Submitted to: Molecular Breeding
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 2, 2007
Publication Date: August 4, 2007
Citation: Skadsen, R.W., Jing, P. 2008. Transcript profile of barley aleurone differs between total and polysomal RNAs: Implications for proteome modeling. Molecular Breeding. 21:261-269. Interpretive Summary: Microarray analysis is frequently used to detect the activities of up to 22,000 genes simultaneously in various plant tissues. This tells which genes are turned on and to what extent. This technique could be very useful in identifying genes associated with barley malting quality. Attempts have been made in several labs but none have been successful. One possible reason is that these labs (like all others) probably use the wrong source of mRNA to probe with the microarrays. When a gene is decoded, it is first copied as an mRNA molecule before it is translated on polysomes to produce a protein or enzyme. Thus, the mRNAs that are bound to polysomes represent the genes that are actively being decoded into proteins and enzymes and also reflect the amounts of those mRNA molecules in use. All labs use total tissue mRNA for microarray analysis, instead of polysomal mRNA. In total RNA, many of the RNAs may not actually be in use by the tissue. Since malting depends on enzymes, it would be more logical to use polysomal mRNA. We compared these two populations in the most active tissue producing malting enzymes in germinating barley seeds. Large qualitative and quantitive differences were found between the genes detected in total mRNA and those detected in polysomal mRNA. The difference are as great as the largest treatment differences that can be found in other microarray studies. This study raises the possibility that the wrong source of RNA is widely used. These findings will impact molecular geneticists as they design experiments to develop gene markers for breeding for complex agronomic traits, such as seed quality.
Technical Abstract: Microarray analysis of mRNA populations is routinely conducted with total RNA. However, such analyses would probably represent the translated genome (proteome) more accurately if conducted with polysomal RNA. In order to determine whether significant variation occurs between these two populations, the relative abundance of transcripts was analyzed in barley aleurones of intact 3.5 day-old germinated seedlings, comparing the total RNA with the polysomal RNA population. A total of 13,744 transcripts was detected; 11,489 were detected in both populations, while 714 were detected only in total RNA, and 1541 were detected only in polysomal RNA. A surprising number (6312 gene calls or 46% of the transcriptome) differed significantly between populations. Almost half of these (2969) were more abundant by at least two-fold, depending on the RNA source. Transcripts encoding hydrolytic enzymes for the mobilization of stored seed macromolecules were more highly represented in total RNA, rather than polysomal RNA. These included proteinases, nucleases and carbohydrases. Genes for ribonucleoprotein complexes, nucleic acid binding and components of ribosomes were more abundant by at least two-fold in the polysomal population. Among genes with signal intensities of 1000 or more, hydrolases were greatly represented in total RNA, whereas ubiquitin, histone and kinase related genes were mainly represented in polysomal RNA. These findings have implications for understanding patterns of gene expression to provide better models of expression at the protein level and more accurate assessments of the metabolome.