Functional Analysis of Singleton and Gene Families Figure 1. Light-Responsiveness of Rice Genes Involved in the Chlorophyll Biosynthesis Pathway. (A) The seven steps in the rice chlorophyll biosynthesis pathway, from magnesium chelatase to chlorophyll b, are shown and mutants of rice corresponding to each step are indicated. Red boxes indicate mutants which have been characterized in rice. CHLH, magnesium-chelatase subunit H family protein (1); CHLI, magnesium-chelatase subunit I family protein (1); CHLD, magnesium chelatase ATPase subunit D protein (1); CHLM, magnesium-protoporphyrin O- methyltransferase (2, and there were two gene family members); MPE, magnesium-protoporphyrin IX monomethyl ester cyclase (3); DVR, divinyl protochlorophyllide reductase (4, and there were five gene family members); POR, protochlorophyllide reductase (5, and there were four gene family members); CHLG, chlorophyll synthase (6, and there were two gene family members); and CAO, chlorophyll a oxygenase (7, and there were two gene family members). oschlh, chlorina-1, and -9 are mutants in rice ChlH, ChlI, ChlD, respectively; xantha-f, g, and h are mutants in barley ChlH, ChlI, ChlD, respectively; cs/ch42 is mutant in Arabidopsis CHLI; gun5 is mutant in Arabidopsis CHLH; chlm is mutant in Arabidopsis CHLM; albino-39 is mutant in rice ChlM; Chl27 is mutant in Arabidopsis MPE; xantha-i is mutant in Barley Mpe; dvr is mutant in Arabidopsis DVR; porb-1porc-1 is double mutant of Arabidopsis PORB and PORC; ygl1 is mutant of rice ChlG; cao is mutant of Arabidopsis CAO; and oscao1 is mutant of rice Cao1. (B) Normalized average expression levels in light and dark and fold changes in expression from dark to light based on results of the NSF45K light vs. dark microarray experiment are shown for genes involved in the 7 steps in the pathway and their gene family members. Unique genes or gene family members predominantly expressed in the light in each step are marked with asterisks. White boxes indicate gene expression levels in light. Black boxes indicate gene expression levels in dark. Gray boxes indicate the fold change ratios of light over dark. Red boxes indicate the gene expression patterns of the genes corresponding to those mutations in red boxes of A. 1a, Os03g20700; 1b, Os03g36540; 1c, Os03g59640; 2-1, Os06g04150; 2-2, Os02g35060; 3, Os01g17170; 4-1, Os03g22780; 4-2, Os02g56690; 4-3, Os08g17500; 4-4, Os08g34280; 4-5, Os09g25150; 5-1, Os04g58200; 5-2, Os10g35370; 6-1, Os05g28200; 6-2, Os03g09060; 7-1, Os10g41780; and 7-2, Os10g41760. (C) RT-PCR results are shown; Ubq1 and Actin1 were used as controls [85]. Numbers of PCR cycles are indicated. IL, leaves from IR24 plants harvested in the light; ID, leaves from IR24 plants harvested in the dark; TL, leaves of Taipei (TP) 309 plants harvested in the light; TD, leaves of TP309 plants harvested in the dark; NL, leaves of Nipponbare plants harvested in the light; ND, leaves of Nipponbare plants harvested in the dark; KL, leaves of Kitaake plants harvested in the light; and KD, leaves of Kitaake plants harvested in the dark. ChlH, Os03g20700; ChlI, Os03g36540; ChlD, Os03g59640; ChlM, Os06g04150, Os02g35060; Mpe, Os01g17170; Dvr1, Os03g22780; Dvr2, Os02g56690; Dvr3, Os08g17500; Dvr4, Os08g34280; Dvr5, Os09g25150; Por1, Os04g58200; Por2, Os10g35370; ChlG1, Os05g28200; ChlG2, Os03g09060; Cao1, Os10g41780; and Cao2, Os10g41760. doi:10.1371/journal.pgen.1000164.g001 to have a function similar to that of its Arabidopsis ortholog, CHL27 [28]. Knockout lines of the two predominantly expressed light- responsive candidate genes (rice magnesium-protoporphyrin O-methyl- transferase (ChlM;Os06g04150; and gene 2-1) and rice chlorophyll a oxygenase (Cao1; Os10g41780;and gene 7-1)) had been previously identified and shown to display with light response-related phenotypes [29,30] (Figure S1). These results indicate that identification of unique genes or the predominantly expressed member of a gene family in the light is an effective method to target genes for further functional analysis. More descriptions on this strategy are shown in Figure S2 and Text S1. (RiceGE, http://signal.salk.edu/RiceGE/RiceGE_Data_Source. html) [4] (Table 1). Refinement of the Candidate Genes List From our NSF45K light vs. dark experiment, we selected 365 candidate genes showing at least an 8-fold induction at the 1024 FDR p-value. We then utilized collections of rice insertional mutants to identify lines carrying mutations in the light-responsive genes identified through expression analysis. To date, some 172,500 sequences have been generated from regions flanking insertional mutants in rice and they are publicly available at the PLoS Genetics | www.plosgenetics.org 3 August 2008 | Volume 4 | Issue 8 | e1000164