Castor plant (Ricinus communis L.) produces a unique seed oil with numerous industrial applications. However, castor seed contains toxin ricin and ...
Engineering New Crops for Safe Castor Oil Production
Grace Q. Chen*, Yeh-Jin Ahn, and Louisa yang U.S. Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan Street, Albany, CA 94710, USA. * Email: QHGCpw.usda.gov
Introduction Castor plant (Ricinus communis L.) produces a unique seed oil with numerous industrial applications. However, castor seed contains toxin ricin and hyper-allergenic 2S albumins detrimental to castor grower and processor. Our project goal is to develop a safe source of castor oil through genetic engineering. The general approach is to generate a safe castor crop by blocking expression of the ricin and 2S albumins in seed. An alternative approach would be transgenic production of ricinoleate from temperate oilseed plants. Morphological criteria for assessing seed developmental age Castor seed consists of a mass of endosperm and an embryo with two thin papery cotyledons lying in the center of the endosperm. The endosperm is not absorbed by the embryo until seed germination. Castor plants produce a racemic, monoecious inflorescence, with male and female flowers blooming asynchronously, so we use pollination time as a common starting point for determining seed developmental age. We examined the external and internal morphological features
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of the seeds associated with specific developmental ages (Chen et al., 2004). We found that the testa color and the endosperm volume were most distinctive in determining the developmental age of castor seed. Based on changes of these two features, we divided seed development into three phases (I to III), each phase spanning approximately 20 days. During phase I of seed development, the capsule and seed developed their basic tissues and grew rapidly to almost full size at 19 days after pollination (DAP). Seeds had ivory white testa color and the endosperm tissue was at the free-nuclear stage and not yet expanded. The majority of the seed volume was filled with ground tissue of inner integument. In phase 11(20-40 DAP), the endosperm underwent cellularization and differentiation, expanding and displacing the inner integument, and ultimately occupied most of the seed volume. The testa started color deposition from the caruncle end to the opposite chalazal end, covering the whole seed with uneven shades of purple and brown color. Seeds in phase III (41-61 DAP) can be distinguished by filled cellular endosperm and sclerified, pigmented testa. After 54 DAP, seed testa was mature showing a shiny mosaic color of chocolate and silver. At about 61 DAP, capsules senesced and desiccated. The method of determining seed developmental stage is critical for drawing accurate comparisons between experiments. The use of morphological markers for determining seed age reduces variability and increases reliability of comparisons among experiments and among cultivars, where maturation times may differ. Expression of ricin and 25 albumin gene during seed development Ricin is a potent water-soluble protein toxin and is only found in castor seed. Its biochemical activity is well characterized as a Type TI ribosome-inactivating enzyme. The ricin molecule is a dimeric glycoprotein composed of a toxic A-chain and a lectin B-chain linked by disulfide bonds. The A-chain is a ribosome-inactivating enzyme which depurinates a specific adenine residue of 28S ribosomal RNA, MR
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thereby inactivating eucaryotic protein synthesis (Endo et al., 1987). Castor 2S albumin proteins were identified as the primary allergenic components based on fractionation studies (Machado et al., 1992, Thorpe et al., 1988, da Silva et al., 1996). Analyzing the sequences of the 2S albumin gene and proteins revealed that a single preprotein produces two heterodimeric 2S albumin proteins (Irwin et al., 1990). As part of a genetic approach to eliminating the ricin and the 2S albumins from castor, we performed comparative Northern analyses between ricin and 2S albumin genes by using the same sets of seeds at specific developmental ages. For the ricin gene (Chen et al., 2005), the mRNA signal was not detectable at early stages before the cellular endosperm emergence (12 and 19 DAP), but increased significantly at the onset of cellular endosperm development (26 DAP) and remained high up to the end of endosperm maturation (54 DAP). When seeds started desiccating (61 DAP), the expression of ricin gene decreased to a trace level. By comparison, low expression levels were detected for the 2S albumin gene in seeds at 12 and 19 DAP before cellular endosperm development (Chen et al., 2004). The mRNA levels rose sharply during 26-40 DAP when the endosperm underwent cellularization and differentiation. Once the cellular endosperm tissue had reached full volume, the level of 2S albumin mRNA started decreasing (47 DAP). After 47 DAP, the mRNA was degraded and did not give a clear signal (data not shown). Our Northern analyses showed that the timing of the major expression of ricin and 2S albumin genes coincided with that of cellular endosperm differentiation. However, we observed different temporal expression patterns between ricin and 2S albumin genes. The ricin gene was up-regulated up to the last stage of the cellular endosperm development, whereas 2S albumin gene had bell-Shaped expression pattern. The different temporal expression patterns between ricin and 2S albumin genes indicate distinctive regulatory mechanisms involved in their mRNA accumulation. The results provide us with critical information to develop promoters and antisense constructs that would optimize timing of transgene expression to suppress the ricin and 2S albumins in a safe castor crop.
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