Os01g40710 and Os08g40160, could not be determined due to confounding somaclonal variations (Figure S4) in the one mutant line available for each of these genes for analysis. We could not analyze the second mutant line corresponding to each of these genes due to poor seed set. Lines with T-DNA insertions in Os03g47610, lines 1A-08723 and 1A-17021, did not produce any homozygous progenies and as a result the phenotypes associated with these mutations were also not determined (Table 2; Table S2). On the other hand, while we did identify homozygous progenies and their siblings among mutants with insertions in Os03g06230 and Os03g17960, we did not observe phenotypic differences between them (Table 2). Targeting functional analysis to unique genes is an effective way to significantly increase the efficiency of identifying genes corresponding to defective phenotypes. Utilizing microarray- derived expression profiles of unique genes can increase the efficiency of functional analyses [1,22,38]. The efficiency (6/11) with which we were able to identify phenotypes associated with mutations in unique genes demonstrates the power of combining knowledge of gene copy number and gene expression patterns. Of the nine predominantly light-induced gene family members that were consistently induced in the light, we found four for which mutant phenotypes co-segregated with a T-DNA insertion in the gene (Figure 3B). These four genes encode carbonic anhydrase 1 (CA1), serine hydroxymethyltransferase 1 (SHMT1), nitrate reductase 1 (NR1), and Vitamin C defective 2 (VTC2), respectively. The rice T-DNA insertional line carrying a mutation in the Shmt1 gene (Os03g52840), line 1D-03944, displayed variegated chlorina leaves (Figure 3B). The line 2B-60065 with a T-DNA insertion in Ca1 (Os01g45274) showed an oxidative stress- related phenotype, necrosis in the middle of the leaf, and a little growth retardation (Figure 3B). Line 4A-50280, with a T-DNA insertion in Nr1 (Os08g36480), displayed dwarfism, and line 3A- 14221, with a T-DNA insertion in the Vtc2 gene (Os12g08810), exhibited a pale green and later photo-bleached leaves phenotype (Figure 2B). The Tos17 line with an insertion in the rice Shmt1 gene exhibited the same phenotype as the corresponding T-DNA mutant, line 1D-03944 (Table 2). The phenotypes associated with mutations in Shmt1 (Os03g52840), Nr1 (Os08g36480), and Vtc2 (Os12g08810) were also reminiscent of the phenotypes associated with mutations in the orthologous genes in Arabidopsis [39,40,41,42]. Mutant phenotypes associated with T-DNA insertions in Os01g08460, and Os05g47540 were not determined due to confounding somaclonal variations (Figure S4). The homozygous progenies of mutant lines with insertions in two other genes, Os03g37830 and Os06g04510, did not show visible phenotypic changes (Table 2). Our success in conducting functional analyses of gene family members that are the predominantly expressed in the light, and consistently so from one microarray experiment to the next, suggests that the functions of gene family members can be successfully analyzed by utilizing data obtained using microarrays representing nearly complete plant transcriptomes. Analyses that consider both sequence similarity and predominance of gene expression has also been reported to be quite effective in functional analysis of yeast genes [14]. Functional Analysis of 17 Non-prioritized Candidate Genes We also carried out functional analysis of 5 genes that showed inconsistent gene expression patterns when we compared our NSF45K light-response experiments and the BGI/Yale light vs. dark experiments. One of them, Os03g48030 (designated U9 in Figure 2 and Figure 3), is a unique gene and the other four genes, Functional Analysis of Singleton and Gene Families encoding lectin protein kinase (Os09g16950), stem-specific protein TSJT1 (Os11g05050), flavin-containing monooxygenase family protein (Os09g37620), and an expressed protein (Os02g58790), were the predominantly light-induced members of their respective families among the NSF45K array-derived data but not in the BGI/Yale light vs. dark dataset. No defective phenotypes were observed among the mutant lines with T-DNA insertions in any of these genes (Table S3). One possible reason for this result is the presence of redundant metabolic networks or the absence of appropriate screening conditions [15,43,44]. Mutants carrying insertions in 12 genes that were not the predominantly expressed light-induced member of their respective gene family were also examined in this study. A visible phenotype was observed in the homozygous mutant progenies associated with only one of these genes, Os07g05000 (R8-2) (Figure S3C and Figure S5), which belongs to the family of genes encoding aldo/ keto reductases. Phenotypic changes were not observed in the homozygous segregants of lines with mutations in any of the other genes (Table S3). The absence of detectable abnormal phenotypes associated with these other genes is generally believed to be due to one (or more) family members compensating for the function of the mutated gene [14,15]. Line 3A-03008, which carries a T-DNA insertion in Os07g05000, showed a weakly pale green phenotype and slight growth retardation (Figure S5). Identification of phenotypes associated with the other mutations may require specific conditions under which there will be no compensatory gene expression from other family members. In cases of gene families without a predominantly expressed member under specific experimental conditions, microarray data can still be used to identify the multiple significantly expressed genes in a family so that they can be subjected to RNA-silencing techniques as has been carried out by Miki et al. [18,45] for the rice genes encoding homologs of mammalian Rac GTPase, OsRac1 and OsRac5. Of our list of non-predominantly light-induced genes, there were two genes from same family expressed under light conditions (Os12g03070 and Os11g03390). Both encode an FHA domain, which is a putative nuclear signaling domain found in protein kinases and transcription factors. However, we did not observe a phenotype in knockout lines of Os11g03390 (R12-1) [46] (Table S3). Similarly, we did not observe phenotypic changes associated with T-DNA insertions in members of the gene families encoding ABC1 proteins (Os02g57160, R3-1 and Os04g54790, R5- 1), S1-RNA binding domain proteins (Os04g54790, R4-1), and glycine dehydrogenases (Os06g40940, R7-1). Further experiments to generate double mutants for these family members and their light-induced relatives will be required to elucidate their functions. In Arabidopsis, the light-responsive functions of genes in gene families such as POR and PHOT have been clarified by using double mutants of two family members, porbporc and phot1phot2, respectively [47,48]. When we consider severity of phenotypes, two of six lines carrying defects in unique genes and one of four lines carrying defects in the gene family member predominantly expressed in the light died at early seedling stage (Figure 3). Therefore, these three genes are essential for survival. Overall, phenotypes of mutants with defects in unique genes were more frequently observed than those of mutants carrying defects in predominantly light-induced gene family members (compare Figure 3A with Figure 3B). We suspect, therefore, that other members in a gene family may carry out a somewhat compensating role [14,15]. Similarly, we did not find phenotypic changes associated with mutations in genes that were not the predominantly light-induced members of their respective families, with the exception of mutations in a gene encoding an aldo/keto PLoS Genetics | www.plosgenetics.org 8 August 2008 | Volume 4 | Issue 8 | e1000164