Figure 3. Overview of expression profiles of sucrose absorp- tion and sugar-phosphate transport genes in developing physic nut seeds. A: Genes involved in the indirect pathway for sucrose absorption. cwINV, cell-wall invertase; PLT, polyol transporter; STP, sugar transport protein. B: Genes involved in the direct sucrose absorption and breakdown pathway. nINV, neutral/alkaline invertase; SuSy, sucrose synthase; UGPP, UDP-glucose pyrophosphorylase. C: Genes encoding plastid envelope transporters. All expression data were transformed to log2 scale for plotting the profiles. GPT, Glc-6-P/ phosphate translocator; NTT, ATP/ADP transporter (Adenine nucleotide translocase); PEP, phosphoenolpyruvate; PPT, phosphoenolpyruvate/ phosphate translocator; TPT, triose phosphate/phosphate translocator; XPT, xylulose 5-phosphate/phosphate translocator. Values less than -2 indicate that no expression of the gene was detected at the indicated point. Genes shown in this figure are listed in Table S2. doi:10.1371/journal.pone.0036522.g003 Gene Expression Profiles in Physic Nut Seeds (JC_C100014296) was expressed at a relatively low level in the seeds (Figures 3C and S1). Central Metabolism-related Genes The glycolysis pathway, oxidative pentose phosphate pathway (OPPP), and C3–C4 intermediary metabolism pathway have been suggested to generate glycerol-3-phosphate and NAD(P)H for FA and TAG synthesis in oilseeds [36–39]. Many enzymes involved in these metabolic pathways are encoded by multiple genes in the physic nut genome. As shown in Figure S2, at least one gene encoding an enzyme catalyzing each step involved in the glycolysis pathway and OPPP was highly expressed during the entire course of seed development. Our data confirm that genes encoding both plastid (Figure 4A) and cytosol (Figure 4B) enzymes of the pathway are expressed at high levels throughout seed development, although the expression of some genes, especially the plastid pathway genes, declined during the late maturing stage, S7. On the other hand, the expression levels of most genes encoding cytosolic glycolytic enzymes were higher than those encoding the plastidial isoforms in the developing seeds. This result is consistent with observations that ESTs for cytosolic glycolytic enzymes are more abundant than those of the plastidial isoforms in developing seeds of several oilseed plants [40]. The expression level of most OPPP enzyme genes (Figure 4C) and C3–C4 intermediary metabolism pathway genes (Figure 4D) peaked at the filling stages, S4 to S6, in the developing physic nut seeds. Starch Metabolism, Fatty Acid, TAG and Oil-body Biosynthesis Genes ADP-glucose pyrophosphorylase (AGPase) catalyzes the first step in starch biosynthesis, yielding the activated glucosyl donor ADP-glucose [41]. It comprises two large subunits (AGPL) and two small subunits (AGPS), each of which is encoded by distinct genes. The AGPS1 (JC_C100009747), AGPL1 (JC_C100011490) and AGPL2 (JC_C100015930) genes were all expressed in developing seeds, but at lower levels at S5 and S6. The expression level of most starch synthesis genes peaked at S3, then declined during the filling stages from S4 or S5 (Figures 5A and S3). The different starch degradation enzyme genes had different expression patterns in the developing seeds (Figures 5B and S3). Two a- amylase genes (JC_C10008238 and JC_C100011196) and two b- amylase genes (JC_C100014576 and JC_C 00002332) were strongly expressed during the filling stages, S4 to S6, while six b- amylase genes and two a-amylase genes were highly expressed in the early developing stage (Figures 5B and S3). Most genes of the fatty acid synthesis pathway [10–11] were expressed at relatively high levels from S4 to S6, and their expression peaked at S5 in the developing seeds (Figures 5C and S4), except for acyl-ACP thioesterase B (FATB, JC_C100013836), expression of which decreased at the filling stage. The expression profiles of TAG assembly pathway genes are shown in Figures 5D and S5. At least one gene encoding an enzyme catalyzing each step of the pathway was highly expressed at the filling stage of the seeds (Figure S5). Of the phosphatidylcholine cycle genes, the oleate desaturase coding gene (FAD2, JC_C100004186) was highly expressed in the developing seeds, the oleic acid hydroxylase coding gene (FAH12, JC_C100003840) was expressed at the filling stage, but no linoleate desaturase coding gene (FAD3, JC_C100006612) transcripts were detected in filling stage seeds (Figures 5D and S5). Two phospholipid:diacylglycerol acyltrans- ferase genes (JC_C100025621 and JC_C100030570) involved in TAG formation were highly expressed at the filling stage (Figure 5D). Expression of most oil-body protein genes began or increased from S4 and remained high during seed maturation PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36522