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Downloaded and a Phylogenetic Consensus Tree Was bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Submission to Genetics 2 3 Title: 4 Mutation of the nuclear pore complex component, aladin1, disrupts 5 asymmetric cell division in Zea mays (maize) 6 7 Authors: Norman B. Best*†‡, Charles Addo-Quaye‡§, Bong-Suk Kim**, Clifford F. 8 Weil††‡‡, Burkhard Schulz†, Guri Johal**‡‡, Brian P. Dilkes‡‡‡ 9 Affiliations: 10 *USDA, Agriculture Research Service, Plant Genetics Research Unit; Columbia, MO, 65211 11 †Department of Horticulture & Landscape Architecture; Purdue University; West Lafayette, IN, 12 47907 13 ‡Department of Biochemistry, Purdue University; West Lafayette, IN, 47907 14 §Natural Sciences and Mathematics, Lewis-Clark State College; Lewiston, ID, 83501 15 **Department of Botany and Plant Pathology, Purdue University; West Lafayette, IN, 47907 16 ††Department of Agronomy, Purdue University; West Lafayette, IN, 47907 17 ‡‡Center for Plant Biology, Purdue University; West Lafayette, IN, 47907 18 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19 Short Running Title: 20 aladin1 controls asymmetric cell division 21 22 Key words: 23 Nuclear Pore Complex 24 Asymmetric cell division 25 Stomata 26 Subsidiary cells 27 Maize 28 29 Corresponding Author: 30 Brian P. Dilkes 31 Address: 32 Whistler Building Room B036B 33 Purdue University 34 West Lafayette, IN 47907 35 36 Phone Number: 37 765-494-2584 38 39 Email Address: 40 [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 41 Abstract: 42 The nuclear pore complex (NPC) regulates the movement of macromolecules between the 43 nucleus and cytoplasm. Dysfunction of many components of the NPC results in human genetic 44 diseases, including triple A syndrome (AAAS) as a result of mutations in ALADIN. Here we 45 report a nonsense mutation in the maize ortholog, aladin1 (ali1-1), at the orthologous amino acid 46 residue of an AAAS allele from humans, alters plant stature, tassel architecture, and asymmetric 47 divisions of subsidiary mother cells (SMCs). Crosses with the stronger nonsense allele ali1-2 48 identified complex allele interactions for plant height and aberrant SMC division. RNA-seq 49 analysis of the ali1-1 mutant identified compensatory transcript accumulation for other NPC 50 components as well as gene expression consequences consistent with conservation of ALADIN1 51 functions between humans and maize. These findings demonstrate that ALADIN1 is necessary 52 for normal plant development, shoot architecture, and asymmetric cell division in maize. bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 53 Introduction 54 The nuclear pore complex (NPC) is a multi-protein complex that is involved in regulating 55 the movement of macromolecules into and out of the nucleus. The NPC also plays important 56 roles in nuclear assembly during cell division (Rasala, Orjalo, Shen, Briggs, & Forbes, 2006). 57 NPC proteins assemble in groups of eight, as spokes, around the opening of the pore to control 58 transport between the nucleus and cytoplasm (Lin et al., 2016; Reichelt et al., 1990). 59 Components of the NPC are conserved across many eukaryotic species, including mammals (J. 60 M. Cronshaw, Krutchinsky, Zhang, Chait, & Matunis, 2002), plants (Tamura, Fukao, Iwamoto, 61 Haraguchi, & Hara-Nishimura, 2010; Tamura & Hara-Nishimura, 2013), fungi (Liu, De Souza, 62 Osmani, & Osmani, 2009; Stuwe et al., 2015), and yeast (Rout et al., 2000). Dysfunction and/or 63 mutation of the genes encoding NPC components has drastic effects on growth, development, 64 and survival of eukaryotic species. In humans, genetic mutations of NPC members have been 65 linked to a number of diseases and illnesses, including different types of cancer, neurological 66 diseases, and autoimmune diseases (Nofrini, Di Giacomo, & Mecucci, 2016; Roth & Wiermer, 67 2012; Sakuma & D'Angelo, 2017). 68 The structure and conserved components of the NPC of plants have been identified 69 (Fiserova, Kiseleva, & Goldberg, 2009; Neumann, Jeffares, & Poole, 2006; Tamura et al., 2010; 70 Tamura & Hara-Nishimura, 2013). Genetic studies have functionally characterized a few NPC 71 members in plant species. The Arabidopsis NPC member, AtMOS7/Nup88, was shown to 72 regulate the nuclear concentrations of a subset of defense proteins, but other proteins tested were 73 not affected in Atmos7 mutants (Cheng et al., 2009). Additional forward and reverse genetic 74 approaches in plant species; primarily Arabidopsis, tobacco, and Lotus japonica, have further 75 characterized the function and role of NPC members in plant defense, plant symbioses, and bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 76 responses to abiotic stress (Binder & Parniske, 2018; Cheng et al., 2009; Dong et al., 2006; Roth 77 & Wiermer, 2012; Wiermer et al., 2012; Zhang & Li, 2005). To date, no member of the NPC has 78 been functionally characterized in Zea mays (maize). It is unclear to what extent the specific 79 function of NPC members can be inferred from phylogeny. In comparisons between mice and 80 human, mutation of WD-40 repeat containing NPC member ALADIN did not produce the same 81 phenotypic outcomes (Huebner et al., 2006). It is unclear whether such species-specific 82 phenotypic impacts will characterize NPC functions in plants. 83 Here we describe the first characterization of an NPC mutant in maize and the first 84 description of an ALADIN mutant in plants. The aladin1-1 (ali1-1) mutant was identified in an 85 ethyl methanesulfonate (EMS) screen as a short plant with unusual tassel architecture and 86 defective subsidiary cell asymmetric division in developing stomata. The ali1-1 mutant results in 87 a weak allele and crosses with a null allele identified allelic dosage effects of the ali1 gene, 88 demonstrating that ali1-1 was haploinsufficient for maintaining normal development especially 89 after phase change. RNA-sequencing identified a compensatory increase in transcripts encoding 90 other NPC members in ali1-1, even during juvenile growth before the defects in SMC 91 asymmetric division were visible. These results implicate this gene in cell division control and 92 NPC homeostasis that may also be true for humans and contribute to AAAS disease. 93 Materials and Methods 94 Plant material and growth conditions 95 In the summer of 2010, the ali1-1 mutant was identified in a B73 background ethyl 96 methanesulfonate (EMS) population as M3 generation plants segregating in the line 97 04IAB73PS007D8 (Weil, 2009). The ali1-2 allele was created by an EMS-targeted mutagenesis 98 approach. B73 pollen was incubated with 6.48 mM EMS (Sigma-Aldrich, St. Louis, MO) in bioRxiv preprint doi: https://doi.org/10.1101/2020.12.16.423105; this version posted December 16, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 99 paraffin oil (Sigma-Aldrich, St. Louis, MO) for 35 mins then squirted onto ~205 ali1-1/ali1-1 100 ears using a 40 mL squeezable bottle (Hobby Lobby, Oklahoma City, OK). ~10,000 M1 seeds 101 were screened in the field in the summer of 2015 in West Lafayette, IN for phenotypes similar to 102 ali1-1. A total of 13 plants resembling ali1-1 were identified. One of the twelve was 103 heterozygous for the causative single nucleotide polymorphism (SNP) in ali1-1. This plant was 104 backcrossed to ali1-1/ali1-1 and B73 to propagate the ali1-2 allele. 105 To map the ali1 gene, an F2 population was created by crossing Mo17 with ali1-1/ali1-1 106 pollen and self-pollinated the F1 generation. The F2 progeny was planted in the field to collect 107 tissue from selected wildtype and ali1-1 phenotypical plants. Wild-type and ali1-1/ali1-1 shoot 108 apical meristem (SAM) and surrounding tissue of 15 days after planting (DAP) plants were 109 grown in the greenhouse in trays with a 2:1 mixture of peat germinating mix (Conrad Fafard Inc., 110 Agawam, MA) to Turface MVP (Profile Products LLC, Buffalo Grove, IL). Total plant height, 111 stomatal index, stomatal density, and percent aberrant stomata measurements of the 8th leaf were 112 collected from plants grown in the field in the summer of 2016 in West Lafayette, IN. Plant 113 height, internode length, tassel length, and rachis length of B73 and ali1-1 were measured on 114 field grown plants in the summer of 2013.
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