BoALG10, an α-1,2 glycosyltransferase, plays an essential role in maintaining leaf margin shape in ornamental kale

Abstract

The morphological diversity of leaf margin shapes is an identifying characteristic of many plant species. In our previous work, BoALG10 (α-1,2 glycosyltransferase) was predicted to be a key regulator of leaf margin shape in ornamental kale (Brassica oleracea var. acephala). An alanine and a leucine residue in the conserved domain of the smooth-margined S0835 were replaced by an aspartate and a phenylalanine, respectively, in the corresponding positions of the feathered-margined F0819. However, the expression pattern and function of this gene remain unclear. Here, we examined the expression patterns of BoALG10 using quantitative real-time PCR, and found that statistically significant differences in expression existed between F0819 and S0835 in nine developmental stages. The BoALG10 protein localized to the endoplasmic reticulum. The function of BoALG10 was then examined using complementary mutant assays. The overexpression strains phenocopied the smooth leaf margin after introduction of BoALG10^S0835 into the feathered-margined inbred line F0819. Simultaneously, irregular dissections appeared in the leaf margins of knockout mutants KO-1 and KO-2, which were generated by CRISPR/Cas9 technology from the smooth-margined inbred line S0835. Microscopic observation showed that the leaf margin cells of the smooth-margined plants S0835 and OE-3 were arranged regularly, while the cells of the feathered-margined plants F0819 and KO-1 were of inconsistent size and distributed in an irregular manner, particularly around the indentations of the leaf. This elucidation of BoALG10 function provides a novel insight into the morphological regulation of leaf margin shape.

References

[1] Tsukaya H . Mechanism of leaf-shape determination. Annu Rev Plant Biol. 2006; 57: 477-96.

[2] Nicotra AB, Leigh A, Boyce CK et al. The evolution and functional significance of leaf shape in the angiosperms. Funct Plant Biol. 2011; 38: 535-52.

[3] Dkhar J, Pareek A . What determines a leaf’s shape? EvoDevo . 2014; 5: 47.

[4] Vogel S. Leaves in the lowest and highest winds: temperature, force and shape. New Phytol. 2009; 183: 13-26.

[5] Rowland SD, Zumstein K, Nakayama H et al. Leaf shape is a predictor of fruit quality and cultivar performance in tomato. New Phytol. 2020; 226: 851-65.

[6] Alvarez JP, Furumizu C, Efroni I et al. Active suppression of a leaf meristem orchestrates determinate leaf growth. eLife. 2016; 5: e15023.

[7] Ori N, Cohen AR, Etzioni A et al. Regulation of LANCEOLATE by miR319 is required for compound-leaf development in tomato . Nat Genet. 2007; 39: 787-91.

[8] Shleizerburko S, Burko Y, Benherzel O et al. Dynamic growth program regulated by LANCEOLATE enables flexible leaf patterning. Development. 2011; 138: 695-704.

[9] Bilsborough GD, Runions A, Barkoulas M et al. Model for the regulation of Arabidopsis thaliana leaf margin development. Proc Natl Acad Sci USA. 2011; 108: 3424-9.

[10] Li Z, Li B, Shen W et al. TCP transcription factors interact with AS2 in the repression of class-I KNOX genes in Arabidopsis thaliana . Plant J. 2012; 71: 99-107.

[11] Bar M, Ori N . Compound leaf development in model plant species. Curr Opin Plant Biol. 2015; 23: 61-9.

[12] Sarvepalli K, Gupta MD, Challa KR et al. Molecular cartography of leaf development-role of transcription factors. Curr Opin Plant Biol. 2019; 47: 22-31.

[13] Wang W, Xu B, Wang H et al. YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development . Plant Physiol. 2011; 157: 1805-19.

[14] Hibara K, Karim MR, Takada S et al. Arabidopsis CUC-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation . Plant Cell. 2006; 18: 2946-57.

[15] Nikovics K, Blein T, Peaucelle A et al. The balance between the MIR164A and CUC2 genes controls leaf margin serration in Arabidopsis . Plant Cell. 2006; 18: 2929-45.

[16] Hasson A, Plessis A, Blein T et al. Evolution and diverse roles of the cup-shaped cotyledon genes in Arabidopsis leaf development. Plant Cell. 2011; 23: 54-68.

[17] Serra L, Perrot-Rechenmann C . Spatiotemporal control of cell growth by CUC3 shapes leaf margins. Development. 2020; 147: 1-29.

[18] Bresso EG, Chorostecki U, Rodriguez RE et al. Spatial control of gene expression by miR319-regulated TCP transcription factors in leaf development. Plant Physiol. 2017; 176: 1694-708.

[19] Saddic LA, Huvermann B, Bezhani S et al. The LEAFY target LMI1 is a meristem identity regulator and acts together with LEAFY to regulate expression of CAULIFLOWER . Development. 2006; 133: 1673-82.

[20] Chen J, Yu J, Ge L et al. Control of dissected leaf morphology by a Cys(2)his(2) zinc finger transcription factor in the model legume Medicago truncatula . Proc Natl Acad Sci USA. 2010; 107: 10754-9.

[21] Vlad D, Kierzkowski D, Rast MI et al. Leaf shape evolution through duplication, regulatory diversification, and loss of a homeobox gene. Science. 2014; 343: 780-3.

[22] Kierzkowski D, Runions A, Vuolo F et al. A growth-based framework for leaf shape development and diversity. Cell. 2019; 177: 1405-1418.e17.

[23] Hajheidari M, Wang Y, Bhatia N et al. Autoregulation of RCO by low-affinity binding modulates cytokinin action and shapes leaf diversity. Curr Biol. 2019; 29: 4183-4192.e6.

[24] Zhang M, Henquet M, Chen Z et al. LEW3, encoding a putative α-1,2-mannosyltransferase (ALG11) in N-linked glycoprotein, plays vital roles in cell-wall biosynthesis and the abiotic stress response in Arabidopsis thaliana . Plant J. 2009; 60: 983-99.

[25] Farid A, Pabst M, Schoberer J et al. Arabidopsis thaliana alpha1,2-glucosyltransferase (ALG10) is required for efficient N-glycosylation and leaf growth . Plant J. 2011; 68: 314-25.

[26] Manzano C, Pallero-Baena M, Silva-Navas J et al. A light-sensitive mutation in Arabidopsis LEW3 reveals the important role of N-glycosylation in root growth and development. J Exp Bot. 2017; 68: 5103-16.

[27] Jiao QS, Niu GT, Wang FF et al. N-glycosylation regulates photosynthetic efficiency of Arabidopsis thaliana . Photosynthetica. 2020; 58: 72-9.

[28] Burda P, Aebi M . The ALG10 locus of Saccharomyces cerevisiae encodes the α-1,2 glucosyltransferase of the endoplasmic reticulum: the terminal glucose of the lipid-linked oligosaccharide is required for efficient N-linked glycosylation . Glycobiology. 1998; 8: 455-62.

[29] Karaoglu D, Kelleher DJ, Gilmore R . The highly conserved Stt3 protein is a subunit of the yeast oligosaccharyltransferase and forms a subcomplex with Ost3p and Ost4p. J Biol Chem. 1997; 272: 32513-20.

[30] Knauer R, Lehle L . The oligosaccharyltransferase complex from yeast. Biochim Biophys Acta Gen Subj. 1999; 1426: 259-73.

[31] Feng X, Li X, Yang X et al. Fine mapping and identification of the leaf shape gene BoFL in ornamental kale. Theor Appl Genet. 2020; 133: 1303-12.

[32] Murray MG, Thompson WF . Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 1980; 8: 4321-6.

[33] Nelson BK, Cai X, Nebenführ A . A multicolored set of in vivo organelle markers for co-localization studies in Arabidopsis and other plants. Plant J. 2007; 51: 1126-36.

[34] Feng X, Zhang Y, Wang H et al. Dihydroflayonol 4-reductase confers leaf color in ornamental kale. Theor Appl Genet. 2021; 134: 159-69.

[35] Saitou NNM, Nei MC . The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987; 4: 406-25.

[36] Kumar S, Stecher G, Tamura K . MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016; 33: 1870-4.

[37] Xing H, Dong L, Wang ZP et al. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014; 14: 327.

[38] Kessler S, Neelima S . Shaping up: the genetic control of leaf shape. Curr Opin Plant Biol. 2004; 7: 65-72.

[39] Israeli A, Capua Y, Shwartz I et al. Multiple auxin-response regulators enable stability and variability in leaf development. Curr Biol. 2019; 29: 1746-1759.e5.

[40] Andres RJ, Coneva V, Frank MH et al. Modifications to a LATE MERISTEM IDENTITY1 gene are responsible for the major leaf shapes of upland cotton (Gossypium hirsutum L.) . Proc Natl Acad Sci USA. 2017; 114: E57-66.

[41] Ni X, Liu H, Huang J et al. LMI1-like genes involved in leaf margin development of Brassica napus . Genetica. 2017; 145: 269-74.

[42] Hu L, Zhang H, Yang Q et al. Promoter variations in a homeobox gene, BnA10.LMI1, determine lobed leaves in rapeseed (Brassica napus L.) . Theor Appl Genet. 2018; 131: 2699-708.

[43] Hu L, Zhang H, Sun Y et al. BnA10.RCO, a homeobox gene, positively regulates leaf lobe formation in Brassica napus L. Theor Appl Genet. 2020; 133: 3333-43.

[44] Chung CY, Majewska NI, Wang Q et al. SnapShot: N-glycosylation processing pathways across kingdoms. Cell. 2017; 171: 258-258.e1.

[45] Aebi M . N-linked protein glycosylation in the ER. Biochim Biophys Acta. 2013; 1833: 2430-7.

[46] Farid A, Malinovsky FG, Veit C et al. Specialized roles of the conserved subunit OST3/6 of the oligosaccharyltransferase complex in innate immunity and tolerance to abiotic stresses. Plant Physiol. 2013; 162: 24-38.