Brassica rapa orphan genes largely affect soluble sugar metabolism

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.

References

[1] Donoghue, M. T., Keshavaiah, C., Swamidatta, S. H. & Spillane, C. Evolutionary origins of Brassicaceae specific genes in Arabidopsis thaliana. BMC Evol. Biol. 11, 47 (2011).

[2] Jiang, M. et al. Mining of Brassica-specific genes (BSGs) and their induction in different developmental stages and under Plasmodiophora brassicae stress in Brassica rapa. Int. J. Mol. Sci. 19, 2064 (2018).

[3] Xu, Y. et al. Identification, characterization and expression analysis of lineage-specific genes within sweet orange (Citrus sinensis). BMC Genomics 16, 995 (2015).

[4] Li, G. et al. Orphan genes are involved in drought adaptations and ecoclimatic-oriented selections in domesticated cowpea. J. Exp. Bot. 28, 3101-3110 (2019).

[5] O’Conner, S. et al. in Engineering Nitrogen Utilization in Crop Plants (eds Shrawat, A., Adel Zayed, A. & Lightfoot, D.) 95-117 (Springer, 2018).

[6] Qi, M. et al. QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests. Plant Biotechnol. J. 17, 252-263 (2019).

[7] Kumar, A., Gates, P. B., Czarkwiani, A . & Brockes, J. P. An orphan gene is necessary for preaxial digit formation during salamander limb development. Nat. Commun. 6, 8684 (2015).

[8] Kondou, Y. et al. Systematic approaches to using the FOX hunting system to identify useful rice genes. Plant J. 57, 883-894 (2009).

[9] Dubouzet, J. G. et al. Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice. Plant Biotechnol. J. 9, 466-485 (2011).

[10] Li, X. et al. Large-scale investigation of soybean gene functions by overexpressing a full-length soybean cDNA library in Arabidopsis. Front. Plant Sci. 9, 631 (2018).

[11] Ling, J. et al. Development of iFOX-hunting as a functional genomic tool and demonstration of its use to identify early senescence-related genes in the polyploid Brassica napus. Plant Biotechnol. J. 16, 591-602 (2018).

[12] Song, X. et al. Genes associated with agronomic traits in non-heading Chinese cabbage identified by expression profiling. BMC Plant Biol. 14, 71 (2014).

[13] Rosa, E., David, M. & Gomes, M. H. Glucose, fructose and sucrose content in broccoli, white cabbage and Portuguese cabbage grown in early and late seasons. J. Sci. Food Agric. 81, 1145-1149 (2001).

[14] Xiang, L. et al. Exploring the neutral invertase-oxidative stress defence connection in Arabidopsis thaliana. J. Exp. Bot. 62, 3849-3862 (2011).

[15] Li, Y. et al. Arabidopsis sucrose transporter SUT4 interacts with cytochrome b5-2 to regulate seed germination in response to sucrose and glucose. Mol. Plant 5, 1029-1041 (2012).

[16] Smeekens, S . Sugar regulation of gene expression in plants. Curr. Opin. Plant Biol. 1, 230-234 (1998).

[17] Fallahi, H. et al. Localization of sucrose synthase in developing seed and siliques of Arabidopsis thaliana reveals diverse roles for SUS during development. J. Exp. Bot. 59, 3283-3295 (2008).

[18] Barratt, D. H. et al. Normal growth of Arabidopsis requires cytosolic invertase but not sucrose synthase. Proc. Natl Acad. Sci. USA 106, 13124-13129 (2009).

[19] Baroja-Fernandez, E. et al. Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production. Proc. Natl Acad. Sci. USA 109, 321-326 (2012).

[20] Ruan, Y. L. Sucrose metabolism: gateway to diverse carbon use and sugar signaling. Annu. Rev. Plant Biol. 65, 33-67 (2014).

[21] Yang, H., Wu, J. J., Tang, T., Liu, K. D. & Dai, C. CRISPR/Cas9-mediated genome editing efficiently creates specific mutations at multiple loci using one sgRNA in Brassica napus. Sci. Rep. 7, 7489 (2017).

[22] Sander, J. D. & Joung, J. K. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat. Biotechnol. 32, 347-355 (2014).

[23] Feng, Z. et al. Efficient genome editing in plants using a CRISPR/Cas system. Cell Res. 23, 1229-1232 (2013).

[24] Jiang, W. Z. et al. Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing. Plant Biotechnol. J. 15, 648-657 (2017).

[25] Braatz, J. et al. CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus). Plant Physiol. 174, 935-942 (2017).

[26] Lawrenson, T. et al. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease. Genome Biol. 16, 258 (2015).

[27] Li, G. et al. Research progress on Agrobacterium tumefaciens-based transgenic technology in Brassica rapa. Hortic. Plant J. 4, 126-132 (2018).

[28] Zhang, X., Henriques, R., Lin, S. S., Niu, Q. W. & Chua, N. H. Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641-646 (2006).

[29] Zhang, C. et al. A thraustochytrid diacylglycerol acyltransferase 2 with broad substrate specificity strongly increases oleic acid content in engineered Arabidopsis thaliana seeds. J. Exp. Bot. 64, 3189-3200 (2013).

[30] Li, L. & Wurtele, E. S. The QQS orphan gene of Arabidopsis modulates carbon and nitrogen allocation in soybean. Plant Biotechnol. J. 13, 177-187 (2015).

[31] Li, L. et al. Identification of the novel protein QQS as a component of the starch metabolic network in Arabidopsis leaves. Plant J. 58, 485-498 (2009).

[32] Fan, S. K. et al. Effects of split applications of nitrogen fertilizers on the Cd level and nutritional quality of Chinese cabbage. J. Zhejiang Univ. Sci. B 18, 897-905 (2017).

[33] Gao, L. W. et al. Genome-wide analysis of auxin transport genes identifies the hormone responsive patterns associated with leafy head formation in Chinese cabbage. Sci. Rep. 7, 42229 (2017).

[34] Jin, C. et al. Carbon dioxide enrichment by composting in greenhouses and its effect on vegetable production. J. Plant Nutr. Soil Sci. 172, 418-424 (2009).

[35] Arendsee, Z. W., Li, L. & Wurtele, E. S. Coming of age: orphan genes in plants. Trends Plant Sci. 19, 698-708 (2014).

[36] Yao, C., Yan, H., Zhang, X. & Wang, R. A database for orphan genes in Poaceae. Exp. Ther. Med. 14, 2917-2924 (2017).

[37] Zhao, Y. Establishment of Agrobacterium-mediated transformation system in Chinese Cabbage. MSc thesis, Shenyang Agricultural Univ. (2017).

[38] Bortesi, L. et al. Patterns of CRISPR/Cas9 activity in plants, animals and microbes. Plant Biotechnol. J. 14, 2203-2216 (2016).

[39] Jeong, S. Y. et al. Generation of early-flowering Chinese cabbage (Brassica rapa spp. pekinensis) through CRISPR/Cas9-mediated genome editing. Plant Biotechnol. Rep. 13, 491-499 (2019).

[40] Zhou, A., Ma, H., Feng, S., Gong, S. & Wang, J. A novel sugar transporter from Dianthus spiculifolius, DsSWEET12, affects sugar metabolism and confers osmotic and oxidative stress tolerance in Arabidopsis. Int. J. Mol. Sci. 19, 497 (2018).

[41] Cabello, S. et al. Altered sucrose synthase and invertase expression affects the local and systemic sugar metabolism of nematode-infected Arabidopsis thaliana plants. J. Exp. Bot. 65, 201-212 (2014).

[42] Dotto, M., Gómez, M. S., Soto, M. S. & Casati, P. UV-B radiation delays flowering time through changes in the PRC2 complex activity and miR156 levels in Arabidopsis thaliana. Plant, Cell Environ. 41, 1394-1406 (2018).

[43] Griffiths, J. et al. Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis. Plant Cell 18, 3399-3414 (2006).

[44] Yu, X. et al. Evaluation of genotypic variation during leaf development in four Cucumis genotypes and their response to high light conditions. Environ. Exp. Bot. 124, 100-109 (2016).

[45] Zhao, D. et al. The role of sugar transporter genes during early infection by root-knot nematodes. Int. J. Mol. Sci. 19, 302 (2018).

[46] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCt method. Methods 25, 402-408 (2001).

[47] Hall, T. A. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95-98 (1999).

[48] Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30, 2725-2729 (2013).

[49] Chen, C. et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 13, 1194-1202 (2020).