Abstract
Orphan genes (OGs), which are genes unique to a specific taxon, play a vital role in primary metabolism. However, little is known about the functional significance of Brassica rapa OGs (BrOGs) that were identified in our previous study. To study their biological functions, we developed a BrOG overexpression (BrOGOE) mutant library of 43 genes in Arabidopsis thaliana and assessed the phenotypic variation of the plants. We found that 19 of the 43 BrOGOE mutants displayed a mutant phenotype and 42 showed a variable soluble sugar content. One mutant, BrOG1OE, with significantly elevated fructose, glucose, and total sugar contents but a reduced sucrose content, was selected for in-depth analysis. BrOG1OE showed reduced expression and activity of the Arabidopsis sucrose synthase gene (AtSUS); however, the activity of invertase was unchanged. In contrast, silencing of two copies of BrOG1 in B. rapa, BraA08002322 (BrOG1A) and BraSca000221 (BrOG1B), by the use of an efficient CRISPR/Cas9 system of Chinese cabbage (B. rapa ssp. campestris) resulted in decreased fructose, glucose, and total soluble sugar contents because of the upregulation of BrSUS1b, BrSUS3, and, specifically, the BrSUS5 gene in the edited BrOG1 transgenic line. In addition, we observed increased sucrose content and SUS activity in the BrOG1 mutants, with the activity of invertase remaining unchanged. Thus, BrOG1 probably affected soluble sugar metabolism in a SUS-dependent manner. This is the first report investigating the function of BrOGs with respect to soluble sugar metabolism and reinforced the idea that OGs are a valuable resource for nutrient metabolism.
Highlights
Orphan genes (OGs), genes that are restricted to a single lineage or species, show either nonsignificant or no sequence similarity with genes in other related species
The overexpression of the transgenes was confirmed by quantitative reverse-transcriptase PCR for both the mutants and wild type (WT), and the results showed that the expression was detected only in the transgenic plants
We observed a change in soluble sugar content in 42 Arabidopsis lines (BrOG19OE was excluded), which suggested that Brassica rapa OGs (BrOGs) could extensively impact soluble sugar metabolism
Summary
Orphan genes (OGs), genes that are restricted to a single lineage or species, show either nonsignificant or no sequence similarity with genes in other related species. OGs have been identified in many species or lineages, such as Arabidopsis thaliana[1], Brassica rapa[2], and Citrus sinensis[3], with the continuing sequencing of several genomes. There is limited information on the functional significance of OGs in a particular species, they are known to play a role in primary metabolism, respond to biotic and abiotic stresses, and influence speciesspecific evolution and species-specific traits. YC4 could reduce Arabidopsis and soybean susceptibility to viruses, bacteria, fungi, aphids, and soybean cyst nematodes[6]. The prediction and characterization of the functions of most OGs are unclear, as they lack identifiable folds, recognizable domains, and functional motifs
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