Functional Analysis of Singleton and Gene Families Figure 5. Schematic Representation of the Role of Rca1, Shmt1, U4, U5, Nr1, Ca1, and Psb28 in Photorespiration, Ammonia Assimilation, Nitrate Assimilation, and Photosynthetic Light Reaction Pathways. Photorespiration occurs in three organelles: the chloroplast, the peroxisome, and the mitochondria. Ammonia association pathway is tightly linked to photorespiration for recycling ammonia, byproduct of photorespiration. Rubisco activase 1 (Rca1) commits photorespiration by helping to incorporate O2 to active site of ribulose bisphosphate carboxylase/oxygenase (RUBISCO) and ribulose bisphosphate (RuBP) is oxidized to 3-phosphoglycerate (glycerate 3-P) and 2- phosphoglycolate (glycolate 2-P). Then, glycolate 2-P is converted to glycine by a series of reactions: 2-phosphoglycolate phosphatase1 (PGP1), glycolate oxidase (GLO1) serine-glyoxylate aminotransferase (SGAT). The decarboxylation of two glycines produces serine, CO2 and NH3 by glycine decarboxlasse complex (GDC) and serine hydroxymethyltransferase 1 (SHMT1, P5-1). In addition, gulatamate dehydrogenase (GDH) in mitochondria deaminates glutamate and generates 2-oxoglutarate (2-OG) and NH3 needed for ammonia assimilation (GS2/Fd-GOGAT cycle). SGAT also generates 2-OG from glutamate. Glutamate is supplied from chloroplast by glutamine synthase 2 (GS2) and ferredoxin dependant glutamine-oxoglutarate aminotransferase (Fd-GOGAT, U11) in ammonia assimilation, and glutamate/malate transporter (DiT2). Function of U5 gene product is suggested to transport 2-oxoglutarate to chloroplast. Serine is further converted to 3-phosphoglycerate by hydroxypyruvate dehydrogenase (HPDH1) and glycerate kinase (GLK). Photorespiration consumes CO2 and energy (ATP) in photosynthetic cells. Photosynthetic light reaction carried out by photosysntem I (PSI) and photsystem II (PSII, U3) supplies O2, ATP and reduncing powers (reduced ferredoxin form, Fdrd). Nitrate (NO32) is another source of nitrogen for plant growth and nitrate assimilation pathway is alternative way to provide ammonia for GS2/Fd-GOGAT cycle. Nitrate reductase 1 (NR1, P9-1) commits this pathway. U4 is suggested to have roles in transcriptional regulation of nitrate assimilation pathway. Glycerate 3- P resulted from photorespiration goes into Calvin cycle for the CO2 fixation. Carbonic anhydrase 1 (CA1, P2-1) might be involved in CO2 fixation in RuBP. Black circles indicate the steps in the pathway which are related to mutants identified in this study. Question marks (?) indicate genes of unknown function (U4 and U5). Purple lines indicate genes belonging to co-expressed cluster III; blue, cluster IV; yellow, cluster V; bright blue, cluster VIII; pure cyan, cluster IX; and dried sage, cluster X in Figure 4. Weak gray lines indicate steps in each pathway not associated with data collected in this study. doi:10.1371/journal.pgen.1000164.g005 activity GO term (GO:0017153) in the molecular function category. Because it is also assigned a membrane GO term (GO: 0016020) in the cellular component category, the U5 gene product is likely to be membrane-bound. The nearly identical expression pattern of the U5 gene with the glycine cleavage system H gene (Gcsh, Os10g37180) encoding a glycine decarboxylase complex H protein subunit in step 6 of the photorespiration pathway suggests that the U5 genes may function as a chloroplast sodium/dicarboxylate transporter[64]. In support of this hypothesis, a mutation in the U5 gene causes a defect in transporting 2 oxaloglutarate, and blocks re-assimilation of ammonia generated by the photorespiratory cycle. This result suggests that the supply of glutamate needed for synthesis glycine by serine-glyoxylate aminotransferase (SGAT, step 4 of photorespiration pathway) is limited. The U5 mutant line displays variegated albino leaves at the early seedling stage and later becomes albino. This phenotype is similar to that observed PLoS Genetics | www.plosgenetics.org 11 August 2008 | Volume 4 | Issue 8 | e1000164