Immunoblotting validates <i>ssp1</i> as an empty container.

<p>Duplicate one microgram aliquots of total seed protein from tester and backcross homozygotes were subjected to SDS-PAGE and immunoblotting with an anti-PHA antibody. Reporter lines 1, 3, 5, and 7 are shown in duplicate as testers (T) and the corresponding backcrosses (B). Aliquots of purified PHA were included as standards. The autoradiographs were scanned by densitometry and quantified by employing Image J software. The bar graphs represent the average PHA values relative to 30 ng of purified PHA. T-test statistical analyses revealed that lines 1, 3, and 5 have p-values less than 0.05, while the line 7 p-value was greater than 0.05. An independent experiment on the previous generation of these tester/backcross lines revealed similar enrichment trends and p-values.</p

<i>ssp1</i> gel profile and seed protein content.

<p>A. Mature seeds from Columbia (WT) and <i>ssp1</i> were extracted in Laemmli buffer and subjected to SDS-PAGE and staining with Coomassie Brilliant Blue. The 12S cruciferins are post-translationally cleaved into stable a (30–35 kD) and b (21–25 kD) polypeptides, and are predominant components of the gel profile, as are the smaller 2S albumins (2S). The arrows point to two cruciferin polypeptides that are missing from the <i>ssp1</i> profile. B. Relative protein levels in mature seed extracts from WT and <i>ssp1</i>. Lowry assays were performed on triplicate aliquots of seed extracts and the optical density values were mathematically transformed to yield micrograms of protein per milligram of dry seed, based on the use of BSA as a protein standard. The data represent the average of values from three independent experiments. T-test statistics for <i>ssp1</i> vs. wildtype reveal a p-value of less than 0.001.</p

The <i>ssp1</i> phenotype is due to a premature stop codon in <i>CRU3</i>.

<p>Aliquots of total RNA from mature seeds were purified and subjected to RNA gel blot analysis with probes prepared from cloned <i>CRU1, CRU2, CRU3</i>, and rRNA genes. <i>CRU1</i> and <i>CRU2</i> are expressed at approximately equal levels in wildtype and <i>ssp1</i>, while there is no <i>CRU3</i> signal from <i>ssp1</i> mRNA. B. Sequence analysis of the <i>CRU3</i> gene in <i>ssp1</i> shows that EMS induced a G to A transition mutation in exon 4, converting a tryptophan codon to a premature stop codon.</p

Characterization of a Cruciferin Deficient Mutant of Arabidopsis and Its Utility for Overexpression of Foreign Proteins in Plants

<div><p>Plant seeds naturally accumulate storage reserves (proteins, carbohydrates, lipids) that are mobilized during germination to provide energy and raw materials to support early seedling growth. Seeds have been exploited as bioreactors for the production to foreign materials, but stable, high level expression has been elusive, in part due to the intrinsic bias for producing the natural reserves in their typical proportions. To identify mutants governing seed filling, we screened a population of mutagenized Arabidopsis plants for a mutant that failed to fill its seeds. Here we report the identification of <i>ssp1</i>, a recessive, viable mutant that accumulates approximately 15% less protein than wildtype seeds. Molecular analyses revealed that <i>ssp1</i> is due to the introduction of a premature stop codon in <i>CRU3</i>, one of the major cruciferin genes. Unlike many other reserve mutants or transgenic lines in which seed storage protein levels are reduced by antisense/RNAi technologies, <i>ssp1</i> exhibits low level compensation by other reserves, and represents a mutant background that might prove useful for high level expression of foreign proteins. To test this hypothesis, we used a bean phytohemagglutinin (PHA) gene as a reporter and compared PHA expression levels in single copy insertion lines in <i>ssp1</i> vs. wildtype. These near isogenic lines allow reporter protein levels to be compared without the confounding and sometimes unknown influences of transgene copy number and position effects on gene expression. The <i>ssp1</i> lines consistently accumulated more PHA than the backcrossed counterparts, with increases ranging from 12% to 126%. This proof of principle study suggests that similar strategies in crop plants may improve the yield of foreign proteins of agronomic and economic interest.</p></div

Lipid profiles of wildtype and <i>ssp1</i> seeds.

<p>Aliquots of wildtype and <i>ssp1</i> were analyzed in triplicate by NMR for oil content, and by gas chromatography for the fatty acid species indicated. The oil content of <i>ssp1</i> is lower than wildtype, but several fatty acid species are slightly enriched. The asterisks indicate pairwise measurements where T-test statistics revealed significant (p<0.05) differences.</p

Strategy for testing the empty container hypothesis.

<p>Homozygous <i>ssp1</i> mutants were transformed with a PHA transgene and single copy insertion lines were selected by Southern blotting (step 1). The primary transgenic lines were selfed and homozygous tester lines were selected by examination of SDS-PAGE profiles of seed extracts (<i>ssp1</i> pattern) and 100% hygromycin segregation in the T3 lines (step 2). The tester lines were crossed to wildtype with the resulting F1 plants being both heterozygous for <i>CRU3 (ssp1)</i> and for the <i>PHA</i> transgene (step 3). Selfing was conducted and F3 lines screened by SDS-PAGE for normal seed protein profiles, by sequencing of <i>CRU3</i> to determine homozygosity, and by hygromycin segregation to determine <i>PHA</i> homozygosity (step 4). The resulting tester and backcross lines are homozygous for a single PHA insertion such that reporter gene copy number and context are fixed in both the tester (<i>cru3</i> background) and the wildtype (<i>CRU3</i>) background.</p

Two dimensional protein profiles of wildtype vs. <i>ssp1</i>.

<p>Total seed proteins were extracted and equivalent amounts of protein subjected to 2D gel analysis. Note the general concordance of spots in each profile. The major differences (for CRU3, <i>ssp1</i> panel; and CRU1, Columbia panel) are highlighted by the arrows.</p

Additional file 4: of Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis

Figure S2. Histogram of p-values and MA plot of log2 fold change vs average expression of each gene. Significantly DE genes (FDR ≤ 0.001) were assessed for significant differential expression between the two cultivars of 1533 and CDC. (PDF 714 kb

Additional file 8: of Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis

Figure S5. Heatmap of DE genes. Expression of genes A) upregulated and B) down regulated in CDC Pintium vs 1533–15 in each biological replicate. (PDF 566 kb

Additional file 3: of Slow darkening of pinto bean seed coat is associated with significant metabolite and transcript differences related to proanthocyanidin biosynthesis

Table S2. List of 922 DE genes (p < 0.001) in CDC Pintium and 1533–15. (XLSX 137 kb