title: Asparagus IRX9, IRX10, and IRX14A Are Components of an Active Xylan Backbone Synthase Complex that Forms in the Golgi Apparatus.
abstract: Heteroxylans are abundant components of plant cell walls and provide important raw materials for the food, pharmaceutical, and biofuel industries. A number of studies in Arabidopsis (Arabidopsis thaliana) have suggested that the IRREGULAR XYLEM9 (IRX9), IRX10, and IRX14 proteins, as well as their homologs, are involved in xylan synthesis via a Golgi-localized complex termed the xylan synthase complex (XSC). However, both the biochemical and cell biological research lags the genetic and molecular evidence. In this study, we characterized garden asparagus (Asparagus officinalis) stem xylan biosynthesis genes (AoIRX9, AoIRX9L, AoIRX10, AoIRX14A, and AoIRX14B) by heterologous expression in Nicotiana benthamiana We reconstituted and partially purified an active XSC and showed that three proteins, AoIRX9, AoIRX10, and AoIRX14A, are necessary for xylan xylosyltranferase activity in planta. To better understand the XSC structure and its composition, we carried out coimmunoprecipitation and bimolecular fluorescence complementation analysis to show the molecular interactions between these three IRX proteins. Using a site-directed mutagenesis approach, we showed that the DxD motifs of AoIRX10 and AoIRX14A are crucial for the catalytic activity. These data provide, to our knowledge, the first lines of biochemical and cell biological evidence that AoIRX9, AoIRX10, and AoIRX14A are core components of a Golgi-localized XSC, each with distinct roles for effective heteroxylan biosynthesis.
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Wei
authors.initials: W
authors.lastname: Zeng
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Edwin R
authors.identifier: 0000-0002-3666-7240
authors.initials: ER
authors.lastname: Lampugnani
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Kelsey L
authors.initials: KL
authors.lastname: Picard
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Lili
authors.initials: L
authors.lastname: Song
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Ai-Min
authors.initials: AM
authors.lastname: Wu
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Isabela M
authors.initials: IM
authors.lastname: Farion
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Jia
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Kris
authors.initials: K
authors.lastname: Ford
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.).
authors.forename: Monika S
authors.identifier: 0000-0002-8921-2725
authors.initials: MS
authors.lastname: Doblin
authors.affiliation: Australian Research Council Centre of Excellence in Plant Cell Walls, School of BioSciences, University of Melbourne, Parkville, Victoria 3010, Australia (W.Z., E.R.L., K.L.P., I.M.F., J.Z., K.F., M.S.D., A.B.);Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Lin'an, Hangzhou 311300, China (L.S.); andState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, South China Agricultural University, Guangzhou 510642, China (A.-M.W.) abacic@unimelb.edu.au.
authors.forename: Antony
authors.initials: A
authors.lastname: Bacic
mesh_terms: D020816:Amino Acid Motifs; D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism; D027761:Asparagus Plant / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D053898:Biosynthetic Pathways / Q000235:genetics; D002473:Cell Wall / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006056:Golgi Apparatus / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D018515:Plant Leaves / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D040901:Proteomics; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics; D014990:Xylans / Q000096:biosynthesis*
chemical_list: D029681:Arabidopsis Proteins; D014990:Xylans; C571990:IRX14 protein, Arabidopsis; C521260:IRX9 protein, Arabidopsis; D010430:Pentosyltransferases; C038331:1,4-beta-D-xylan synthase