Gene Expression Profiles in Physic Nut Seeds in importing sugar and energy into plastids for fatty acid synthesis in physic nut seeds. declined, while the starch breakdown rate remained high, resulting in a net reduction of starch levels in the seeds. Activities of Three Sugar Metabolic Pathways in Developing Physic Nut Seeds The hexose phosphates generated by sucrose cleavage can be metabolized through glycolysis or the oxidative pentose phos- phate pathway (OPPP). This central pathway is a key component of a metabolic network, providing inputs of precursors for fatty acid and TAG synthesis, namely glycerol- 3-phosphate and NAD(P)H. Each of the glycolysis enzymes is present in both the cytosol and plastids, and both the cytosolic [49–50] and plastidial glycolytic [36–37,39,48,51] pathways have been suggested to play central metabolic roles during oilseed development. The role of the OPPP in fatty acid production has been described in several reports [52–54]. OPPP enzymes have been detected in proteomic analyses of filling castor seeds [36], while metabolic feeding of purified organelles and in vivo metabolic flux analyses strongly implicate involve- ment of the C3–C4 intermediary metabolism pathway in castor bean and soybean filling, respectively [55–56]. In the latter pathway, phosphoenolpyruvate can be carboxylated to oxaloac- etate by phosphoenolpyruvate carboxylase (PEPC). Expression levels of most genes of the cytosolic and plastidial glycolysis, OPPP and C3–C4 intermediary metabolism pathways remained high during the S4 to S6 stages, but those of all but the cytosolic glycolysis pathway declined at S7 (Figure 4), when oils rapidly accumulate in developing physic nut seeds (Figures 1C and 5C). The decline in expression of genes of the plastid glycolysis, OPPP and C3–C4 intermediary metabolism pathways is consistent with a low rate of oil accumulation at the S7 stage. Hence, our results strongly suggest that these three metabolic pathways play important roles in oil synthesis in physic nut seeds, while the continuing high expression of most genes of the cytosolic glycolysis pathway during S7 indicates that cytosolic glycolysis plays other roles during the maturation stage of the seeds. Starch Metabolism in Developing Physic Nut Seeds In oilseed plants such as B. napus and Arabidopsis, starch deposition occurs transiently in the early stages of embryo development. Starch accounts for approximately 8% to 10% of the seed dry weight in these stages, and is absent in mature seeds [57–58]. Phosphoglucomutase, which catalyzes the readily revers- ible interconversion of Glc-1-P and Glc-6-P, is essential for starch synthesis and plays an essential role in the degradation of assimilatory starch. Seeds of plastidial phosphoglucomutase gene knockdown mutants reportedly accumulate 40% less oil than wild type Arabidopsis seeds, indicating that starch is positively linked to oil accumulation in seeds [59]. Further, ADP-Glc pyropho- sphorylase (AGPase) catalyzes the first step in the starch biosynthesis pathway, and knockdown of the small subunit of AGPase reduced the starch content and rate of starch synthesis, with a consequent 50% reduction in lipid contents, in B. napus seeds examined by Vigeolas et al. [60]. The starch content is ca. 11% of the dry weight in early stage kernels and, following a net reduction in starch content of the seeds after S5 (Figure 1C, 1D), ca. 2% in kernels of mature physic nut seeds. Accordingly, starch synthesis genes and some starch breakdown genes were highly expressed before S5 in the seeds (Figures 5A and S3). In contrast, several starch breakdown genes were highly expressed at the filling stage (Figures 5B and S3). These results suggest that starch is actively turned over during early stages of physic nut seed development. During the filling stage, the starch synthesis rate Changes in the Fatty Acid Composition of Physic Nut Oils are Consistent with Relative Expression Levels of Different Types of Acyl-ACP Thioesterase and Fatty Acid Desaturase Genes The ability of a biological resource to meet specified criteria for use as a biodiesel fuel is largely determined by its FA composition [61–63]. Thus, identification of means to manipulate the FA composition of candidate resources would be highly valuable, and the coinciding changes in FA composition and expression patterns of genes involved in FA biosynthesis and modification in developing physic nut seeds provide a convenient model for exploring and potentially refining FA biosynthesis in plants. Physic nut oil (Figure 2B) contains more C18:1, but less C18:3 and C20, than Arabidopsis oil [64]. In Arabidopsis seeds the fatty acid elongase 1 (FAE1, At4G34520) of the TAG synthesis pathway is required for synthesis of very long chain fatty acids [65], the FAD2 gene encodes an endoplasmic reticulum 18:l desaturase [66], while the FAD3 gene encodes an endoplasmic reticulum 18:2 desaturase [67]. We detected no FAE1 subfamily homologous gene in the physic nut genome, and no FAD3 transcripts in physic nut seeds at the filling stage (Figures 5D and S5), although FAE1 and FAD3 genes are reportedly highly expressed during this stage in Arabidopsis seeds [8]. Acyl-ACP thioesterase A (FATA) has a high specificity for 18:1- ACP and a lower activity toward 18:0- and 16:0-ACP, whereas, FATB more efficiently catalyzes the reaction with ACPs carrying saturated fatty acyl chains [68–69]. During the development of the physic nut seed, the content of C18:1 increased while C16:0 and C18:0 contents decreased (Figure 2C). In accordance with the changes in levels of these fatty acids, the expression level of stearoyl-ACP desaturase (SAD) genes and FATA increased, whereas FATB expression decreased in filling stage seeds (Figure S4). Overall, the fatty acid composition of the oil in the developing physic nut seeds was consistent with the relative expression levels of different isoforms of both acyl-ACP thioesterase and fatty acid desaturase genes. Thus, differences in the expression profiles of FAE1 and fatty acid desaturase genes appear to make major contributions to the differences in fatty acid composition of TAGs between physic nut and Arabidopsis oils. This information provides valuable indications of possible ways to optimize the FA composition of oils in seeds of both physic nut and other oilseed plants in order to improve their fuel properties using genetic and transgenic methods. About 90% of Castor bean oil consists of ricinoleate (12- hydroxy-oleate), produced from oleoyl-phosphatidycholine by the catalytic activity of a FAD2 family protein, oleic acid hydroxylase (FAH12) [70]. The expression patterns of fatty acid synthesis genes and FAH12 in developing physic nut seeds (Figures 5C–D, S4 and S5) were similar to those in developing castor bean seeds [70], except that the FAD2 gene was highly expressed in the physic nut seeds, but is very weakly expressed in castor bean endosperm [71]. A castor bean phospholipid:diacylglycerol acyltransferase (PDAT) gene, RcPDAT1A, also specifically enhances the reaction that generates ricinoleate in vivo and generates castor-like TAGs in RcFAH-expression Arabidopsis (CL37 parental line) seeds [72–73]. The physic nut genome has both a FAH12 gene (JC_C100003840) and a PDAT1A gene (JC_C100025621). Further, the presence of ricinoleate in its seed oil suggests that the physic nut FAH12 family gene probably encodes an active oleic acid hydroxylase (Figure 2A). However, physic nut seed oil contains very little ricinoleate (no PLoS ONE | www.plosone.org 8 May 2012 | Volume 7 | Issue 5 | e36522