Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism

Abstract

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.

Figure 1.

Characterization of castor seed development during early seed-filling phase. A, Castor seeds at five experimental stages of development. Whole castor seeds were harvested at exactly 2, 3, 4, 5, and 6 WAF (left to right). Harvested seed corresponded to morphological stages II/III, IV, V, VI, and VII reported by Greenwood and Bewley (1981). B, Average fresh and dry seed mass at each experimental stage. Seed harvested at each experimental stage were weighed before and after drying. Values are the average of three biological replicates. sds are shown. C, Percentage of seed fresh mass. Percentages were calculated using the average fresh and dry masses. Average dry mass was subtracted from the average fresh mass to determine water content. Percentages of average dry mass and water content equal 100% of the average fresh mass. D and E, Total fatty acid and protein content of castor seed. Total fatty acids and protein are noted in milligrams/seed. Values are the average of three biological replicates. sds are shown. [See online article for color version of this figure.]

Figure 2.

2-DGE of Cy5-labeled and unlabeled proteins. Total protein was isolated from castor seeds harvested at 4 WAF. A, Protein extracts (0.05 mg) were labeled with fluorescent dye, Cy5, fractionated by 2-DGE, and detected using fluorescence imaging. B, Unlabeled protein extracts (1 mg) were fractionated by 2-DGE and stained with colloidal Coomassie Brilliant Blue. Migrating positions of isoelectric point (pI) and molecular mass (kD) markers are shown. Circles highlight corresponding regions.

Figure 3.

Summary of experimental approach. A, Expression profile workflow. Protein extracts from each experimental stage were fractionated by 2-DGE in biological triplicate. Gels were imaged and analyzed to detect, quantify, and match spots. Spots represented in all three gels and at least two experimental stages were matched and labeled as spot groups. Average relative volumes, sds, and expression profiles were developed for the 660 spot groups. B, Protein identification workflow. Six hundred and sixty spots were excised from colloidal Coomassie Brilliant Blue reference gels. Each protein spot was digested, and the peptides were analyzed by LC-MS/MS. Spectra were searched against the castor TIGR database using SEQUEST. All protein assignments and expression profiles were deposited in the oilseeds proteomics Web site.

Figure 4.

Functional classification of identified proteins from castor, soybean, and rapeseed. Castor proteins were identified and grouped into 14 functional classes as described by Bevan et al. (1998) and modified by Hajduch et al. (2006). These data were compared with parallel studies of seed filling in soybean and rapeseed (Hajduch et al., 2006; Agrawal et al., 2008). The histogram represents percentage of identified proteins in each functional class. Numbers in parentheses correspond to functional classifications in Supplemental Table S3.

Figure 5.

Functional subclasses grouped according to expression pattern during castor seed development. Composite expressions of protein belonging to functional subclasses were grouped according to their similarity (Hajduch et al., 2005). Only subclasses with 10 or more proteins are shown. Number of proteins (right) and maximum relative volumes (left) are shown above the expression profile. Numbers in parentheses correspond to functional subclassifications in Supplemental Table S4.

Figure 6.

Characterization of carbon assimilation during seed filling in oilseeds. Proteins involved in sugar breakdown that lead to carbon sources for biosynthetic pathways are displayed. Expression profiles show the relative abundance for each enzyme at each stage of the early seed-filling phase. C (castor), S (soybean), or R (rapeseed) above the expression profile represents the origin of the protein (Hajduch et al., 2006; Agrawal et al., 2008). The number above the graph shows the number of identified proteins. The number to the left of the graph shows the maximum value (y axis) of the relative volume. Solid lines represent proteins identified in parallel proteomic studies of castor, soybean, or rapeseed, while unidentified proteins are represented by dashed lines. Checked squares indicate identified proteins with no expression profiles due to low volumes or presence in less than three stages. Brackets indicate the number of proteins identified by Sec-MudPIT. Gray boxes highlight differences in number (≥2) or relative abundance (≥2). Abbreviations not defined in the text: SuSy, Suc synthase; UGP, UDP-Glc pyrophosphorylase; PGM, phosphoglucomutase; PGI, phosphoglucose isomerase; FK, Fru kinase; PFK, phosphofructose kinase; TPI, triose-P isomerase; PDC, pyruvate dehydrogenase complex.

Figure 7.

Characterization of malate synthesis pathways. Expression profiles show the relative abundance (left), species of origin (C, castor; S, soybean; R, rapeseed), and number of identified proteins (top). Solid lines represent proteins identified in parallel proteomic studies of castor, soybean, or rapeseed, while unidentified proteins are represented by dashed lines. Brackets indicate proteins identified by immunoblot analysis. The proteins shown are listed as follows: PEPC, MDH, ME, PK, and PDC (pyruvate dehydrogenase complex).