suggesting that metabolic alterations in the AP could be caused by lack of GAPCp activity in roots or in other AP cell types. In this sense, GAPCp1 expression under the control of the PHT promoter improved growth of the AP in gapcp1gapcp2 and modified its metabolite profile. We found that PHT1.2 was not only expressed in roots as described elsewhere (Mudge 2002; Nussaume 2011; Kirchsteiger but also in the leaf epidermal cells of gapcp1gapcp2 . The PHT:GAPCp1 expression in the AP may be related to the P i -deficient phenotype of gapcp1gapcp2 as this promoter is induced by P i starvation in WT roots (Mudge et 2002) although not in the AP, as was observed in gapcp1gapcp2 . Given that GAPCp1 expression in photosynthetic