Identification and characterization of lysine-rich proteins and starch biosynthesis genes in the opaque2 mutant by transcriptional and proteomic analysis

Abstract

Background: The opaque2 mutant is valuable for producing maize varieties with enhanced nutritional value. However, the exact mechanisms by which it improves protein quality and creates a soft endosperm texture are unclear. Given the importance of improving nutritional quality in grain crops, a better understanding of the physiological basis for these traits is necessary.

Results: In this study, we combined transcript profiling and proteomic analysis to better understand which genes and proteins are altered by opaque2 in the W64A inbred line. These analyses showed that the accumulation of some lysine-rich proteins, such as sorbitol dehydrogenase and glyceraldehyde3-phosphate dehydrogenase, was increased in mature kernels and may contribute substantially to the lysine content of opaque2 endosperm. Some defense proteins such as beta-glucosidase aggregating factor were strongly down regulated and may be regulated directly by opaque2. The mutant also had altered expression of a number of starch biosynthesis genes and this was associated with a more highly crystalline starch.

Conclusions: The results of these studies revealed specific target genes that can be investigated to further improve nutritional quality and agronomic performance of high lysine maize lines, particularly those based on the presence of the opaque2 mutation. Alteration of amylopectin branching patterns in opaque2 starch could contribute to generation of the soft, starchy endosperm.

Figure 1

GO classification for genes with altered expression in o2. Genes were assigned to GO molecular function (A) and biological process (B). The plots on the left are genes down regulated in o2 and the plots on the right are genes up regulated in o2.

Figure 2

2D SDS-PAGE analysis of W64A + and W64Ao2. Non-zein proteins from W64A + (A) and W64Ao2 (B) lines were extracted from mature endosperm flour and separated by 2D SDS-PAGE. Circled, numbered spots were excised from gels and protein identities were determined by MALDI-TOF peptide mass mapping and correspond to the rows in Table 1.

Figure 3

Confirmation of genes or proteins altered in W64Ao2 by qRT-PCR. Expression of the indicated genes was analyzed in 22 DAP endosperms of W64A + and W64Ao2 by GeneCalling transcript profiling (A) or qRT-PCR to confirm the difference in expression in the transcript profiling, or to measure the expression levels of genes that were differentially expressed by 2D SDS-PAGE analysis (B). All expression values are normalized relative to the expression of RRB1. Asterisks indicate significantly different expression using the two-tailed t-test at a level of p < 0.05. Note that the Y-axis is logarithmic to accommodate the wide differences in gene expression levels among the transcripts. Missing columns in A indicate that the gene was not among the genes that had a confirmed identity in the transcript profiling data.

Figure 4

Western blot analysis of selected proteins in W64A + and W64Ao2 . Western blots were performed using antisera against the proteins indicated on the left. Three replicate samples for each line were obtained from independent ears frozen at 22 DAP and 25 μg of protein from each was separated by SDS-PAGE followed by western blotting. Each band was analyzed by densitometry and the fold change values calculated for each protein and the values are presented in Table 2.

Figure 5

Amylopectin branch length analysis. Solubilized starch was de-branched in the presence of isoamylase and the resulting glucan chains were separated by capillary electrophoresis. (A) Histograms of the distribution of glucan chains were similar but W64Ao2 was shifted toward a higher degree of polymerization. (B) Difference plot was calculated by subtracting the W64A + values from the W64Ao2 values showed a substantial increase in chains with a degree of polymerization between 15 and 25 glucose subunits. The histograms represent the average of three replicates for each genotype.