Author Summary Rice, a model monocot, is the first crop plant to have its entire genome sequenced. Although genome-wide tran- scriptome analysis tools and genome-wide, gene-indexed mutant collections have been generated for rice, the functions of only a handful of rice genes have been revealed thus far. Functional genomics approaches to studying crop plants like rice are much more labor- intensive and difficult in terms of maintaining the plants than when studying Arabidopsis, a model dicot. Here, we describe an efficient method for dissecting gene function in rice and other crop plants. We identified light response- related phenotypes for ten genes, the functions for which were previously unknown in rice. We also carried out co- expression analysis of 72 genes involved in specific biochemical pathways connected in lines carrying muta- tions in these ten genes. This analysis led to the identification of a novel set of genes likely involved in these pathways. The rapid progress of functional genomics in crops will significantly contribute to overcoming a food crisis in the near future. the complicated physiological networks with which plants adapt to environmental changes. Toward that end, plant researchers have carried out microarray experiments with various plant species in efforts to identify light-regulated genes [9,10,11]. One of the major challenges facing scientists in the field of functional genomics, even those working with relatively simple model organisms like Arabidopsis, is the prevalence of gene families [12,13]. Such family members often encode redundant functions [14,15]. Because of the presence of gene families with functional redundancy, it is often the case that a gene, when mutated, will display no detectable phenotype. For example, an Arabidopsis line carrying a T-DNA insertion in a gene encoding a protein with a putative R3H domain, At5g05100, hypothesized to be of importance in seed organ development, was reported to have no observable mutant phenotype [13]. Because Arabidopsis also has three genes, At2g40960, At3g10770, and At3g56680, the lack of a mutant phenotype in this line is probably due to one or more of these genes encoding a function redundant of At5g05100 [13]. Efforts to overcome such functional redundan- cy have mostly focused on generating mutants that disrupt more than one gene family member simultaneously [16,17]. For example, RNA interference (RNAi) technology has also been used to simultaneously silence multiple members of a gene family [18]. Rice researchers encounter even larger gene families, more difficulty in scaling up experiments and greater time constraints associated with rice’s longer life cycle than do researchers working with Arabidopsis. For example, whereas Arabidopsis has more than 600 genes in its receptor-like kinase (RLK) family, 80 in the bric-a-brac/tramtrack/broad (BTB) protein family, and 37 genes in the GARS family, rice has more than 1131, 149, and 52 members in those families, respectively [19,20,21]. Accordingly, a strategy for overcoming these difficulties and accelerating functional genomics analysis is especially helpful in crops like rice. Interestingly, the presence of multiple gene family members does not always mask phenotypic changes in gene ‘‘knockout’’ mutants. For example, 43.9% of the Arabidopsis seedling-lethal mutants and 45.2% of the Arabidopsis embryo-defective mutants phenotypically identified in two Arabidopsis studies carried defects in genes that were members of gene families [22,23]. These results suggest that all members of gene families are not necessarily Functional Analysis of Singleton and Gene Families functionally equivalent and individuals among them may play predominant roles [24]. Here we report the use of a microarray that covers nearly the entire rice transcriptome, the National Science Foundation- supported 45K microarray (NSF45K, http://www.ricearray.org/), to identify light-responsive genes in rice and subsequent functional analysis of a subset of those genes through screening gene-indexed mutant lines of rice. We describe the advantages of utilizing candidate genes derived from rice gene expression profiles to screen rice insertional mutant collections and discuss the biological significance of our findings. Results/Discussion Light-Responsive Genes Were Identified in Rice from Four Different Genetic Backgrounds We performed expression-profiling experiments with a newly- developed NSF45K microarray (http://www.ricearray.org/) using two weeks-old rice leaf tissues harvested from plants grown under light and dark conditions (See Materials and Methods). Because genetic backgrounds can affect expression profiles [25] and we wanted to select light-responsive genes that behaved similarly in different genetic backgrounds, identical experiments were carried out with four different rice varieties: Kitaake, Nipponbare, Tapei309 and IR24. We used the LMGene Package developed by Rocke (2004) to identify 10361, 4962, 1933, and 453 genes differentially expressed in response to light versus dark treatment at FDR p-values of 0.01, 1024, 1026, and 1028, respectively (Table S1). To assess the difference in gene expression profiles among the varieties, we calculated correlation coefficients. Correlation coefficient values were 0.89–0.92 between the three japonica varieties (i.e. Kitaake, Nipponbare, and Tapei309) whereas correlation coefficients values between subspecies (i.e. japonica and indica) were 0.80–0.82. This result indicates that differences in genetic background clearly affect the expression patterns. Accordingly, by focusing on genes whose expression pattern is similar between varieties, we can avoid genes that show cultivar-specific light responses. Identification of Rice Insertional Mutants Corresponding to Microarray-Derived Candidate Genes To assess the usefulness of our microarray data we surveyed the expression patterns we obtained through microarray analysis for seventeen genes, including gene family members, which encode proteins for the seven steps in the well-characterized chlorophyll biosynthetic pathway (Figure 1). The expression patterns of all of the candidate genes in the seven steps of this pathway were validated using reverse transcriptase- (RT-) PCR (Figure 1). Based on this analysis we identified four unique genes (Figure 1B, 1a, 1b, 1c, and 3), and two predominantly expressed light-responsive candidate genes (Figure 1B, 2-1 and 7-1) as good targets for studying the biological functions of genes involved in rice chlorophyll biosynthesis. A predominantly expressed light-respon- sive candidate gene is the one that is most significantly induced by light (compared to other members of the multi-gene family). Previous studies in rice showed that the knockout mutants of three of the unique genes (encoding the subunits comprising magnesium chelatase complexes in rice: magnesium chelatase subunit H (CHLH, 1a), magnesium-chelatase subunit I family protein (CHLI, 1b), and magnesium chelatase ATPase subunit D protein (CHLD, 1c) exhibited chlorina phenotypes [26,27]) (Figure S1). The remaining unique gene (magnesium-protopor- phyrin IX monomethyl ester cyclase, MPE) at step 3 is predicted PLoS Genetics | www.plosgenetics.org 2 August 2008 | Volume 4 | Issue 8 | e1000164