more than 2% of the total FAs) (Figure 2B), although both the FAH12 and FAD2 genes were highly expressed at the filling stage of physic nut seeds (from S4 to S6) (Figures 5D and S5). The hydroxy fatty acid content (total HFA) is reduced from ca. 17% in the Arabidopsis CL37 parental line to less than 5% in the most- extreme FAD2 transgenics [73]. Thus, transcriptional regulation of FAD2 and FAH12 genes may be one of the mechanisms that contribute to a high level of ricinoleate accumulation in castor endosperm [73], and the low content of ricinoleate in physic nut oils may be due to the high expression of the FAD2 gene in the seeds. Alternatively, the physic nut FAH12 enzyme may have lower oleic acid hydroxylation activity and the PDAT1A enzyme lower specificity for ricinoleate than the corresponding castor bean enzymes. Hence, further study of the enzymatic activities and substrate specificities of physic nut FAH12 and PDAT1A enzymes should provide interesting information on the differences in evolution and roles of lipid synthesis enzymes between physic nut, castor bean and other oilseed plants. Storage Protein Genes Shown Different Expression Patterns in Developing Physic Nut Seeds Seed storage proteins in seeds are important source of amino acids for animal feed. The protein composition of physic nut seed meal has been analyzed, and shown to compare favorably with soybean meal, containing a good balance of essential amino acids, with the exception of lysine [74–75]. Physic nut seed storage proteins, 11S globulins and 2S albumins, but not 7S globulins, contain less Lys than soybean proteins according to the deduced amino acids of their coding genes (Table S4). The expression of the 11S globulin and 2S albumin genes began before the 7S globulin genes during filling of the physic nut seeds (Figures 5F and S6), and their longer duration of high level expression in the developing seeds is likely to be an important contributor to the relatively low Lys content of the mature seeds’ proteins. Conservative Transcriptional Regulation between Physic Nut and Arabidopsis Seed Reserve Accumulation In previous Arabidopsis studies a model for coordinated transcriptional regulation of the synthesis of seed storage reserves has been proposed [11,20]. The present study provides the first profiling of transcription factors throughout the development of physic nut seeds. The detected genes include many putative orthologs of genes involved in embryogenesis and seed reserve synthesis in Arabidopsis according to amino acid sequence alignments. In Arabidopsis, the transcription factor WRINKLED1 (WRI1) is involved in the regulation of seed storage metabolism, and expression of genes involved in glycolysis and lipid synthesis in developing seeds is compromised in wri1 mutants [11–12]. WRI1 functions downstream of LEC1 and LEC2, and ectopic expression of LEC1, LEC2 or WRI1 in vegetative tissues up-regulates the expression of genes involved in FA synthesis [13,76]. Storage protein gene transcription is promoted by the combined action of LEC2, FUSCA3 (FUS3) and ABSCISIC ACID INSENSITIVE-3 (ABI3). The synthesis of these factors is in turn activated by LEC1 and LEC1L [19–20,76–77], while ABI4 acts as a repressor of lipid breakdown in Arabidopsis seed germination [21]. At the end of seed filling, ABI5/EEL has been implicated in shutting down seed storage protein synthesis during seed desiccation, and activating several late embryogenesis-abundant genes, including AtEm1 and AtEm6, during seed maturation [23–24,78]. In developing physic nut seeds, the expression sequence of these genes is as follows: LEC1 gene at S1; LEC2 and LEC1L genes at S3; FUS2, ABI3, ABI4, and WRI1 genes at S4 (Figure 6; Table S3). As in Gene Expression Profiles in Physic Nut Seeds Arabidopsis, this expression pattern of TF genes is consistent with the decreased expression of starch synthesis genes and increased expression of fatty acid and TAG synthesis and storage protein genes during the filling stages in the developing seeds (Figure 5C– F). The expression level of the ABI5 gene was highest at the last stage, S7, in accordance with the decline in expression of genes encoding 11S globulin and 2S albumin storage proteins, and up- expression of late embryo abundant protein genes (Figures 5F, 6 and S6). In contrast to Arabidopsis seeds, in which the storage compounds are deposited in cotyledons, physic nut seeds deposit storage compounds in the endosperm. These findings suggest that a similar regulatory network of TFs controls oil and seed storage protein accumulation in the endosperm of physic nut seed as in the embryos of Arabidopsis seeds. Thus, our data provide an important step toward identifying the regulatory gene networks responsible for programming the development of physic nut seeds. However, the reasons and roles of differences in the compart- mentation of storage reserves (in endosperms of physic nut and cotyledons of Arabidopsis seeds) and seed-specific TF genes in this process remain to be determined. In conclusion, the presented study of seed development, storage reserve accumulation and gene expression, provide integrative information for understanding the relationships of these processes during physic nut seed development. Our results show that the phase between 17 and 29 DAP is associated with rapid increases in seed volume and seed coat dry matter, while the seeds fill from 29 to 41 DAP during physic nut seed development (under our growth conditions). As proposed in Arabidopsis, the WRI1, FUS3 and ABI3transcription factor genes were expressed from the onset of seed-filling (S4), which shifted the metabolic network towards the production of storage fatty acids and oils and seed storage proteins in the developing seeds. Expression levels of FATA, SAD and FAD2 genes were high, and the expression of FATB and FAD3 genes low in filling stage seeds, in accordance with the high contents of C18:1 and C18:2 lipids detected in physic nut seed oils. These data should facilitate understanding of the molecular basis of fatty acid and lipid biosynthesis, and identification of the key genes involved in regulation of the fatty acid composition, oil content and storage reserve biosynthesis during physic nut seed development. Further investigations of the functions of these genes and the scope for using them to manipulate the fatty acid composition and oil contents of seeds by genetic engineering means could substantially contribute to the refinement of biofuels. Materials and Methods Ethics Statement No specific permits were required for the described field studies, because the experimental field is owned by South China Botanical Garden, Chinese Academy of Sciences, and the Key Laboratory of Plant Resources Conservation and Sustainable Utilization per- forms the management. No specific permits were required for these locations/activities, because the location is not privately- owned or protected in any way and the field studies did not involve endangered or protected species. Plant Materials and Chemicals Physic nut (J. curcas L.) trees of the inbred cultivar GZQX0401 were grown in a natural environment on farmland in the South China Botanical Garden (Guangzhou, Guangdong Province, China). Seeds were sampled from artificially pollinated 4-year- old plants from 14 days after pollination (DAP) during the period of July-September, defining the pollination day as day 0. Thirty to one hundred seeds (after and before 24 DAP, respectively) were PLoS ONE | www.plosone.org 9 May 2012 | Volume 7 | Issue 5 | e36522