Functional Analysis of Singleton and Gene Families Figure 4. Co-Expression Analysis of 72 Genes in the Pathways Related to 10 Mutants Identified in This Study. Hierarchical clustering analysis was carried out for 72 genes related to the 10 mutants shown in Figure 3; cluster results are indicated on the far left side of this figure. In the left panel, the log2 fold change values of the 10 datasets used to perform hierarchical clustering analysis are shown (for details of the 10 datasets and how they were analyzed see Materials and Methods). The middle panel indicates the average of all spot intensities for an oligo in the light (av_NSF45K_light intensity) and dark (av_NSF45K_dark intensity) from NSF 45K light vs. dark datasets as another indicator of gene expression levels. Oligo_id indicates the name of the oligos in NSF45K array; Putative function describes the annotation assigned to that gene; Pathway indicates the biochemical pathway and step in the pathway associated with the gene; Type indicates status of the gene in the rice genome: U, unique sequence; P, predominantly expressed gene family member in the light; and NP, not the predominantly expressed gene family member in the light; CC-GO_term, indicates GO terms within the cellular component category; Rice Mutant indicates the phenotype associated with a mutation in the gene; and Reference provides citations to published evidence for the phenotypes described in the previous column. More information regarding U1, U3, U4, U5, U10, U11, P2-1, P5-1, P9-1, and P13-1 is contained in Table 2. In the column labeled CC-GO_term: Ch indicates chloroplast; Cy, cytoplasm; ER, endoplasmic reticulum; Mi, mitochondrion; Me, membrane; N, nucleus; and P, peroxisome. Data used for clustering analysis and more detailed information regarding the 72 genes on which the analysis was performed are contained in Table S4. doi:10.1371/journal.pgen.1000164.g004 Of the genes in cluster III, the phenotypes of a mutant line with a T-DNA insertion in Fd-GOGAT (U11) and a mutant lines under- expressing the Rca1 gene (Os11g47970) were characterized (Figure 4, Figure 5 and Figure S7) [59]. The Rca1 under-expressed mutant displays chlorotic leaves [59]. The mutant (3A-01082, this study) with a T-DNA insertion in Fd-GOGAT gene displayed pale green leaves shortly after germination (Figure 3). The same mutant displayed chlorotic leaves four weeks after germination and is similar to the phenotype of one of the Rca1 under-expressed mutants (data not shown) [59]. These results indicate that co-expressed groups of genes carry out closely related functions as reported in other species [1,33,60]. Therefore we can predict that mutations in other genes in this cluster will display similar phenotypes to those observed in the Fd- GOGAT and Rca1 mutant lines. Those predicted to display such phenotypes include phosphoglycolate phosphatase (Pgp, Os04g41340) [61] at step 2 of the photorespiration pathway. In support of this hypothesis, a mutant with defects in Arabidopsis (PGLP1) displayed chlorotic leaves at the early seedling stage, the phenotype was similar to that observed for Rca1 and U11 mutant lines [61]. Assigning Functions of Genes Unlinked to Known Biochemical/Metabolic Pathways Function of U5 (Unknown Gene). Gene ontology can help assign putative functions to unknown genes [62,63]. For example, Os03g04470 (unknown gene, U5) has a sodium/dicarboxylate cotransporter PLoS Genetics | www.plosgenetics.org 10 August 2008 | Volume 4 | Issue 8 | e1000164