Quantitative Proteomics of Seed Filling in Castor: Comparison with Soybean and Rapeseed Reveals Differences between Photosynthetic and Nonphotosynthetic Seed Metabolism1[C][W][OA]

Plant Physiology, Oct 2009 by Houston, Norma L, Hajduch, Martin, Thelen, Jay J

Seed maturation or seed filling is a phase of development that plays a major role in the storage reserve composition of a seed. In many plant seeds photosynthesis plays a major role in this process, although oilseeds, such as castor (Ricinus communis), are capable of accumulating oil without the benefit of photophosphorylation to augment energy demands. To characterize seed filling in castor, a systematic quantitative proteomics study was performed. Two-dimensional gel electrophoresis was used to resolve and quantify Cy-dye-labeled proteins expressed at 2, 3, 4, 5, and 6 weeks after flowering in biological triplicate. Expression profiles for 660 protein spot groups were established, and of these, 522 proteins were confidently identified by liquid chromatography-tandem mass spectrometry by mining against the castor genome. Identified proteins were classified according to function, and the most abundant groups of proteins were involved in protein destination and storage (34%), energy (19%), and metabolism (15%). Carbon assimilatory pathways in castor were compared with previous studies of photosynthetic oilseeds, soybean (Glycine max) and rapeseed (Brassica napus). These comparisons revealed differences in abundance and number of protein isoforms at numerous steps in glycolysis. One such difference was the number of enolase isoforms and their sum abundance; castor had approximately six times as many isoforms as soy and rapeseed. Furthermore, Rubisco was 11-fold less prominent in castor compared to rapeseed. These and other differences suggest some aspects of carbon flow, carbon recapture, as well as ATP and NADPH production in castor differs from photosynthetic oilseeds.

Castor (Ricinus communis) is a model heterotrophic oilseed that contains up to 60% fatty acids compared to approximately 20% and 40% in autotrophic oilseeds soybean (Glycine max) and rapeseed (Brassica napus), respectively (Weiss, 2000). Castor oil is a major component of many industrial lubricants and is currently of special interest, as it is being considered for biofuel use (Baldwin and Cossar, 2008; Dyer et al., 2008; Scholz and da Silva, 2008).With its high fatty acid content and recently sequenced genome, castor is a model plant for studying carbon assimilation in nonphotosynthetic oilseeds.

In oilseeds, fatty acid reserves are synthesized mainly during the seed-filling phase of seed development (Norton and Harris, 1975; Ruuska et al., 2002). Seed filling is a phase defined by morphological, cellular, and metabolic changes in the endosperm and embryo that coincide with rapidly increasing storage reserves, such as fatty acids and protein (Norton and Harris, 1975; Ruuska et al., 2002). Metabolic hallmarks of the seed-filling phase of castor seed development have been reported (Simcox et al., 1977; Greenwood and Bewley, 1981; Weiss, 2000). During development, seed length increases from 6 to 14 mm, while the seed coat color changes from white to red to speckled (Greenwood and Bewley, 1981). Fatty acid synthesis (FAS) begins in reddish-white seeds at approximately stage IV (3 weeks after flowering [WAF] for this study) and increases rapidly until stage VII (6 WAF) when the seed is speckled (Weiss, 2000). During this period, approximately 75% of fatty acid reserves are produced (Weiss, 2000).

De novo FAS requires carbon (acetyl-CoA), energy (ATP), and reducing equivalents (NADH/NADPH). In photosynthetic seeds, light energy can be used to produce ATP and NADPH within the plastid (Allen et al., 2009). In the plastids of heterotrophic seed, the origin of these components is unclear. Unlike ATP and NADH/NADPH, acetyl-CoA is not readily shuttled across the plastid membrane, where de novo FAS occurs (Bender-Machado et al., 2004). Therefore, precursors for acetyl-CoA synthesis must be produced in the plastid or imported from the cytosol. For example, Glc-6-P, pyruvate, and malate, may be imported via transporters or produced in the plastid (Smith et al., 1992; Eastmond et al., 1997; Pleite et al., 2005). When labeled carbon sources were supplied to isolated plastids from developing sunflower (Helianthus annuus) and castor seed, malate produced the highest rate of FAS (Smith et al., 1992; Eastmond et al., 1997; Lara-Nuez and Rodrguez-Sotres, 2004; Pleite et al., 2005). However, Alonso et al. (2007) suggested that malate only produces 5% to 9% of the carbon necessary for FAS in sunflower. Using metabolic flux analyses, they concluded that 91% to 95% of the carbon required for FAS was supplied by triose-Ps produced from hexoses imported into the plastid. The origin of energy and reducing equivalents needed for FAS also remains under investigation for nonphotosynthetic seeds. Studies conducted on heterotrophic plastids suggest two pathways are capable of providing adequate levels of reducing equivalents. One such pathway is the malate pathway described previously. Pleite et al. (2005) suggest that NADP-malic enzyme (ME) and the pyruvate dehydrogenase complex produce adequate levels of acetyl-CoA and reducing equivalents to support FAS in isolated plastids from sunflower embryos. Another pathway is the oxidative pentose-P pathway (OPPP),where threemolecules of Glc-6-P enter the pathway and are oxidized to three molecules of ribulose-5-P and CO2 while forming six NADPH molecules (Kruger and von Schaewen, 2003). Alonso et al. (2007) showed that OPPP produces the majority of reducing equivalents for FAS in sunflower, compared to ,4% produced by ME
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