for a mutation in the gene controlling step 5 (SHMT1, P5-1) of the photorespiration pathway (Figure 3 and Figure 5). A phenotype exhibited by lines carrying mutations in the U5 and Shmt1 (P5-1) genes suggests that blockage of 2-OG transport by the defect in the U5 gene causes a shortage of glycine, which is a substrate for serine biosynthesis controlled by the P5-1 gene product (Figure 5) [42]. Function of U4 (Unknown Gene). Based on its GO terms (GO:0009579 and GO:0006355), Os02g57030 (unknown gene, U4) is predicted to be located on the thylakoid membrane and carry out transcription. The nitrate reductase 1 gene (Nr1, Os08g36480) con- trolling step 1 of the nitrate assimilation pathway displays an expression pattern most similar to that of U4 (in Figure 4 and 5). Thus a probable role for U4 is to carry out light-dependent trans- cription regulation of nitrate assimilation (Figure 4 and Figure 5). Function of CA1 Unlinked to Known Pathways. Unlike the above two genes, which previously had no known function, the Ca1 gene (P2-1, Os01g45274) is predicted to catalyze the reversible conversion of bicarbonate (HCO32) to CO2 [65]. However, CA1 is still not directly linked to the known biochemical/metabolic pathways shown in Figure 5 and Figure S7. We have now determined that Ca1 (P2-1) is co-expressed with glycerate kinase gene (Glk, Os01g48990) that controls step 8 of the photorespiratory pathway (Figure 5 and Figure S7). This result suggests that rice CA1 carries out roles associated with RubisCO (ribulose-1,5- biphosphate carboxylase/oxygenas) and the Calvin cycle as reported in Cyanobacterium and Algae [65,66]. Transcriptional Hierarchy of Genes in the MEP Pathway The MEP pathway is a unique and essential process for plants, algae and bacteria [67,68,69]. The final metabolites of the pathway are isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), which are used for the synthesis of isoprenoids (such as isoprene), carotenoids, plastoqui- nones, phytol conjugates (such as chlorophylls and tocopherols), and hormones (such as gibberellins and abscisic acid) [60,70,71, 72,73,74]. Co-expressed Cluster I consists of genes (Os05g33840, Os07g36190 and Os05g33840) controlling steps 1, 2, and 8 of the MEP pathway, genes (Os04g37619 and Os07g10490) controlling steps step 3 and 6 of the carotenoid biosynthetic pathway, genes (Os03g36540 and Os03g59640) encoding two components (CHLI and CHLD) controlling step 1 of the chlorophyll biosynthetic pathway, gene (AK059143) encoding Psba of photosystem II, and gene (OS09g29070) controlling step 1 of the vitamin C biosynthetic pathway. Co-expression of genes in the MEP pathways with those in the chlorophyll and carotenoid biosynthesis pathways supports the hypothesis that the syntheses of pigments mediated by metabolite(s) resulted from the MEP pathway are dependent on photosynthesis (Figure 4 and Figure S7). In Arabidopsis, isopentenyl-PP or dimethylallyl-PP cause feedback regulation of step 1 (1-deoxy-D-xylulose-5-phosphate- synthase, DXS) of the MEP pathway (Figure 6) [72]. The co- regulation of genes controlling step 1 and step 8 suggest the existence of feedback regulation between these two steps in rice as reported in Arabidopsis [72] (Figure 4, Figure 6, and Figure S7). In an effort to deduce the hierarchical sequence of the enzymes involved in this pathway, we assessed the expression patterns of 12 genes controlling all steps in the MEP pathway in the homozygous mutant dxr (step2; progenies of Line 1A-14224, Ho-1 and Ho-2 in Figure 6) and its wild-type segregants (WT1 and WT2 in Figure 6). This analysis revealed that a defect at step 2 inhibits the previous step (step 1) in this pathway. These results indicate that the gene products controlling step2 and step8 of the MEP pathway cause feedback regulation of step 1 and that these three steps might be Functional Analysis of Singleton and Gene Families controlled by the same regulatory molecule (Figure 4, Figure 6, and Figure S7). In addition to the predicted feedback regulation of step 2 to step 1, transcription analysis of 12 candidate genes in MEP pathway in the dxr mutant revealed probable compensating rotes that could be taken to make up for the defect in the family member, Dxs1, that is predominantly expressed in the light. There are three gene family members (Dxs1, Dxs2, and Dxs3) associated with step 1 (DXS). The induction in the dxr mutant of the two other gene family members of step1, Dxs2 and Dxs3, supports a compensating roles for gene family members. The observed expression patterns of genes in other steps in this mutant facilitated predictions as to how these compensating routes would affect hierarchical sequence of the enzymes involved in this pathway. For example, as predicted, the Dxr (Os01g01710) gene at step 2 was not expressed in the mutant. The C-methyl-D-erythritol4-phosphate cytidylyl transferase (Cmt,Os01g66360) gene at step 3 was also repressed and the effect of a mutation in the Dxr gene might be extended to step 3 (Figure 6). However, the Cmk (Os01g58790) gene at step 4 (2-C- methyl-D-erythritol4-phosphate cytidylyl transferase 1, Cmk) was up-regulated in the rice dxr mutants suggesting that a compensat- ing route might be associated with this step as well. The predominantly light-induced gene family members in the four steps following the Cmt1 (Step 4), 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase (Mcs, Os02g45660), 4-hydroxy-3-methylbut-2- en-1-yl diphosphate synthase (Hds, Os02g39160), 4-hydroxy-3-methylbut- 2-enyl diphosphate reductase 1 (Hdr1, Os03g52170), and Isopentenyl- diphosphate delta-isomerase 1 (Idi1, Os07g36190), exhibited expression levels similar to those observed for the wild-type segregants. Interestingly, Hdr2 and Idi2 (in step 7 and step 8 of Figure 6, respectively), which are not the predominantly expressed family members in light-grown wild-type rice, were up-regulated in the dxr mutants (Figure 6). This result suggested that there might be another compensation mechanism occurring to cope with the upstream blockage of the MEP pathway. As a consequence of up- regulation in step 7 (Hdr2, Os03g52180), the gene expression of step 8 (Idi2, Os05g34180) would be increased. However, these compensating routes must not to be predominant because the original route has evolved to be the most effective. Finally, plants homozygous for this mutant locus were lethal despite the functioning of these two putative compensating pathways. The specifics of these proposed compensating routes await validation through further analyses. Roles of the MEP Pathway in the Light Response Most of genes involved in the carotenoid, abscisic acid, chlorophyll, and the tocopherol (Vitamin E) biosynthesis pathways, predicted to be downstream of the MEP pathway [60,75], are light-responsive in various light vs. dark experiments (Figure 4 and Figure S7). These results indicate a probable metabolic connections between the MEP pathway and these four downstream pathways. The connections are likely made through intermediates synthesized via the MEP pathway in the light. In support of the importance of this pathway in metabolism, we observed an albino leaf phenotype in homozygous progenies of lines 1A-14224 and 1C-07431, carrying T-DNA insertions in the Dxr gene at step 2 and 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (Mcs, Os02g45660) at step 5, respectively (Figure 3, Figure 6, and Figure S8). A mutation in Arabidopsis Mcs1 revealed a similar albino phenotype. These plants died at the early seedling stage [76]. In Arabidopsis or tobacco, lines carrying mutations in other genes of this pathway displayed albino- or chlorotic-leaf pheno- types [72,77,78,79,80,81]. Thus, our results indicate that the MEP PLoS Genetics | www.plosgenetics.org 12 August 2008 | Volume 4 | Issue 8 | e1000164