J. Gen. Appl. Microbiol. Doi 10.2323/Jgam.2020.02.003 ©2020 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation

Advance Publication

1 Short Communication

2 Establishment of a firefly luciferase reporter assay system in the unicellular red alga 3 Cyanidioschyzon merolae

4 (Received January 25, 2020; Accepted February 11, 2020; J-STAGE Advance publication date: September 16, 2020)

5 Running Head: Luciferase reporter system in C. merolae

6 Baifeng Zhou1,2, Sota Takahashi1,3, Tokiaki Takemura1,2, Kan Tanaka1,*, Sousuke Imamura1,*

7 1Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute 8 of Technology, Nagatsuta, Midori-ku, Yokohama, Japan 9 2School of Life Science and Technology, Tokyo Institute of Technology, Nagatsuta, Midori- 10 ku, Yokohama, Japan 11 3Interdisciplinary Graduate School of Science and Engineering, Tokyo Institute of 12 Technology, Nagatsuta, Midori-ku, Yokohama, Japan 13

14 Key Words: Cyanidioschyzon merolae; luciferase; nitrogen; reporter assay; transcription 15 factor

16 *To whom correspondence should be addressed. E-mail: [email protected] (K. 17 Tanaka); [email protected] (S. Imamura)

18 The firefly luciferase (Luc) reporter assay is a powerful tool used to analyze promoter 19 activities in living cells. In this report, we established a firefly Luc reporter assay system in 20 the unicellular model red alga Cyanidioschyzon merolae. A nitrite reductase (NIR) promoter- 21 Luc fusion gene was integrated into the URA5.3 genomic region to construct the C. merolae 22 NIR-Luc strain. Luc activities in the NIR-Luc strain were increased, correlating with the 23 accumulation of endogenous NIR transcripts in response to nitrogen depletion. Luc activity 24 was also significantly increased by the overexpression of the MYB1 gene, which encodes a

25 transcription factor responsible for NIR promoter activation. Thus, our results demonstrate 26 the utility of the Luc reporter system in C. merolae.

27 Abbreviations: GFP, green fluorescent protein; GUS, β-glucuronidase; kbp, kilobase pair; 28 kDa, kilodalton; Luc, firefly luciferase; ORF, open reading frame; PCR, polymerase chain 29 reaction; PEG, polyethylene glycol; SD, standard deviation; TFs, transcription factors; −/+ N, 30 nitrogen depleted/replete condition

31 Reporter genes are marker genes used to study the regulatory elements of the genes of 32 interest, where the expression of reporter genes can be easily detected and measured. Several 33 reporter genes, such as firefly luciferase (Luc), β-glucuronidase (GUS), and green fluorescent 34 protein (GFP), are widely used in plants (Jefferson, 1987; Koncz et al., 1990; Millar et al., 35 1992; Naylor, 1999). The GUS system has the advantage of no background noise in most 36 plant species and is easily quantifiable using a substrate. Therefore, the GUS reporter system 37 has been used for the quantitative analysis of promoter activity (Quaedvlieg et al., 1998). 38 GUS activity can also be used for histochemical localization of GUS-tagged target proteins 39 (Guivarc’H et al., 1996). The GFP system is used for analyzing the subcellular localization of 40 target proteins since fluorescence from GFP can be directly detected in living cells (Haseloff 41 and Amos, 1995; Moriguchi et al., 2005). On the other hand, the Luc system is used to study 42 gene transcription regulation because of its high sensitivity, time resolution, and accurate 43 quantitative characteristics (Velten et al., 2008). 44 Cyanidioschyzon merolae is a unicellular red alga with a completely sequenced and 45 annotated genome (Matsuzaki et al., 2004; Nozaki et al., 2007). Because of the small number 46 of transcription factors (TFs; less than 100 TFs are estimated for the 16.5 Mbp of the nuclear 47 genome) as well as several genetic and molecular biology related tools, C. merolae has been 48 considered as an ideal photosynthetic model eukaryote for studying fundamental 49 transcriptional networks (Imamura et al., 2009, 2010; Matsuzaki et al., 2004). However, to 50 date, only the GFP reporter system has been established in C. merolae for monitoring the 51 expression of nitrogen (N) assimilation genes (Fujiwara et al., 2015). In the case of the 52 unicellular model green alga, Chlamydomonas reinhardtii, the luciferase system has been 53 established and used for monitoring expression of nuclear genes (Fuhrmann et al., 2004; 54 Ruecker et al., 2008; Shao and Bock, 2008). In this study, we constructed the Luc reporter 55 system to analyze transcriptional regulation in C. merolae.

56 To examine the utility of the Luc reporter system in C. merolae, we used MYB1, an 57 R2R3-type MYB TF, and the nitrite reductase (NIR) promoter region, since MYB1 58 positively regulates the expression of N assimilation genes, including NIR, under the N 59 depleted (−N) condition (Imamura et al., 2009). To examine MYB1-dependent NIR 60 transcription through Luc activity, we first constructed the NIR-Luc strain; in this strain, the 61 NIR promoter-driven Luc gene was used to replace the UMP synthase gene URA5.3 in the 62 genome of C. merolae (Fig. 1A). The NIR-Luc strain exhibits uracil auxotrophic phenotype 63 and, therefore, can be used as a host strain for expressing exogenous TFs, including MYB1. 64 The NIR promoter fragment (−1,200 to +61; +1 represents the translation start site) was 65 generated by a polymerase chain reaction (PCR) using the primers NIR_pKTL_F1 (5′- 66 CGCGAAGATCTCATATGGATTTACCGTCGTTCAACTCAAA-3′) and NIR_pKTL1_R1 67 (5′-ACCGGAATGCCAAGCTAGATTGGTGGGTGCCAAACCTCTGC-3′), C. merolae 68 genomic DNA (template), and KOD-Plus Neo DNA polymerase (Toyobo, Tokyo, Japan). 69 The PCR product was then cloned into EcoRV-digested pKTL1 vector (Imamura et al., 2017), 70 which contains the open reading frame (ORF) of Luc, URA5.3 upstream region (URA5.3 up), 71 and URA5.3 downstream region (URA5.3 down), using Gibson assembly (Gibson et al., 72 2009) to create the pKTL1-NIR construct. Finally, a fragment containing URA5.3 up, NIR 73 promoter, Luc, URA5.3 down, in this order from 5′ to 3′ (hereafter this fragment is referred to 74 as NIR-Luc), was amplified by PCR with the primers URA_F1 and URA_R1 (Imamura et al., 75 2017) using pKTL1-NIR DNA as the template. The purified 7.1 kbp fragment was used for 76 polyethylene glycol (PEG)-mediated transformation of C. merolae wild-type (WT) (Taki et 77 al., 2015). To confirm the replacement of the URA5.3 gene by the NIR-Luc fragment, 78 transformants were further screened by PCR using two primer sets. First, primers F1 and R1 79 (Imamura et al., 2017), which anneal outside of the integration region, were used to obtain a 80 7.2 kbp fragment from the positive stain and a 6.1 kbp fragment from the WT strain, as 81 predicted (Fig. 1B, left). Then, primers F2 (5′-ACGGAAAAAGAGATCGTGGATTAC-3′) 82 and R1 were used to amplify a 2.3 kbp fragment from the positive stain, where no 83 amplification was obtained from the WT strain, as predicted (Fig. 1B, right). Taken together, 84 these data indicated that the NIR-Luc fragment was integrated into the URA5.3 locus in the 85 positive strain; this strain is hereafter referred to as the NIR-Luc strain. 86 To examine NIR promoter activity, we performed a Luc reporter assay using proteins 87 extracted from the NIR-Luc stain and T1 strain (lacking the URA5.3 gene) before and after 88 exposure to -N conditions. The T1 strain, which carries a complete deletion of the URA5.3 89 gene was used as a control (Taki et al., 2015). Protein samples were prepared as previously 3

90 described (Imamura et al., 2008). The total protein (120 µg) in 100 µl of lysis buffer 91 (Imamura et al., 2008) was incubated with 100 µl of luciferin-containing ONE-Glo™ 92 Reagent (Promega, Tokyo, Japan) for 5 min in the dark. Luc activity was then measured by 93 Lumat LB 9507 (EG & G Berthold) and estimated as relative fluorescence units (RFU) 94 divided by the amount of total input protein (RFU/Protein). The results showed that Luc 95 activity was significantly increased at 2 h after exposure to -N and peaked at 4 h in the NIR- 96 Luc strain; however, the T1 strain showed no increase of the luminescence (Fig. 2A). To 97 compare the Luc activity with mRNA levels of Luc and endogenous NIR in the NIR-Luc 98 strain, we performed quantitative reverse transcription PCR (qRT-PCR) using mRNA 99 extracted from cells under the same conditions as those described in Fig. 2A with sequence- 100 specific primers (for NIR, NIR_F1: 5′-ATCCGTTGACCGAGGTACTG-3′ and NIR_R1: 5′- 101 TGCAGTCATCGGAGATGAAG-3′; for Luc, Luc_F1: 5′- 102 GGTTTTGGAATGTTTACTACACTCG-3′ and Luc_R1: 5′- 103 CTCAGAAACAGCTCTTCTTCAAATC-3′). The transcript levels of both genes were 104 increased at 2 h after exposure to -N, and their patterns corresponded well with the Luc 105 activity (Fig. 2A and 2B). These results indicate that the NIR-Luc strain can be used to 106 monitor NIR promoter activity. Furthermore, these results also demonstrate that the NIR 107 promoter region used in this study contains a cis-acting element for the -N responsive 108 transcription. 109 Next, to evaluate whether the Luc reporter assay system can be used to identify trans- 110 acting factor(s), we constructed a FLAG-tagged MYB1 overexpression (MYB1-OE) strain 111 using NIR-Luc as the host strain. Previously, we showed that MYB1 specifically binds to the 112 promoter of N assimilation genes, including NIR, under the -N condition as a positive 113 transcriptional regulator (Imamura et al., 2009). Therefore, we hypothesized that 114 overexpression of MYB1 would increase Luc activity in the MYB1-OE strain compared with 115 that in the control strain (MYB1-C, see below) even under the +N condition. 116 We constructed the pO250-CmMYB1 plasmid to construct the MYB1-OE strain. 117 Briefly, fragments 1 and 2 were PCR amplified from the C. merolae genomic DNA as the 118 template using two primer sets (for fragment 1, O250_Pro_F: 5′- 119 ACCGATGGAGTAGCCTTGTG-3′ and O250_Pro_R_J282: 5′- 120 CTCCACGTCGTCCATGGTCAACGAACGAAGAAACACAGAG-3′; for fragment 2, 121 J282_F_O250_Pro: 5′-CTTCGTTCGTTGACCATGGACGACGTGGAGCCTTTCAC-3′ and 122 J282_R_FLAG: 5′-ATGGTCTTTGTAGTCGACGCCACTCAGGAGCCAGC-3′), and

123 fragment 3 was PCR amplified from the pO250-FKBP12 construct (Imamura et al., 2013) 124 using primers FLAG_F_J282 (5′- 125 CTCCTGAGTGGCGTCGACTACAAAGACCATGACGGTG-3′) and M13_Primer_RV-N 126 (5′-TGTGGAATTGTGAGCGG-3′). Then, a fragment required for MYB1 overexpression 127 was generated by joint-PCR using primers O250_Pro_F_EcoRI and O250_Ter_R_EcoRI 128 with all three fragments as the DNA template, as previously described (Imamura et al., 2013). 129 Finally, the resultant PCR product was digested by EcoRI and cloned into EcoRI-digested 130 pKFURACm-Gs by T4 DNA ligase (Takara Bio, Shiga, Japan), as previously described 131 (Imamura et al., 2013), to construct pO250-CmMYB1. The NIR-Luc strain was transformed 132 with pO250-CmMYB1, as previously described (Imamura et al., 2013; Takemura et al., 133 2018), to obtain the MYB1-OE strain. The cloned FLAG-tagged MYB1 gene was expressed 134 under the control of the strong APCC promoter (Fujii et al., 2013). The empty vector

135 pKFURACm-Gs was introduced into the NIR-Luc strain to obtain the MYB1-C control strain. 136 Both strains were grown under the +N condition, and the expression of MYB1 in the MYB1- 137 OE strain was examined by northern blot analysis, as previously described (Imamura et al., 138 2009, 2018). The results showed that MYB1 transcripts in MYB1-OE were dramatically 139 increased compared with those in MYB1-C (Fig. 3A). Furthermore, immunoblot analysis 140 using anti-DYKDDDDK (FLAG) antibody (FUJIFILM Wako Pure Chemical Corporation, 141 Tokyo, Japan) revealed the FLAG-tagged MYB1 protein at the predicted molecular size 142 (approximately 75 kDa) in MYB1-OE but not in MYB1-C (Fig. 3B). In addition to the band 143 corresponding to the FLAG-tagged MYB1 protein, a few additional bands were detected in 144 MYB1-OE (Fig. 3B). Although the reason for an intense band at approximately 37 kDa is 145 unknown, these bands might be nonspecifically recognized by the antibody since the same 146 molecular weight proteins were also detected in MYB1-C (Fig. 3B). Thus, these data 147 confirmed the successful construction of the FLAG-tagged MYB1 overexpression strain, 148 MYB1-OE. We measured the Luc activity in MYB1-OE and MYB1-C strains under the +N 149 condition. As predicted, Luc activity in the MYB1-OE strain was approximately 4,000-fold 150 higher than that in MYB1-C strain (Fig. 3C). Under the same conditions, the results of 151 northern blot analysis indicated that transcripts of the endogenous NIR gene as well as 152 another MYB1 target gene, NRT, were also increased in the MYB1-OE strain (Fig. 3A). As 153 the control, transcript levels of histone H3 (control) were confirmed to be similar in the two 154 strains (Fig. 3A). These results showed that overexpression of FLAG-tagged MYB1 induced 155 the MYB1 target genes under the +N condition and that NIR promoter activity could be 156 successfully monitored using the Luc reporter assay. 5

157 In conclusion, we succeeded in constructing the Luc reporter assay system in C. 158 merolae. Luc activity in the NIR-Luc strain correlated well with the transcript levels of Luc 159 and endogenous NIR under the -N condition, indicating that the NIR promoter region contains 160 cis-acting element(s) for the -N-responsive NIR transcription. The NIR-Luc strain can be 161 used as a host strain for transformation experiments to analyze trans-acting TFs. In this report, 162 although we have focused on only one gene (NIR) and its identified regulator (MYB1), this 163 system can be used to study any promoter region and TF in this microalga. Thus, in the future, 164 combining this Luc reporter assay system with transcriptome data generated from DNA 165 microarray and RNA-seq analyses would enable the screening and identification of novel TFs 166 and/or cis-acting elements. 167 168 Acknowledgments 169 The authors thank the Biomaterials Analysis Division, Tokyo Institute of Technology, 170 for DNA sequencing analysis. This study was supported by JSPS KAKENHI Grant Number 171 18F18084 and Ohsumi Frontier Science Foundation. 172 173 References 174 Fuhrmann, M., Hausherr, A., Ferbitz, L., Schödl, T., Heitzer, M. et al. (2004) Monitoring 175 dynamic expression of nuclear genes in Chlamydomonas reinhardtii by using a synthetic 176 luciferase reporter gene. Plant Mol. Biol., 55, 869–881. 177 Fujii, G., Imamura, S., Hanaoka, M., and Tanaka, K. (2013) Nuclear-encoded chloroplast 178 RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes 179 in a red alga, Cyanidioschyzon merolae. FEBS Lett., 587, 3354–3359. 180 Fujiwara, T., Kanesaki, Y., Hirooka, S., Era, A., Sumiya, N. et al. (2015) A nitrogen source- 181 dependent inducible and repressible gene expression system in the red alga 182 Cyanidioschyzon merolae. Front Plant Sci., 6, 657. 183 Gibson, D., Young, L., Chuang, R., Venter, J., Hutchison, C. et al. (2009) Enzymatic 184 assembly of DNA molecules up to several hundred kilobases. Nat Methods., 6, 343. 185 Guivarc’H, A., Caissard, J.C., Azmi, A., Elmayan, T., Chriqui, D. et al. (1996) In situ 186 detection of expression of the gus reporter gene in transgenic plants: Ten years of blue 187 genes. Transgenic Res., 5, 281–288. 188 Haseloff, J., and Amos, B. (1995) GFP in plants. Trends Genet., 11, 328–329. 189 Imamura, S., Hanaoka, M., and Tanaka, K. (2008), The plant-specific TFIIB-related protein, 190 pBrp, is a general transcription factor for RNA polymerase I, EMBO J., 27, 2317–2327. 6

191 Imamura, S., Kanesaki, Y., Ohnuma, M., Inouye, T., Sekine, Y. et al. (2009) R2R3-type 192 MYB transcription factor, CmMYB1, is a central nitrogen assimilation regulator in 193 Cyanidioschyzon merolae. Proc. Natl. Acad. Sci. USA., 106, 12548–12553. 194 Imamura, S., Terashita, M., Ohnuma, M., Maruyama, S., Minoda, A. et al. (2010). Nitrate 195 assimilatory genes and their transcriptional regulation in a unicellular red alga 196 Cyanidioschyzon merolae: genetic evidence for nitrite reduction by a sulfite reductase- 197 like enzyme. Plant Cell Physiol., 51, 707–717. 198 Imamura, S., Ishiwata, A., Watanabe, S., Yoshikawa, H., and Tanaka, K. (2013) Expression 199 of budding yeast FKBP12 confers rapamycin susceptibility to the unicellular red alga 200 Cyanidioschyzon merolae. Biochem. Biophys. Res. Commun., 439, 264–269. 201 Imamura, S., Taki, K., and Tanaka, K. (2017) Construction of a rapamycin-susceptible strain 202 of the unicellular red alga Cyanidioschyzon merolae for analysis of the target of 203 rapamycin (TOR) function. J. Gen. Appl. Microbiol., 63, 305–309. 204 Imamura, S., Nomura, Y., Takemura, T., Pancha, I., Taki, K. et al. (2018) The checkpoint 205 kinase TOR (target of rapamycin) regulates expression of a nuclear-encoded chloroplast 206 RelA–SpoT homolog (RSH) and modulates chloroplast ribosomal RNA synthesis in a 207 unicellular red alga, Plant J., 94, 327–339. 208 Jefferson, R.A., (1987). Assaying chimeric genes in plants: the GUS gene fusion system. 209 Plant Mol. Biol. Rep., 5, 387–405. 210 Koncz, C., Langridge, W.H.R., Olsson, O., Schell, J., and Szalay, A.A. (1990) Bacterial and 211 firefly luciferase genes in transgenic plants: Advantages and disadvantages of a reporter 212 gene. J. Craniofac. Genet. Dev. Biol., 11, 224–232. 213 Matsuzaki, M., Misumi, O., Shin-I, T., Maruyama, S., Takahara, M. et al. (2004) Genome 214 sequence of the ultra-small unicellular red alga Cyanidioschyzon merolae. Nature, 428, 215 653–657. 216 Millar, A.J., Short, S.R., Hiratsuka, K., Chua, N.H., and Kay, S.A. (1992) Firefly luciferase 217 as a reporter of regulated gene expression in higher plants. Plant Mol. Biol. Rep., 10, 218 324–337. 219 Moriguchi, K., Suzuki, T., Ito, Y., Yamazaki, Y., Niwa, Y. et al. (2005) Functional isolation 220 of novel nuclear proteins showing a variety of subnuclear localizations. Plant Cell, 17, 221 389–403. 222 Naylor, L.H., (1999) Reporter gene technology: the future looks bright. Biochem. Pharmacol., 223 58, 749–757.

224 Nozaki, H., Takano, H., Misumi, O., Terasawa, K., Matsuzaki, M. et al. (2007) A 100%- 225 complete sequence reveals unusually simple genomic features in the hot spring red alga 226 Cyanidioschyzon merolae. BMC Biol., 5, 28. 227 Quaedvlieg, N.M., Schlaman, H.M., Admiraal, P., Wijting, S., Stougaard, J. et al. (1998) 228 Fusions between green fluorescent protein and β-glucuronidase as sensitive and vital 229 bifunctional reporters in plants. Plant Mol. Biol., 38, 861–873. 230 Ruecker, O., Zillner, K., Groebner-Ferreira, R., and Heitzer, M. (2008) Gaussia-luciferase as 231 a sensitive reporter gene for monitoring promoter activity in the nucleus of the green alga 232 Chlamydomonas reinhardtii. Mol. Genet. Genomics, 280, 153–162. 233 Shao, N., and Bock, R. (2008) A codon-optimized luciferase from Gaussia princeps 234 facilitates the in vivo monitoring of gene expression in the model alga Chlamydomonas 235 reinhardtii. Curr. Genet., 53, 381–388. 236 Takemura, T., Imamura, S., Kobayashi, Y., and Tanaka, K. (2018) Construction of a 237 selectable marker recycling system and the use in epitope tagging of multiple nuclear 238 genes in the unicellular red alga Cyanidioschyzon merolae. Plant Cell Physiol., 59, 239 2308–2316. 240 Taki, K., Sone, T., Kobayashi, Y., Watanabe, S., Imamura, S. et al. (2015) Construction of a 241 URA5.3 deletion strain of the unicellular red alga Cyanidioschyzon merolae: A 242 backgroundless host strain for transformation experiments. J. Gen. Appl. Microbiol., 61, 243 211–214. 244 Velten, J., Pogson, B., and Cazzonelli, C.I. (2008) Luciferase as a Reporter of Gene Activity 245 in Plants. Transgenic Plant J., 2, 1–13.

246 Figure Legends 247 Fig. 1. Construction of the nitrite reductase (NIR) promoter-driven firefly luciferase (Luc) 248 expression strain. 249 (A) Schematic representation of the URA5.3 locus in the wild-type (WT; top panel) and NIR- 250 Luc (bottom panel) strains. “URA5.3 up” and “URA5.3 down” indicate 2.2 kbp upstream and 251 2.0 kbp downstream regions of URA5.3, respectively, used for homologous recombination. 252 The 1.2 kbp NIR promoter (−1200 to +61, where +1 represents the translation start site) is 253 indicated. Arrows indicate the open reading frames (ORFs) of URA5.3 (1.4 kbp) and Luc (1.7 254 kbp). The positions of primers used for PCR analysis are indicated by arrowheads. F1 and R1 255 primers anneal outside of the integration region. (B) Confirmation of the replacement of 256 URA5.3 by the NIR-Luc fragment. Genomic DNAs were used as templates for PCR analyses 257 using F1/R1 and F2/R1 primer pairs. The PCR products were resolved by gel electrophoresis 258 on a 1.0% agarose gel. Positions based on a molecular size marker are indicated in kilobase 259 pairs on the left. 260 261 Fig. 2. Monitoring NIR promoter activity in the NIR-Luc strain under the nitrogen depleted (- 262 N) condition. 263 (A) Luc reporter assay. NIR-Luc and T1 cells were harvested at the indicated times after 264 exposure to the -N condition, and the total protein was extracted from the cells. Aliquots of 265 the total protein extract were then used for the Luc reporter assay. Luc activity was estimated 266 as the relative fluorescence units (RFU) divided by the amount of the total input protein 267 (RFU / Protein). Values represent the average Luc activity in three independent experiments 268 at the specified time points. Error bars indicate the standard deviation (SD). Asterisks 269 indicate the statistical significance of differences between the 0 h sample vs. the other time 270 points in each group (** P < 0.01; Student’s t-test). (B) Quantitative reverse transcription 271 PCR (qRT-PCR) analysis of the expression levels of Luc and NIR. Experiments were 272 performed thrice independently, and data represent transcript levels relative to the 0 h time 273 point (the value at time 0 was set at 1.0). Asterisks indicate the statistical significance of 274 differences between the 0 h sample vs. the other time points in each group (* P < 0.05, *** P 275 < 0.001; Student’s t-test). 276 277 Fig. 3. Effect of MYB1 overexpression on NIR promoter activity under the N replete (+N) 278 condition.

279 (A) Transcript levels of N assimilation genes and MYB1 under the +N condition. Cells of the 280 FLAG-tagged MYB1 overexpression (MYB1-OE) or MYB1 control (MYB1-C) strains were 281 harvested during the logarithmic growth phase. Total RNA (4.5 µg) extracted from the cells 282 was then subjected to northern blot analysis using gene-specific probes. Molecular marker 283 positions are indicated in nucleotides (nt) on the left. Histone H3 gene (CMN176C) and 284 rRNA stained with methylene blue are shown as the loading controls. (B) Confirmation of the 285 expression of exogenous FLAG-tagged MYB1. Total protein extract (4 µg) prepared from 286 the MYB1-OE or MYB1-C strains in the logarithmic growth phase were separated by 10% 287 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by 288 immunoblot analysis using monoclonal anti-DYKDDDDK (FLAG) antibody. Molecular 289 marker positions are indicated in kilodaltons (kDa) on the left. The arrowhead indicates the 290 expected position of the FLAG-tagged MYB1 protein. After the detection of a signal at 291 approximately 75 kDa, the membrane was stained with Coomassie Brilliant Blue, which was 292 used as a loading control (lower panel). (C) Analysis of NIR promoter activity via the Luc 293 reporter assay in MYB1-OE and MYB1-C strains. Luc activity was estimated as RFU / 294 Protein. Experiments were performed thrice independently. Error bars represent SD. 295 Asterisks indicate the statistical significance of the differences between MYB1-OE with 296 MYB1-C strains (*** P < 0.001; Student’s t-test). 297

Figure 1

URA5.3 up URA5.3 URA5.3 down WT F1 R1 NIR promoter Luc NIR-Luc F1 F2 R1

F1/R1 F2/R1 Luc Luc - - WT WT NIR NIR (kbp) (kbp)

19.3 19.3 7.7 7.7 6.2 6.2 3.5 3.5 2.7 2.7 1.9 1.9 1.5 1.5 Figure 2

T1 NIR-Luc 10000 ** ** ** 1000

100 RFU/Protein

1 0 2 4 8 Time after –N (hr)

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Relative transcript level 1

0.1 0 2 4 8 Time after –N (hr) Figure 3

A B C OE C - OE - - - MYB1 MYB1 MYB1

(nt) MYB1 3000 (kDa) 2000 MYB1 1500 100 75 3000 NIR 2000 50

3000 NRT 37 2000

1000 H3 500 75 CBB staining rRNA 50

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1000

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1 MYB1 EmptyVec-C CmMYB1MYB1-OE OE