title: RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture.
abstract: Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Gokhan
authors.initials: G
authors.lastname: Kir
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Huaxun
authors.identifier: http://orcid.org/0000-0001-9878-1447
authors.initials: H
authors.lastname: Ye
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Hilde
authors.identifier: http://orcid.org/0000-0001-7494-1290
authors.initials: H
authors.lastname: Nelissen
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Anjanasree K
authors.initials: AK
authors.lastname: Neelakandan
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Andree S
authors.initials: AS
authors.lastname: Kusnandar
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Anding
authors.identifier: http://orcid.org/0000-0002-2603-8854
authors.initials: A
authors.lastname: Luo
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Dirk
authors.initials: D
authors.lastname: Inzé
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Anne W
authors.identifier: http://orcid.org/0000-0001-7282-4189
authors.initials: AW
authors.lastname: Sylvester
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.).
authors.forename: Yanhai
authors.initials: Y
authors.lastname: Yin
authors.affiliation: Genetics, Development, and Cell Biology Department (G.K., H.Y., A.K.N., A.S.K., Y.Y., P.W.B.), Interdepartmental Genetics and Genomics Program (G.K., H.Y., A.S.K., Y.Y., P.W.B.), and Agronomy Department (P.W.B.), Iowa State University, Ames, Iowa 50011;Department of Plant Systems Biology, Vlaams Instituut voor Biotechnologie, B-9052 Ghent, Belgium (H.N., D.I.);Department of Plant Biotechnology and Bioinformatics, Ghent University, B-9052 Ghent, Belgium (H.N., D.I.); andDepartment of Molecular Biology, University of Wyoming, Laramie, Wyoming 82071-2000 (A.L., A.W.S.) becraft@iastate.edu.
authors.forename: Philip W
authors.identifier: http://orcid.org/0000-0002-3299-2126
authors.initials: PW
authors.lastname: Becraft
mesh_terms: D000595:Amino Acid Sequence; D060406:Brassinosteroids / Q000378:metabolism* / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D055785:Gene Knockdown Techniques; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D018515:Plant Leaves / Q000166:cytology / Q000187:drug effects / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D030821:Plants, Genetically Modified; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects; D013260:Steroids, Heterocyclic / Q000378:metabolism* / Q000494:pharmacology; D003313:Zea mays / Q000033:anatomy & histology* / Q000187:drug effects / Q000235:genetics*
chemical_list: D060406:Brassinosteroids; D010940:Plant Proteins; D013260:Steroids, Heterocyclic; C023623:brassinolide