bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424898; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 1 An amiRNA screen uncovers redundant CBF & ERF34/35 transcription 2 factors that differentially regulate arsenite and cadmium responses 3 4 Qingqing Xie1,2,3,6, Qi Yu1,4,6, Timothy O. Jobe1,5,6, Allis Pham1, Chennan Ge1, Qianqian 5 Guo3, Jianxiu Liu3, Honghong Liu3, Huijie Zhang3, Yunde Zhao1, Shaowu Xue3, Felix 6 Hauser1, Julian I. Schroeder1* 7 8 1 Division of Biological Sciences, Cell and Developmental Biology Section, University of California, 9 San Diego, La Jolla, California 92093-0116, USA 10 2 Plant Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies, La Jolla, CA 11 92037, USA 12 3 College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, Hubei, 13 P.R. China 14 4 School of Life Sciences, and Hubei Key Laboratory of Genetic Regulation and Integrative Biology, 15 Central China Normal University, Wuhan 430079, Hubei, P. R. China 16 5Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of 17 Cologne, 50674 Cologne, Germany 18 19 6These authors contributed equally to this work. 20 *Corresponding authors: Julian I. Schroeder E-mail: [email protected] 21 22 Funding information: National Institute of Environmental Health Sciences of the National Institutes of 23 Health under Award Number P42ES010337; National Natural Science Foundation of China 24 (31770283). 25 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424898; this version posted January 2, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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-NC-ND 4.0 International license. 26 Running title: amiRNA screen uncovers regulators of arsenite and cadmium responses 27 Abstract 28 Arsenic stress causes rapid transcriptional responses in plants. However, transcriptional 29 regulators of arsenic-induced gene expression in plants remain less well known. To date, 30 forward genetic screens have proven limited for dissecting arsenic response mechanisms. 31 We hypothesized that this may be due to the extensive genetic redundancy present in 32 plant genomes. To overcome this limitation, we pursued a forward genetics screen for 33 arsenite tolerance using a randomized library of plants expressing >2,000 artificial 34 microRNAs (amiRNAs). This library was designed to knock-down diverse combinations 35 of homologous gene family members within sub-clades of transcription factor and 36 transporter gene families. We identified six transformant lines showing an altered 37 response to arsenite in root growth assays. Further characterization of an amiRNA line 38 targeting closely homologous CBF and ERF transcription factors show that the CBF1,2 39 and 3 transcription factors negatively regulate arsenite sensitivity. Furthermore, the 40 ERF34 and ERF35 transcription factors are required for cadmium resistance. Generation 41 of CRISPR lines, higher-order T-DNA mutants, and gene expression analyses, further 42 support our findings. These ERF transcription factors differentially regulate arsenite 43 sensitivity and cadmium tolerance. 44 45 Key Words: amiRNA, redundancy, ERF transcription factors, heavy metal, arsenic 46 47 48 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424898; this version posted January 2, 2021. 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-NC-ND 4.0 International license. 49 50 Introduction 51 Arsenic (As) is a carcinogenic metalloid found widely in the environment (Clemens & 52 Ma, 2016; Cooper et al., 2020). Arsenic toxicity decreases crop yields, and accumulation 53 of arsenic in edible crop tissues can cause contamination of the food chain and lead to 54 severe human health problems (Clemens, 2019; Rai, Lee, Zhang, Tsang, & Kim, 2019; 55 Abedi & Mojiri, 2020; Palma-Lara et al., 2020) . For example, rice grain can accumulate 56 up to 2.24 mg/kg As and has become a dominant dietary exposure route to arsenic (As) 57 (Gilbert-Diamond et al., 2011; Pan, Wu, Xue, & Hartley, 2014). Thus, understanding the 58 molecular mechanisms of As uptake and accumulation in plants is an urgent priority for 59 food security worldwide. 60 Recent studies have identified genes involved in the uptake, transport, and 61 sequestration of arsenic in Arabidopsis, rice, and other plant species (Mendoza-Cozatl, 62 Jobe, Hauser, & Schroeder, 2011; Abbas et al., 2018; Kumari, Rastogi, Shukla, 63 Srivastava, & Yadav, 2018; Garbinski, Rosen, & Chen, 2019; Shri et al., 2019; Abedi & 64 Mojiri, 2020). These include nutrient transporters that transport arsenite (As(III)) across 65 plant membranes and can function in arsenic accumulation, including Nodulin 26-like 66 Intrinsic Proteins (NIPs), Plasma membrane Intrinsic Proteins (PIPs), Natural Resistance- 67 Associated Macrophage Protein OsNRAMP1, and Tonoplast Intrinsic Proteins (TIP) 68 (Bienert et al., 2008; Isayenkov & Maathuis, 2008; Kamiya & Fujiwara, 2009; Kamiya et 69 al., 2009; Mosa et al., 2012; Tiwari et al., 2014; Xu et al., 2015; Yang et al., 2015; Duan 70 et al., 2016b; Lindsay & Maathuis, 2016). Furthermore, phosphate transporters (PHTs) 71 play a role in arsenate (As(V)) uptake (Shin, Shin, Dewbre, & Harrison, 2004; Catarecha 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424898; this version posted January 2, 2021. 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-NC-ND 4.0 International license. 72 et al., 2007; Remy et al., 2012; LeBlanc, McKinney, Meagher, & Smith, 2013; Fontenot 73 et al., 2015), and arsenate reductase enzymes are responsible for the reduction of As(V) 74 to arsenite As(III) (Chao et al., 2014; Shi et al., 2016; Xu et al., 2017). The enzymes 휸- 75 glutamylcysteine synthetase (g-ECS), glutathione synthetase (GS), and phytochelatin 76 synthase (PCS) are critical enzymes for As detoxification (Schmoger, Oven, & Grill, 2000; 77 Li et al., 2004; Picault et al., 2006; Gasic & Korban, 2007; Guo, Dai, Xu, & Ma, 2008; 78 Herschbach et al., 2010; Wojas, Clemens, Sklodowska, & Maria Antosiewicz, 2010; 79 Hayashi et al., 2017). Additionally, the ATP Binding Cassette (ABC) transporters ABCC1 80 and ABCC2 transport As-GSH and As-PC complexes into vacuoles as a mechanism for 81 arsenic accumulation (Song et al., 2010; Song et al., 2014; Hayashi et al., 2017). The 82 inositol transporters (AtINT2 and AtINT4) in A. thaliana and OsNIP6;1 and OsNIP7;1 in 83 rice play a role in arsenic transport into seeds (Duan et al., 2016a; Lindsay & Maathuis, 84 2017). 85 Moreover, several regulatory proteins involved in plant responses to arsenic have 86 been identified. For example, glutaredoxins (Grx) regulate As(V) reduction (Verma et al., 87 2017), and the transcription factors WRKY6, WRKY45, and OsARM1 (Arsenite- 88 Responsive Myb1) have been implicated in the regulation of arsenic transporters 89 (Castrillo et al., 2013; Wang et al., 2014; Wang et al., 2017). The calcium-dependent 90 protein kinase (CPK31) was reported to regulate AtNIP1;1 (Ji et al., 2017), and miR528 91 was found to be necessary for As(III) responses (Liu et al., 2015). Despite the above list 92 of genes identified as being involved in arsenic responses in plants, many genes and 93 mechanisms have yet to be determined. 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.30.424898; this version posted January 2, 2021. 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-NC-ND 4.0 International license. 94 Arsenic is known to cause large transcriptional responses in plants (Chakrabarty et 95 al., 2009; Jobe et al., 2012; Castrillo et al., 2013; Srivastava, Srivastava, Sablok, 96 Deshpande, & Suprasanna, 2015; Zvobgo et al., 2018; Huang et al., 2019). Research 97 has suggested that transcriptional activators and transcriptional repressors play essential 98 roles in arsenic-induced gene expression (Jobe et al., 2012; Castrillo et al., 2013; Wang 99 et al., 2014; Wang et al., 2017). While WRKY transcription factors have been shown to 100 regulate phosphate transporters that contribute to arsenic tolerance (Castrillo et al., 2013; 101 Wang et al., 2014; Wang et al., 2017), additional transcription factors that mediate 102 arsenic-induced gene expression and repressors remain unknown. 103 Forward genetic screens are powerful tools for identifying new genes. However, 104 forward genetic screens have limitations due to the extensive over-lapping functions of 105 closely related genes (“redundancy”) mediated by large gene families found in plant 106 genomes (Arabidopsis Genome, 2000) . Phenotypes linked to a single gene loss-of- 107 function mutation have been found for less than 15% of the genes encoded in the 108 Arabidopsis genome (Lloyd & Meinke, 2012; Cusack et al., 2020). A genome-wide 109 analysis showed that approximately 75% of Arabidopsis genes are members of gene 110 families (Hauser et al., 2013). Thus, partially overlapping gene functions, referred to as 111 functional redundancies, have hampered the identification of new gene functions in 112 forward genetic screens. In previous research, we computationally designed artificial 113 microRNAs (amiRNAs) (Schwab, Ossowski, Riester, Warthmann, & Weigel, 2006) , and 114 synthesized 22,000 amiRNAs designed to combinatorially co-silence closely homologous 115 gene clade members (Hauser et al., 2013).