The Zea mays mutants opaque-2 and opaque-7 disclose extensive changes in endosperm metabolism as revealed by protein, amino acid, and transcriptome-wide analyses

Abstract

Background: The changes in storage reserve accumulation during maize (Zea mays L.) grain maturation are well established. However, the key molecular determinants controlling carbon flux to the grain and the partitioning of carbon to starch and protein are more elusive. The Opaque-2 (O2) gene, one of the best-characterized plant transcription factors, is a good example of the integration of carbohydrate, amino acid and storage protein metabolisms in maize endosperm development. Evidence also indicates that the Opaque-7 (O7) gene plays a role in affecting endosperm metabolism. The focus of this study was to assess the changes induced by the o2 and o7 mutations on maize endosperm metabolism by evaluating protein and amino acid composition and by transcriptome profiling, in order to investigate the functional interplay between these two genes in single and double mutants.

Results: We show that the overall amino acid composition of the mutants analyzed appeared similar. Each mutant had a high Lys and reduced Glx and Leu content with respect to wild type. Gene expression profiling, based on a unigene set composed of 7,250 ESTs, allowed us to identify a series of mutant-related down (17.1%) and up-regulated (3.2%) transcripts. Several differentially expressed ESTs homologous to genes encoding enzymes involved in amino acid synthesis, carbon metabolism (TCA cycle and glycolysis), in storage protein and starch metabolism, in gene transcription and translation processes, in signal transduction, and in protein, fatty acid, and lipid synthesis were identified. Our analyses demonstrate that the mutants investigated are pleiotropic and play a critical role in several endosperm-related metabolic processes. Pleiotropic effects were less evident in the o7 mutant, but severe in the o2 and o2o7 backgrounds, with large changes in gene expression patterns, affecting a broad range of kernel-expressed genes.

Conclusion: Although, by necessity, this paper is descriptive and more work is required to define gene functions and dissect the complex regulation of gene expression, the genes isolated and characterized to date give us an intriguing insight into the mechanisms underlying endosperm metabolism.

Figure 1

Two-dimensional Analysis of Alpha-Zein Polypeptides. Genotypes are indicated above each panel. Relative molecular weights derived from size standards are indicated as kDa values within each panel.

Figure 2

Distribution of maize EST Unigenes amongst functional categories. Gene-ontology categories were assigned to ESTs through curator-revised categorization. Eight thousand nine hundred and fifty endosperm preferred ESTs were classified. Gene-ontology terms (http://www.geneontology.org) were assigned based on similarity to known protein sequences in several databases using the BLAST2GO software (http://www.blast2go.org).The percentage of ESTs in each category is indicated next to the corresponding map sector.

Figure 3

Analysis of differential gene expression in A69Ywt, o2, o7, and o2o7 endosperms. Signal correlation plots were used to examine mutant effects on the EST pool taken into consideration in endosperms at 14 DAP. (A) Signals derived for each EST in wild-type (x-axis) and o2 (y-axis) backgrounds were plotted using a logarithmic scale. Similar graphs were made to compare the expression of (B): wild-type (x-axis) vs. o7 (y-axis), (C): wild-type (x-axis) vs. o2o7 (y-axis) and (D): o2 (x-axis) vs. o7 (y-axis), respectively.

Figure 4

Differential expression of ESTs in A69Yo2, o7, and o2o7 endosperms with respect to A69Ywt endosperm at 14 DAP. Diagrams show the number of down- and up-regulated genes in the mutant endosperms with respect to wt endosperm.

Figure 5

Schematic representation of amino acid biosynthetic pathways. Leu, Ile, Val, Thr, Met, Cys, Ser, and Gly (A) and Phe, Tyr, and Trp (B) biosynthesis pathways are indicated. Those steps associated with enzymes exhibiting an altered expression profile in the mutant endosperms analyzed with respect to the wt endosperm are marked. ESTs revealing an altered expression profile in A69Yo2 (1), A69Yo7 (2) endosperm, and both the A69Yo2 and A69Yo7 endosperms (3) are indicated.

Figure 6

Schematic representation of metabolic pathways. The citrate cycle (A), glycolysis (B) and starch (C) metabolisms are represented. Steps associated with enzymes exhibiting an altered expression profile in the mutant endosperms analyzed with respect to the wt endosperm are marked. ESTs revealing an altered expression profile in A69Yo2 (1), A69Yo7 (2) endosperm, and both the A69Yo2 and A69Yo7 endosperms (3) are indicated.