title: Cis-Regulatory Elements Determine Germline Specificity and Expression Level of an Isopentenyltransferase Gene in Sperm Cells of Arabidopsis.
abstract: Flowering plant sperm cells transcribe a divergent and complex complement of genes. To examine promoter function, we chose an isopentenyltransferase gene known as PzIPT1. This gene is highly selectively transcribed in one sperm cell morphotype of Plumbago zeylanica, which preferentially fuses with the central cell during fertilization and is thus a founding cell of the primary endosperm. In transgenic Arabidopsis (Arabidopsis thaliana), PzIPT1 promoter displays activity in both sperm cells and upon progressive promoter truncation from the 5'-end results in a progressive decrease in reporter production, consistent with occurrence of multiple enhancer sites. Cytokinin-dependent protein binding motifs are identified in the promoter sequence, which respond with stimulation by cytokinin. Expression of PzIPT1 promoter in sperm cells confers specificity independently of previously reported Germline Restrictive Silencer Factor binding sequence. Instead, a cis-acting regulatory region consisting of two duplicated 6-bp Male Gamete Selective Activation (MGSA) motifs occurs near the site of transcription initiation. Disruption of this sequence-specific site inactivates expression of a GFP reporter gene in sperm cells. Multiple copies of the MGSA motif fused with the minimal CaMV35S promoter elements confer reporter gene expression in sperm cells. Similar duplicated MGSA motifs are also identified from promoter sequences of sperm cell-expressed genes in Arabidopsis, suggesting selective activation is possibly a common mechanism for regulation of gene expression in sperm cells of flowering plants.
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Jinghua
authors.identifier: http://orcid.org/0000-0002-0542-0704
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Tong
authors.initials: T
authors.lastname: Yuan
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Duan
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wei
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Tao
authors.identifier: http://orcid.org/0000-0002-6340-266X
authors.initials: T
authors.lastname: Shi
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.).
authors.forename: Jia
authors.initials: J
authors.lastname: Li
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.) gouxp@lzu.edu.cn srussell@ou.edu.
authors.forename: Scott D
authors.identifier: http://orcid.org/0000-0001-8302-4160
authors.initials: SD
authors.lastname: Russell
authors.affiliation: Ministry of Education Key Laboratory of Cell Activities and Stress Adaptations, School of Life Sciences, Lanzhou University, Lanzhou 730000, China (J.Z., X.D., T.S., J.L., S.D.R., X.G.); Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma 73019 (T.Y., X.W., S.D.R.) gouxp@lzu.edu.cn srussell@ou.edu.
authors.forename: Xiaoping
authors.identifier: http://orcid.org/0000-0002-8391-0258
authors.initials: X
authors.lastname: Gou
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D003583:Cytokinins / Q000494:pharmacology; D018506:Gene Expression Regulation, Plant / Q000187:drug effects / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics; D010937:Plant Growth Regulators / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics*; D030821:Plants, Genetically Modified; D029606:Plumbaginaceae / Q000235:genetics; D011058:Pollen / Q000166:cytology / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D024363:Transcription Initiation Site
chemical_list: D003583:Cytokinins; D010937:Plant Growth Regulators; D010940:Plant Proteins; D049452:Green Fluorescent Proteins; D019883:Alkyl and Aryl Transferases; C024274:adenylate isopentenyltransferase