Functional Analysis of Singleton and Gene Families Figure 6. Altered Expression Patterns of Genes Associated with the MEP Pathway in the Rice dxr Mutant. (A) Validation of microarray data corresponding to genes in this pathway using RT-PCR amplifications. Ubq1 and Actin1 were used as controls for RT-PCR [85]. Numbers of PCR cycles are indicated. See legend for Figure 1 for explanation of column heading abbreviations. Dxs1, Dxs2, Dxs3: gene family members encoding 1- deoxy-D-xylulose-5-phosphatesynthase; Dxr: gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase (2 in B); Cmt: gene encoding 2-C- methyl-D-erythritol4-phosphate cytidylyl transferase (3 in B); Cmk: gene encoding 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (4 in B); Mcs: gene encoding 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (5 in B); Hds: gene encoding 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (6 in B); Hdr1, Hdr2: gene family members encoding 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (7 in B) and there were two gene family members; and Idi1, Idi2: gene family members encoding isopentenyl-diphosphate delta-isomerase (8 in B) and there were two gene family members. (B) Proposed pathway model for explaining altered expression patterns. The MEP pathway was generated using RiceCyc (http://www. gramene.org/pathway/). Twelve candidate genes were assigned to 8 steps in the pathway. Blue colored letters and arrows indicate the suppression of gene expression in dxr mutant; black ones indicate not significantly changed gene expression patterns in dxr mutant; and red ones indicate significantly induced gene expression patterns in dxr mutant. Phenotypes in T-DNA insertional homozygous progenies in Mcs gene is displayed in Figure S8. (C) RT-PCR analysis of twelve candidate genes in this pathway in the rice dxr mutant (homozygous siblings) and its wild-type segregants. Genes indicated in red were up-regulated in the dxr mutant. Genes indicated in blue were down-regulated in the dxr mutant. Expression of genes indicated in black was not significantly changed in the dxr mutant. WT1, wild-type segregant 1; WT2, wild-type segregant 2; Ho-1, dxr homozygote 1; Ho-2, dxr homozygote 2. Ubq1 were used as controls for RT-PCR. RT-PCR amplifications were carried out as described in Material and Methods. doi:10.1371/journal.pgen.1000164.g006 pathway in rice performs key roles in generating pigments such as chlorophyll or carotenoid as it does in other plant species [31,67,82]. Conclusion and Perspective This study provides a method for identifying sets of candidate genes involved in specific biochemical pathways. Our results reveal that light regulated gene expression controls diverse metabolic networks [83,84] and that co-expression analysis is an effective strategy for elucidating the plant response to light. Materials and Methods Sample Collection Nipponbare, Kitaake, TP309, and IR24 rice seeds were germinated and grown in the greenhouse. Nipponbare, Kitaake, PLoS Genetics | www.plosgenetics.org 13 August 2008 | Volume 4 | Issue 8 | e1000164