Functional Analysis of Singleton and Gene Families Figure 2. Strategy Used to Refine the List of 37 Candidate Genes Prior to Functional Validation and Summary of Phenotype Analysis. (A) The flow chart of functional analysis using microarray and gene-indexed mutants. (B) Gene expression patterns of selected 37 candidate genes and summary of functional analyses using gene-indexed mutants of these genes. Figure 2A is adapted from a previous review article [4]. Dotted lines in Figure 2A indicate the cut region of rice seedlings used for this array experiment. The NSF45K and BGI/Yale light vs. dark array datasets were used to refine the list of 37 candidate genes prior to functional validation. Unique genes (U), genes without gene family members, are distinguished from genes (GF) with gene family members. The gene among the gene family members predominantly expressed in the light was marked as P, distinguished from non predominant genes (NP). The consistency of a gene’s expression pattern among the NSF45K and BGI/Yale light vs. dark array data was noted with a Yes or No or 45K; 45K indicating the unavailability of any other reference array data for comparison. The genes for which functional analyses was performed are marked Yes. Co-segregation of the observed phenotype and a T-DNA insertion is indicated by Yes, No or ND; ND indicates that co-segregation was not determined due to somaclonal variation or problems with primers. Yellow box indicates rice light vs. dark microarray data derived using NSF45K and BGI/Yale array. Red box indicates gene expression data in leaf, seedling shoot, and young leaf derived using the Affymetrix array. doi:10.1371/journal.pgen.1000164.g002 genes that we analyzed for function from our list of top candidate candidates. Of these, the phenotypes of knockout lines associated with Os01g01710 (1-deoxy-D-xylulose 5-phosphate reductoisomerase, Dxr) and Os03g04470 (Expressed protein) were albino and displayed chlorotic leaves, respectively, 2 weeks post-sowing (Figure 3A). Knockouts of Os01g71190 (Photosystem II subunit 28, Psb28), Os02g57030 (Expressed protein), and Os07g46460 (ferredoxin-dependent glutamine:2-oxoglutarate aminotransferase, Fd-GOGAT) displayed pale green phenotypes (Figure 3A). Of these, a mutation in the Fd- GOGAT gene displayed photo-bleached leaves two weeks later after revealing pale green leaves (Data not shown). Knockouts of Os04g37619 (Zeaxanthin epoxiydase, Aba1) produced dwarf mutants (Figure 3A). We noted that the mutant phenotypes associated with three of these 6 genes, those encoding DXR, Fd-GOGAT, and ABA1, were similar to those associated with their Arabidopsis orthologs [31,32,33]. The Arabidopsis ortholog of Os01g71190 encodes photosystem II (PSII) reaction center Psb28 protein (Psb28) that was first identified from PSII of Synechocystis 6803 with Psb27 [34]. The function of this PSB28 has not yet been well- characterized, although it is predicted to serve a role as a regulatory protein based on its substoichiometric amount [34,35]. Similarly, Arabidopsis lines carrying a mutation in Psb27 did not display a severe phenotype. Recovery of PSII activity after photoinhibition was delayed in the Arabidopsis psb27 mutant supporting a role in PSII for this gene [36]. The mild phenotype displayed by the Psb28 T-DNA insertional rice plants in Psb28 gene suggests that this gene product might serve as a regulatory protein to stabilize PSII activity. The other two unique genes for which we identified corresponding mutant phenotypes as a result of our analysis encode as yet unidentified proteins. PLoS Genetics | www.plosgenetics.org 5 August 2008 | Volume 4 | Issue 8 | e1000164