Figure 2. Changes in fatty acid composition during physic nut seed development. A: Gas chromatography of fatty acids in mature physic nut seeds. The peaks were identified by mass spectrometry. B: Fatty acid composition in mature physic nut seeds. Each data point represents the mean (6SD) of three biological replicates. C: Changes in fatty acid composition during seed development. Fatty acids were extracted from whole seeds sampled 14 and 19 days after pollination (DAP) and from kernels of seeds sampled 25 to 45 DAP. Each data point represents the mean (6SD) of three biological replicates. C17:0 was used as both a seed lipid extraction and transmethylation internal standard. C16:0, palmitic acid; C16:1 palmitoleic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; C20:0, arachidic acid; C20:1, gondoic acid; 12-OH C18:1, 12-hydroxyoleic acid (ricinoleic acid). doi:10.1371/journal.pone.0036522.g002 Gene Expression Profiles in Physic Nut Seeds Strategy for the Global Analysis of Gene Expression Profiles of Developing Seeds For the genome-wide analysis of gene expression in the developing physic nut seeds, we examined changes in the transcriptome of seeds at seven developmental points using next- generation sequencing-based Digital Gene Expression tag profiling [27]. The seven points were during the following stages: early seed development (histodifferentiation) (S1, 14 DAP), early increase of seed dry-weight (S2, 19 DAP), rapid increase of seed coat dry- weight (S3, 25 DAP), early increase of kernel dry-weight (S4, 29 DAP), rapid increase of kernel dry-weight (S5, 35 DAP), late kernel dry-weight increase (S6, 41 DAP), and desiccation (S7, 45 DAP) (Figure 1A, 1B). ANOVA identified nine clusters of the relative abundance of 11412 detected genes during the seven stages (Table S1). We then examined in detailed changes in the expression of genes pertaining to carbon flow, storage reserve accumulation and their associated transcription factors. Sucrose Hydrolysis and Sugars/Sugar-phosphate Translocation-related Genes Sucrose is either directly transported into sink cells by the sucrose transporter (SUT) or, following its cleavage by an extracellular invertase (cwINV), the monosaccharides glucose and fructose are taken up by sugar transport proteins (STPs) and polyol transporters (PLTs) [28–29]. In cells, sucrose cleavage can be catalyzed by sucrose synthase (SuSy) and intracellular invertases (vacuolar invertases, vINV, or neutral/alkaline invertases, nINV). Physic nut has five putative cell wall-type invertase (cwINV) genes, eighteen putative STP and seven putative PLT genes, according to alignments with Arabidopsis and poplar orthologs [29–30]. Expression levels of two cwINV genes (JC_C100020497 and JC_C100025743), three STP genes (JC_C100009119, JC_C100014694 and JC_C100026553) and two PLT genes (JC_C100027064 and JC_C100007576) are highest before the filling stage, while those of three PLT genes (JC_C100000725, JC_C100025470 and JC_C 100025471) are highest during the filling stage in the seeds (Figures 3A and S1). Physic nut has only three SUT genes, all expressed at relatively high levels in developing seeds, but the expression level of two decreased during the filling stage. Physic nut has six SuSy genes: one (JC_C100010353) maximally expressed in the filling stage, and two (JC_C100024259 and JC_C100025110) that are strongly expressed but their expression level declines in the filling stage. The expression level of vINV genes also declines during the filling stage, while expression levels of seven of the eight nINV genes remain high in the filling stage (Figures 3B and S1). These results suggest that different sugar importing pathways are active during different developmental stages in physic nut seeds. Sugar-phosphates transported across the plastid envelope membrane by phosphate/glucose-6-phosphate transporter (GPT), phosphate/phosphoenolpyruvate translocator (PPT), phosphate/triose-phosphate transporter (TPT), and xylulose-5- phosphate/phosphate translocator (XPT) [31–33]. Another key enzyme in translocation and assimilation processes is the plastidic ATP/ADP transporter (NTT), which plays a crucial role in supplying storage plastids with ATP [34–35]. GPT, PPT, and TPT are each encoded by two genes, while XPT and NTT are both encoded by a single gene in the physic nut genome. Expression levels of GPT (JC_C100019452), TPT (JC_C100026660), PPT (JC_C100021622) and NTT (JC_C100013456) genes remained high during the entire seed development process and peaked at S3–S5, while the XPT gene PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36522