Gene Expression Profiles in Physic Nut Seeds Figure 6. Expression profiles of transcription factor homologues corresponding to those known to regulate reserve synthesis in Arabidopsis. doi:10.1371/journal.pone.0036522.g006 developing physic nut seeds. The work has also revealed global insights into the regulatory network of FA metabolism of the physic nut seed, and will thus be helpful for oil-crop engineering. 29–41 DAP is the Key Period for Seed Storage Reserve Synthesis in Physic Nut Seeds The histodifferentiation and accumulation of dry matter in the seed coat of the physic nut seeds occurred before 29 DAP, under our field conditions (Figure 1A). Rapid increase of the kernel dry mass started at 29 DAP and ended at 41 DAP (Figure 1A). During this period, the starch content declined from ca. 11% to ca. 2%, while the lipid content increased from ca. 3% to ca. 55% of kernel dry weight (Figure 1D), the storage protein level increased (Figure 1C) and the fatty acid composition significantly changed (Figure 2C). Expression analysis revealed that opposite shifts in expression levels of starch and fatty acid synthesis genes also occurred from 29 to 41 DAP (Figure 5A and 5C, respectively). Oil-body protein genes (Figure 5E) and seed storage protein genes (Figure 5F) were highly expressed at this stage. Thus, 29–41DAP is the critical seed storage reserve synthesis and accumulation period in developing physic nut seeds. In addition, the crucial period for the transition of seed-to-sink tissue was around 29 DAP. At this point, expression of several seed storage reserve synthesis regulation genes, such as WRI1, FUS3, and ABI3 began (Figure 6), the expression of starch synthesis genes decreased (Figure 5A) and that of fatty acid synthesis genes increased (Figure 5C). Sucrose Import and Breakdown Sucrose import and breakdown are important processes for seed production and development. In B. napus L. seeds, the expression levels of SUT1 control some of the phenotypic variation for branch number and seed weight [43]. The rice GIF1 (GRAIN INCOMPLETE FILLING 1) gene encodes a cell- wall invertase required for carbon partitioning during early grain-filling [44]. The acid invertases are targeted to the cell wall (cwINV) and vacuole (vINV), neutral/alkaline invertases (nINV) function in the cytosol [45], and the monosaccharides glucose and fructose are taken up by sugar transport proteins (STP) and polyol transporters (PLT) [29–30]. In our developing physic nut seeds, cwINV genes were highly expressed at the early filling stage (Figures 3A and S1). The STP and PLT genes were also expressed, at low levels, at the late filling stage (Figures 3A and S1). In contrast, the total expression level of sucrose transporter (SUT), sucrose synthase (SuSy) and nINV genes was high not only during the early developing stage but also during the filling stage (Figures 3B and S1). These results imply that the direct sucrose importing pathway plays the major role in sucrose import, while the monosaccharide importing pathway plays secondary role during the filling stage in developing physic nut seeds. Phosphoenolpyruvate and triose-phosphate are the key carbon sources imported into the plastid, by the phosphoenolpyruvate translocator (PPT) and triose-phosphate translocator (TPT), respectively, for FA synthesis, while the glucose-6-phosphate (Glc-6-P) required for starch biosynthesis and the oxidative pentose phosphate pathway (OPPP) is imported by the Glc-6-P transporter (GPT). Mutation of GPT1 alters the expression of genes involved in starch turnover and the oxidative pentose phosphate pathway, and results in seed abortion at the end of the globular stage of embryo development, in Arabidopsis [46]. In the PPT1 Arabidopsis mutant cue1, the oil content of mature seeds is reportedly 10 to 20% lower than in wild type [47]. The xylulose 5-phosphate/phosphate translocator (XPT) accepts xylulose 5-phosphate, ribulose 5-phosphate and erythrose 4- phosphate as counter exchange substrates for Pi between the plastid and cytosol [33]. In addition, the nucleoside triphosphate transporter (NTT) provides the plastid stroma with ATP from the cytosol. Arabidopsis possesses two NTTs, NTT1 and NTT2, with similar transport characteristics. Loss of NTT2, but not NTT1, impairs the supply of cytosolic ATP to plastids, resulting in a reduced seed oil phenotype [34–35]. In both Arabidopsis and B. napus, PPT-encoding genes are reportedly expressed strongly at all stages of seed development, including later stages, while GPT genes are expressed at high levels during early stages [8,48]. Genes encoding GPT (JC_C100019452), PPT (JC_C100021622) and TPT (JC_C100026660) were all strongly expressed during all stages of physic nut seed development until maturation (Figures 3C and S1). The NTT gene was also strongly expressed, while the XPT gene was weakly expressing during development of the seeds (Figure 3C). These results indicate that C3 and C6 sugar phosphates, but not C4 and C5 sugar phosphates, are the major sugar phosphates imported into plastids in developing physic nut seeds. The results also imply that the GPT, PPT, TPT and NTT genes play important roles PLoS ONE | www.plosone.org 7 May 2012 | Volume 7 | Issue 5 | e36522