diacylglycerol and acyl-CoA) under low-N conditions [22]. ABI5 activates several late embryogenesis-abundant genes during seed maturation [23–24]. Transcriptome analysis of developing seeds can provide fundamental molecular understanding of processes such as embryogenesis, seed filling, maturation, and seed quality. Several such analyses of developing physic nut seeds, using expressed sequence tags (ESTs), have provided limited information on storage accumulation in the seeds [25–26]. To extend this information we have characterized seed developmental stages in the species. We then exploited the abundant genomic resources available for physic nut to analyze transcriptomic changes in developing seeds using next-generation sequencing-based digital gene expression tag profiling to establish timeframes of key metabolic processes related to reserve accumulation and their regulators. Results Physiology of Physic Nut Seed Development To establish a framework for global analysis of gene expression profiles of the developing seeds, we initially defined four developmental stages, from embryogenesis to seed dispersal, characterized by distinct physiological events and associated changes in seed coat color, seed size, whole seed dry weight, kernel dry weight, and moisture status (Figure 1A). The first phase is the histodifferentiation or embryogenesis stage, during the first 17 days after pollination (DAP), under growth conditions at the harvesting site. At this stage the seeds have a water content of ca. 90% (fresh weight), and are milky white or faintly yellow. The second phase, between 17 and 29 DAP, is associated with rapid increases in seed volume and seed coat dry matter, the seeds are yellow to saffron yellow in color, and the seed coat hardens. The third (seed-filling) stage phase, from 29 to 41 DAP, is associated with a rapid increase in kernel dry mass, and a change in the seed coat color to black. Finally, during the late-maturing or desiccation stage there is a sharp declined in seed water content (Figure 1A, 1B). Under our field conditions, starch rapidly accumulated from 25 to 35 DAP, and then declined during the seed-filling stage (Figure 1C). The amount of starch, as a percentage of kernel mass, fell from ca. 11% at 35 DAP to ca. 4% at 39 DAP and ca. 2% at 45 DAP (Figure 1D). The oil and protein accumulation rates were high between 29 and 41 DAP (Figure 1C), in accordance with the rapid increase in kernel dry weight observed during this period (Figure 1A). The percentage contribution of total oils to the kernel mass increased from ca. 3% at 29 DAP to ca. 55% at 41 DAP (Figure 1D). Our fatty acid analyses confirm previous findings [4] that the most important fatty acids in physic nut oil are palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1) and linoleic acid (C18:2) (Figure 2A, 2B). Minor amounts (accounting for no more than 3% of the total fatty acids in oils of the mature seeds) of palmitoleic acid (C16:1), linolenic acid (C18:3), arachidic acid (C20:0), gondoic acid (C20:1), and 12-hydroxyoleic acid (ricinoleic acid, 12-OH C18:1) were also detected (Figure 2A, 2B). However, the fatty acid composition varied dramatically in different developmental stages of the seeds. Levels of C18:1 increased, while levels of C16:0 and C18:3 decreased as the seeds developed, and C18:3 contents were very low in mature seeds. C18:2 levels initially increased as the seeds developed, then declined during the late maturation stage. 12-hydroxyoleic acid appeared during the late seed-filling stage (Figure 2C). Gene Expression Profiles in Physic Nut Seeds Figure 1. Characterization of physic nut seed development stages. A: Changes in dry weight per seed, dry weight per kernel and water content of whole seeds from 14 to 55 days after pollination (DAP). The seed coat and kernel can be hardly separated before 24 DAP. The four following phases are indicated: I, histodifferentiation; II, seed coat development; III, seed filling; and IV, desiccation. Data are means 6SD of three replicates of 15 seeds. B: Appearance and size of physic nut seeds from 14 to 45 DAP. C: Accumulation kinetics of the storage compounds in kernels during seed development. Values are means (6SD) of three biological replicates. D: Changes in percentage contents of starch and oil in kernels during seed development. Values are means (6SD) of three biological replicates. doi:10.1371/journal.pone.0036522.g001 PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36522