a rice chloroplast‐localized abc transporter arg1 ...2020/06/19 · 5 6 7 article type : regular...

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1

2 PROF. KANG CHONG (Orcid ID : 0000-0001-7336-4225)

3 DR ZHUKUAN CHENG (Orcid ID : 0000-0001-8428-8010)

4 DR ZHENG MENG (Orcid ID : 0000-0002-9372-5179)

5

6

7 Article type : Regular Manuscript

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10 A rice chloroplast-localized ABC transporter ARG1 modulates cobalt and nickel

11 homeostasis and contributes to photosynthetic capacity

12 Haixiu Li1,2,6, Yuan Liu1,6,*, Huihui Qin1,2, Xuelei Lin1, Ding Tang3, Zhengjing Wu1, Wei

13 Luo1,2, Yi Shen3, Fengqin Dong1, Yaling Wang4, Tingting Feng1,2, Lili Wang1,2, Laiyun Li1,2,

14 Doudou Chen1,2, Yi Zhang5, Jeremy D. Murray4, Daiyin Chao4, Kang Chong1, Zhukuan

15 Cheng3,*, and Zheng Meng1,*

16

17 1Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of

18 Sciences, Beijing 100093, China; 2University of Chinese Academy of Sciences, Beijing

19 100049, China; 3State Key Laboratory of Plant Genomics and Center for Plant Gene

20 Research, Institute of Genetics and Developmental Biology, Chinese Academy of

21 Sciences, Beijing 100101, China; 4National Key Laboratory of Plant Molecular Genetics,

22 Chinese Academy of Sciences Center for Excellence in Molecular Plant Sciences,

23 Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences,

24 Chinese Academy of Sciences, Shanghai 200032, China; 5State Key Laboratory of Crop

25 Biology, College of Life Sciences, Shandong Agricultural University, Taian, Shandong

26 271018, China

27

28 Authors for correspondence: Zheng Meng, Zhukuan Cheng, Yuan Liu Acc

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https://doi.org/10.1111/NPH.16708



http://crossmark.crossref.org/dialog/?doi=10.1111%2Fnph.16708&domain=pdf&date_stamp=2020-05-28

This article is protected by copyright. All rights reserved

29 Email: Zheng Meng (Email: [email protected]; Tel: +86 10 62836556); Zhukuan

30 Cheng (Email: [email protected]; Tel: +86 10 64806551); Yuan Liu (Email:

31 [email protected]; Tel: +86 10 82105317).

32 6These two authors contributed equally to this work.

33

34 Received: 16 March 2020

35 Accepted: 9 May 2020

36

37

Total word count (excluding

summary, references,

acknowledgements and

legends):

6330 Discussion: 1520

Summary: 198 Acknowledgements 112

Introduction: 755 No. of figures: 7 (Figs 1–7 in

colour)

Materials and Methods: 1619 No. of Tables: 1

Results: 2436 No of Supporting

Information files:

17 (Fig. S1–

S11; Table

S1–S6)

38

39 Summary

40 • Transport and homeostasis of transition metals in chloroplasts, which are accurately

41 regulated to ensure supply and to prevent toxicity induced by these metals, is thus crucial

42 for chloroplasts function and photosynthetic performance. However, the mechanisms that

43 maintain the balance of transition metals in chloroplasts remain largely unknown.

44 • We have characterized an albino-revertible green 1 (arg1) rice mutant. ARG1 encodes Acc

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45 an evolutionarily conserved protein belonging to the ATP-binding cassette (ABC)

46 transporter family. Protoplast transfection and immunogold-labelling assays showed that

47 ARG1 is localized in the envelopes and thylakoid membranes of chloroplasts.

48 • Measurements of metal contents, metal transport, physiological and transcriptome

49 changes revealed that ARG1 modulates cobalt (Co) and nickel (Ni) transport and

50 homeostasis in chloroplasts to prevent excessive Co and Ni from competing with essential

51 metal cofactors in chlorophyll and metal binding proteins acting in photosynthesis. Natural

52 allelic variation in ARG1 between indica and temperate japonica subspecies of rice is

53 coupled with their different capabilities for Co transport and Co content within chloroplasts.

54 This variation underpins the different photosynthetic capabilities in these subspecies.

55 • Our findings link the function of the ARG1 transporter to photosynthesis, and potentially

56 facilitate breeding of rice cultivars with improved Co homeostasis and consequently

57 improved photosynthetic performance.

58

59 Keywords: chloroplast, rice, cobalt, nickel, transporter, homeostasis, natural variation,

60 photosynthetic capacity

61

62 Introduction

63 Chloroplasts are the site of photosynthesis and require substantial quantities of transition

64 metals (Fe, Mn, Cu and Zn) as cofactors or components of metal binding/dependent

65 proteins, which are essential for photosynthetic electron transport, metabolic pathways,

66 and protein structure (Blaby et al., 2013; Lopez-Millan et al., 2016). Homeostasis of

67 transition metals in chloroplasts is tightly regulated to ensure their sufficient supply but to

68 avoid toxic concentrations of these metals from inducing severe oxidative damage

69 resulting from the excessive accumulation of reactive oxygen species (ROS). Cobalt (Co)

70 and nickel (Ni), have some similar chemical properties to other transition metals and are

71 considered to be able to compete with or replace metal cofactors of chlorophyll and metal

72 binding proteins found in biochemical and physiological processes in many organisms Acc

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73 including plants (Wildner & Henkel, 1979; Morel & Price, 2003; Küpper & Kroneck, 2007;

74 Ranquet et al., 2007; Thorgersen & Downs, 2007; Chen et al., 2009; Jayakumar et al.,

75 2009; Anjum et al., 2015). Given that Co and Ni have been documented to be present in

76 chloroplasts (Terry, 1981; Chen et al., 2009), maintaining their correct levels is thus crucial

77 for chloroplast function and photosynthetic performance.

78 Co is a beneficial element for plants and low concentration of Co increases plant

79 growth, biochemical constituents, mineral yield (Cheruth et al., 2009; Anjum et al., 2015).

80 Ni, as a cofactor of urease, is a necessary nutrient for plants. Ni-deficient plants show

81 abnormal symptoms resulting from defective nitrogen metabolism, such as chlorosis or

82 necrotic spots on leaves (Eskew et al., 1983; Chen et al., 2009). Excessive Co and Ni

83 affect plant growth, photosynthesis, oxidative stress, and metabolic activity (Chen et al.,

84 2009; Anjum et al., 2015). Co hyperaccumulator-Haumaniastrum robertii contains even

85 above 1% cobalt in dried leaves (Brooks et al., 1980), and many Ni hyperaccumulators

86 from Alyssum genus accumulate about 2.5% Ni in dry leaves (Broadhurst et al., 2009).

87 However, there are few studies on the molecular mechanisms of the toxic effects of these

88 metals in plants.

89 Metal transporters play central roles in metals homeostasis in plants. Currently, many

90 broad-range metal transporters have been functionally identified. ZmYS1 transports

91 Fe-nicotianamine/Ni-nicotianamine and IRT1 transports Fe2+/Mn2+/Zn2+/Co2+/Ni2+ into

92 plasma membrane (Schaaf et al., 2004; Morrissey et al., 2009). AtMGT1, a plasma

93 membrane protein, has high affinity for Mg2+ but also probably transports Co2+ and Ni2+ ( Li

94 et al., 2001). AtIREG2/FPN2 transports Fe2+/Co2+/Ni2+ and AtHMA3 transport

95 Cd2+/Zn2+/Co2+/Pb2+ into vacuolar membrane involved in detoxification of these metals

96 (Schaaf et al., 2006; Morel et al., 2009; Morrissey et al., 2009). No specific transporters for

97 Co2+ and/or Ni2+ have yet been characterized in plants.

98 ABC transporters are responsible for the transport of a wide range of substrates across

99 membranes, including amino acids, vitamins, sugars, lipids, and metals (Rea, 2007; Bailly

100 et al., 2008; Rees et al., 2009). The canonical ABC transporters consist of transmembrane Acc

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101 domains (TMDs) and nucleotide binding domains (NBDs), which contains the conserved

102 ATP binding domains and the ABC signature motifs (Verrier et al., 2008). In plants,

103 non-intrinsic ABC transporters that have orthologs from bacterial ABC transporters belong

104 to ABC group I. In prokaryotes ABCI proteins can assemble into multi-subunit complexes,

105 designated as an energy-coupling factor (ECF) (Slotboom, 2014). Among ECFs,

106 CbiMNQO transports Co and Ni in bacteria (Rodionov et al., 2006; Kirsch & Eitinger,

107 2014; Bao et al., 2017). However, the function of ABCI subfamily members in plants

108 remains largely uncharacterized.

109 Asian cultivated rice (Oryza sativa) consists of two main subspecies, indica and

110 japonica (temperate japonica or tej and tropical japonica or trj), which have different

111 physiological traits (Kovach et al., 2007; Ma et al., 2015). Previous investigations revealed

112 that indica and japonica accessions differ in their concentrations of metals, foliage color

113 and photosynthetic capacity, including the photosynthetic rate and the regulation of

114 non-photochemical quenching (Chang & Bardenas, 1965; Cook & Evans, 1983; Kasajima

115 et al., 2011; Campbell et al., 2017).

116 In this study, we characterized a rice albino-revertible green mutant possessing a

117 mutation in the ABCI member gene ARG1. We provide evidence that chloroplast-localized

118 ARG1 mediates Co and Ni homeostasis by transporting Co and Ni from chloroplast, as

119 well as influencing photosynthesis in a way that Co and Ni compete with or replace metal

120 cofactors in chlorophyll and metal binding proteins acting in photosynthesis. Natural allelic

121 variation in ARG1 between indica and temperate japonica rice subspecies results in

122 differences in Co transport and photosynthetic performance. These results show how

123 excessive Co and Ni can affect chloroplast function and photosynthetic performance, and

124 provide a way to develop rice varieties with finely tuned Co concentration and improved

125 photosynthetic capacity.

126

127 Materials and MethodsAcc

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128 Plant materials and growth conditions

129 A γ-ray radiation mutagenized rice albino-revertible green mutant arg1 was isolated from

130 the Oryza sativa L. ssp. japonica cultivar Nipponbare. The wild-type (WT) and mutant rice

131 were germinated and transferred after 14 days to a paddy field at the Institute of Botany,

132 Chinese Academy of Sciences, Beijing, China.

133 To investigate the effects of metal ions on the phenotype of WT and arg1 mutant, rice

134 seeds were sterilized and grown on ½ MS solid media or Hoagland nutrient solution

135 (nitrogen source: NH4NO3 and KNO3 in MS; KNO3, CaNO3 and NH4PO4 in Hoagland)

136 supplemented with or without different concentrations of Co2+, Ni2+, Fe2+, and Fe3+ under a

137 16 h white light (25°C) and 8 h dark (25°C) cycle with approximately 150 μM m−2 s−1

138 photon density in a culture chamber.

139

140 Transmission electron microscopy (TEM) analyses

141 Rice leaf pieces (∼1 mm × 5 mm) were immediately fixed with 3% glutaraldehyde

142 containing 0.1 M phosphate buffer (pH 7.2), post-fixed in 1% osmium tetroxide (in 0.2 M

143 PBS, pH 7.2), dehydrated in an alcohol series, gradually transferred to acetone and then

144 embedded in Spurr’s resin (Sigma-Aldrich). Ultrathin sections (70-nm thick) were then

145 mounted on a grid, stained with 2% uranyl acetate, and imaged on a transmission electron

146 microscope (JEM-1230; JEOL).

147

148 Map-based cloning

149 For map-based cloning of ARG1, a total of 142 F2 and 164 F3 population plants showing

150 albino-revertible green phenotype were selected for genetic mapping. Polymorphic

151 markers developed based on rice simple sequence repeat (SSR) and InDel data were

152 used for fine mapping. ARG1 was narrowed down to a 108 kb region located on

153 chromosome 5, and then the corresponding fragments from the arg1 mutant and WT

154 plants were amplified and sequenced to discover the mutated locus. Primers used for Acc

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155 map-based cloning and sequencing are listed in Table S1.

156

157 Complementation and rice transformation

158 To generate complementation vectors, the ~1.5 kb promoter region of ARG1tej (ARG1

159 from temperate japonica) or ARG1ind (ARG1 from indica) was amplified and inserted into

160 the Pst I/BamH I sites of the pCAMBIA1301 vector, and the coding region and ~1 kb

161 downstream region of the ARG1tej and ARG1ind were amplified and then introduced into

162 the BamH I/Sac I sites of the above vectors. The resultant constructs gARG1tej and

163 gARG1ind were introduced into the arg1 mutant by Agrobacterium tumefaciens-mediated

164 transformation as described (Hiei et al., 1994).

165

166 RT-PCR and qRT-PCR analyses

167 Total RNAs were extracted from various rice tissues of WT, the arg1 mutant, and

168 transgenic lines at different developmental stages using TRIzol (Invitrogen), digested with

169 DNase I, and then reverse transcribed into cDNA samples using M-MLV reverse

170 transcriptase (Promega). qRT-PCR analyses were performed using SYBR Premix ExTaq

171 Mix (Takara) on an AB StepOne Plus system, and relative expression levels were

172 calculated with 2-ΔΔCT method (Livak & Schmittgen, 2001) using ACTIN1 as the reference

173 gene. The primer sequences are listed in Table S1.

174 Phylogenetic analyses

175 Fifty-one ARG1 homologous protein sequences were retrieved from the databases of

176 NCBI (http://www.ncbi.nlm.nih.gov), Phytozome v7.0 (http://www.phytozome.net), Uniprot

177 (http://www.uniprot.org), and Ensembl (http://ensembl.gramene.org) using the BLAST

178 search (Table S2). Phylogenetic analyses were reconstructed based on the alignment of

179 protein sequences using the maximum likelihood method by PHYML with the GTR + I + G

180 model, and the bootstrap test was performed with 1,000 replications (Guindon & Gascuel,

181 2003). Acc

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http://www.ncbi.nlm.nih.gov/

http://www.phytozome.net/

http://www.uniprot.org

http://ensembl.gramene.org/


182

183 Protoplast transient transfection assays

184 For subcellular localization of ARG1, the CDSs of ARG1tej and ARG1ind were amplified and

185 recombined into the P2GWF7.0 vector. For the subcellular localization of OsABCI7 and

186 OsABCI8, the eGFP fragment was amplified and cloned into the Kpn I/Sac I sites of

187 pUN1301 to yield pUN1301-eGFP. The CDSs of OsABCI7 and OsABCI8 in the

188 pEASY-Blunt were digested with BamH I/Kpn I and ligated into pUN1301-eGFP to

189 generate pUN1301-OsABCI7-eGFP and pUN1301-OsABCI8-eGFP, respectively. The

190 OsTRX m5-GFP and OsRpl6-2-GFP constructs were used as chloroplast and

191 mitochondrial localization markers (Kubo et al., 2008; Zhang et al., 2011), respectively.

192 The constructs described above and the empty vector were transformed into Arabidopsis

193 or rice protoplasts as described (Chen et al., 2006; Yoo et al., 2007).

194

195 Immunogold electron microscopy analyses

196 To generate Ubipro:ARG1-Myc vector, the CDS of ARG1 was inserted into the Kpn I/BamH

197 I sites of the pUN1301-Myc vector (a derivative of pCAMBIA1301 carrying the sequences

198 encoding a C-terminal Myc tag). The resultant construct was transformed into rice to

199 generate transgenic plants.

200 The leaf pieces (<2 mm) of ARG1-Myc and WT were immediately put into 4%

201 formaldehyde and 0.5% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.3). Samples

202 were placed in 30% ethanol on ice and then transferred into flow-through capsules for

203 further processing in a Leica AFS2 (Leica, UK) following a progressively lowering of

204 temperature protocol as described (Wells, 1985). Ultrathin sections (∼90 nm) were

205 mounted on grids and incubated with the primary antibody Myc (rabbit polyclonal

206 anti-c-Myc in PBS containing 0.1% actetylated BSA, pH7.3). Grids were washed in

207 incubation buffer, and subsequently detected by the goat anti-rabbit secondary antibody

208 conjugated to 15 nm gold. Grids were stained with 2% uranyl acetate and imaged on a Acc

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209 transmission electron microscope (Thermo Fisher, Netherlands).

210

211 Split ubiquitin-based yeast two-hybrid assays

212 The CDSs of OsABCI8 and OsABCI7 were individually inserted into EcoR I/Cla I sites of

213 pDSL-Nx and recombined into pDSL-Nx prey vector, respectively, while the CDS of ARG1

214 was recombined into pNCW bait vector. The split ubiquitin-based yeast two-hybrid assay

215 was performed as described (Ouyang et al., 2011). Transformed yeast cells containing

216 pNCW-ARG1 and pDSL-Nub I were used as a positive control, while those containing

217 pNCW-ARG1 and pDSL-Nub G were used as a negative control. Primers used for CSSSL

218 identification are listed in Table S1.

219

220 Chloroplast isolation

221 Rice leaves were harvested and homogenized using a blender in 25 ml buffer (0.33 M

222 sorbitol, 20 mM Tricine-KOH, pH 8.4, 5 mM EGTA, 5 mM EDTA, and 10 mM NaHCO3).

223 Samples were filtered through Miracloth and then centrifuged for 5 min at 4,200 g. The

224 pellets were suspended in 1 ml resuspension buffer (0.3 M sorbitol, 20 mM HEPES/KOH,

225 pH 8.0, 5 mM MgCl2, 2.5 mM EDTA), loaded on a Percoll gradient (40/85%), and

226 centrifugated at 2,000 g for 10 min. The intact chloroplasts appeared at the interface

227 between the 40% and 85% layers, were recovered and washed with HEPES-sorbitol

228 buffer (50 mM HEPES/KOH, pH 7.3, 0.3 M sorbitol), and subsequently centrifuged at 750

229 g for 5 min. Most of the pellets were purified chloroplasts and the intactness of

230 chloroplasts is assessed using ferricyanide reduction assays (Gee et al., 1965; Zhong et

231 al., 2020). All the chloroplast isolation procedures were performed at 4°C.

232

233 Determination of metal content

234 After drying in an oven at 70°C for 2 d, the samples of rice leaves and chloroplasts were

235 digested with 2 ml of concentrated HNO3 at 180°C for 2 h or 120°C for 4 h. The metal Acc

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236 concentration was determined by ICP-MS (Agilent 7700; NexION 350D) and ICP-ES

237 (ICAP6300).

238

239 Detection of ROS

240 The protoplasts dissociated from leaves in WT and arg1 mutants were incubated with 30

241 μM H2DCFDA in 10 mM Tris-HCl (pH 7.2) for 2 min at room temperature as described

242 (Joo et al., 2005). H2DCFDA fluorescence was measured using a MoFlo XDP high-speed

243 flow cytometer (Beckman-Coulter, USA), and fluorescence intensity was analyzed by

244 MoFlo XDP flow cytometer and Summit 5.2 software (Beckman Coulter Inc., USA).

245

246 RNA-seq analyses

247 WT and arg1 mutant seedlings at the late one-leaf stage were collected, and total RNAs

248 were extracted as described above. Three biological replicates were performed for each

249 sample. A total of 6 mRNA libraries were prepared according to the Illumina RNA

250 sequencing protocols, and sequenced on the Illumina HiSeqTM platforms. Gene

251 expression levels were estimated using HTSeq (Anders et al., 2015). Differentially

252 expressed genes (DEGs) were identified using the DeSeq program (Anders & Huber,

253 2010), and genes showing |log2FoldChange| > 0.5 and Padj < 0.05 were considered

254 differentially expressed. The gene ontology (GO) and Kyoto Encyclopedia of Genes and

255 Genomes (KEGG) enrichment analysis for the DEGs were performed using GOseq

256 (Young et al., 2010) and KOBAS (Mao et al., 2005), respectively.

257

258 Immunoblot analyses

259 Chloroplasts of WT and arg1 mutant seedlings at the late one-leaf stage were purified,

260 resuspended with buffer (25 mM BisTris-HCl, pH 7.0, 1% n-dodecyl-beta-D-maltoside,

261 20% glycerol), and centrifuged at 12,000 g for 10 min at 4°C, with the supernatant used as

262 protein samples. Electrophoretic separation, electro-transference, hybridization with the Acc

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263 Os02g0103800 (ferredoxin-NADP reductase) and Os07g0556200 (Rieske iron-sulfur

264 protein) polyclonal antibodies, and detection by enhanced chemiluminescence were then

265 performed.

266

267 MM281 strain metal-transport assays

268 The CDSs of ARG1tej and ARG1ind were inserted into the Sac I/Xho I and EcoR I/Hind III

269 sites of pET32 vector, respectively. For site directed mutagenesis assays, ARG1tej

270 (I156→V156) and ARG1tej (E308→K308) generated by PCR method were inserted into the

271 EcoR I/ Xho I sites of the pET32 vector. Salmonella enterica sv. typhimurium strain

272 MM281 cells were then transformed with vectors pET32, pET32-ARG1tej, pET32-ARG1ind,

273 ARG1tej (I156→V156), and ARG1tej (E308→K308). MM281 assay was performed as described

274 (Li et al., 2001).

275

276 Chromosome single-segment substitution line (CSSSL) identification

277 The CSSSL lines were generated from a back-cross population of temperate japonica

278 Nipponbare and indica 93-11. N9 5-1 including a fragment of chromosome 5 from

279 Nipponbare was identified from a BC5F2 population based on polymorphism Indel markers.

280 Primers used for CSSSL identification are listed in Table S1.

281

282 Measurement of chlorophyll content and photosynthetic parameters

283 Chlorophyll of the leaves from temperate japonica Nipponbare, indica 93-11, gARG1tej,

284 and gARG1ind transgenic plants was extracted with 80% acetone, and then the content

285 was measured using a Hitachi spectrophotometer. Photosynthetic parameters were

286 measured using a Dual-PAM-100 chlorophyll fluorometer (Heinz-Walz, Effeltrich,

287 Germany) as described (Yang et al., 2017).

288 Acc

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289 Data availability

290 The RNA transcriptome data has been deposited to the NCBI BioProject under the

291 accession number PRJNA510168. All other essential data supporting the findings of this

292 study are available in the main text or the Supporting Information.

293

294 Results

295 Phenotypic characterization of rice arg1 mutant

296 A rice mutant showing albino-revertible green phenotype, designated as arg1, was

297 isolated from a japonica variety Nipponbare. The seedlings exhibited albinism during seed

298 sprouting (Fig. 1a), but the albino seedlings began to turn green after the one-leaf stage

299 (Fig. S1), and recovered to green at the two-leaf stage (Fig. 1b). From the five-leaf stage

300 onward, leaves and stems of arg1 mutant plants gradually appeared chlorotic with albino

301 stripes (Fig. S1) or completely albino when grown in paddy fields (Fig. 1c). Finally, a

302 fraction of the albino arg1 mutants died before flowering (50:126, 39.7%), but the others

303 (76:126, 60.3%) recovered as yellowish green plants with reduced height (Fig. 1d).

304 The ultrastructures of the chloroplasts of WT and arg1 plants were investigated using

305 transmission electron microscopy (TEM). TEM revealed that the outer and inner

306 membrane structures of chloroplasts in arg1 albino leaves were intact at the early one-leaf

307 stage, but the stacked structures of grana thylakoids were rudimentary or defective and

308 only a few lamellar membranes were present. At the end of the one-leaf stage,

309 examination of the chloroplasts revealed that the granal stacks and thylakoid membranes

310 of the mutant were less abundant compared to WT plants. At the two-leaf stage, arg1

311 chloroplasts exhibited well-organized internal membrane structures with normal granal

312 stacks and thylakoids. Subsequently, the development of the grana thylakoids was

313 perturbed after the five-leaf stage in mutant chloroplasts, and the arg1 chloroplasts lacked

314 of grana stacks or lamellar structures at the six to eight-leaf stage (Fig. 1e,f and Fig. S1).

315

316 Map-based cloning of the ARG1 locusAcc

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317 To identify the gene responsible for the arg1 phenotype, map-based cloning was

318 performed. Using F2 and F3 mapping populations from a reciprocal cross between arg1

319 and indica var. Guangluai 4, the mutated gene locus was narrowed down to a 108 kb DNA

320 region on chromosome 5, which contained 19 putative open reading frames (Fig. S2a).

321 DNA sequencing revealed a 53-bp deletion in the first exon of Os05g0400600 in arg1,

322 with no sequence differences found in other genes. The 53-bp deletion in arg1 resulted in

323 a frame-shift mutation and produced a premature stop codon. The RT-PCR expression

324 analysis confirmed that the Os05g0400600 transcript was truncated in the arg1 mutant

325 (Fig. S2b).

326 To verify that Os05g0400600 was the gene mutated in arg1, a transgenic construct,

327 gARG1, containing the entire genomic fragment of the Os05g0400600 gene from

328 Nipponbare, was transformed into the arg1 mutant. As a result, the arg1 mutant

329 phenotype was completely rescued in gARG1 complemented transgenic plants (Fig.

330 S2c,d). Genomic PCR confirmed that both the truncated and normal length ARG1 genes

331 were present in the transgenic plants (Fig. S2e). These results confirmed that

332 Os05g0400600 indeed corresponded to the ARG1 gene and a 53-bp deletion in ARG1

333 was responsible for the mutant phenotype.

334

335 Phylogenetic analyses of the ARG1 homologous genes

336 ARG1 homologous sequences were retrieved from a broad variety of organisms, including

337 plants, archaea, and bacteria, but none were detected from animals and fungi (Table S2).

338 After all these sequences were aligned, phylogenetic analysis was performed using the

339 maximum likelihood method. The phylogenetic tree revealed that ARG1 shared homology

340 with bacterial protein CbiQ, which is a transmembrane component of the CbiMNQO

341 complex and functions in Co and Ni transport (Kirsch & Eitinger, 2014; Bao et al., 2017).

342 Moreover, the most closely related sequences were from cyanobacteria. According to the

343 endosymbiosis hypothesis, cyanobacteria are the ancestors of chloroplasts, so it seems

344 likley that plant ARG1 originated from cyanobacterial homologous genes. Other ARG1 Acc

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345 homologs from algae, lycophytes, mosses, gymnosperms, monocots, and eudicots

346 clustered into subgroups reflecting their known phylogeny (Fig. 1h and Fig. S3).

347

348 Expression patterns and subcellular localization of ARG1

349 The expression profile of ARG1 was determined by RT-PCR. ARG1 is widely expressed in

350 various tissues, including roots, seedlings, leaves, panicles, and seeds at different

351 developmental stages (Fig. S2f). ARG1 belongs to a member of the ABCI transporter

352 subfamily, and was predicted to be a membrane protein with five transmembrane domains

353 by the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM-2.0) and 3D-structure

354 analysis (Fig. 2a). A chloroplast transit peptide was identified in ARG1 by ChloroP

355 (Emanuelsson et al., 1999) and TargetP (Emanuelsson et al., 2000). To investigate the

356 actual subcellular localization of the ARG1 protein, a plasmid containing a ARG1-eGFP

357 fusion construct was transformed into rice protoplasts. The green fluorescent signals of

358 ARG1-eGFP fusion protein colocalized with the auto-fluorescence of chlorophyll in the

359 chloroplasts, suggesting that the ARG1 protein was indeed localized in the chloroplasts

360 (Fig. 2b).

361 The precise location of ARG1 in the chloroplasts, was investigated by

362 immunogold-labelling which showed that ARG1-Myc was located on the chloroplast

363 envelope and thylakoid membranes (Fig. 2c,d), implying that the protein likely functions as

364 a transporter in chloroplasts.

365

366 ARG1-mediated Co and Ni transport and homeostasis

367 We next measured the metals content in arg1 and WT field-grown plants using ICP-MS

368 and ICP-ES to determine whether ARG1 is involved in Co and Ni transport. Chloroplasts

369 were isolated and purified and the intactness of chloroplasts is 81.8% average in three

370 biological replicates by ferricyanide reduction assays (Table S3). The results

371 demonstrated that the contents of both Co and Ni in the chloroplasts of arg1 mutant were

372 much higher than in the WT (Fig. 3a,b), while other elements in the chloroplasts of the Acc

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373 arg1 mutants were comparable to WT (Fig. S4a,b). These findings suggested that ARG1

374 mediates Co and Ni homeostasis, and that loss of function of ARG1 results in excessive

375 accumulation of Co and Ni in arg1 chloroplasts.

376 The effects of Co and Ni on the phenotype of WT and arg1 mutant were subsequently

377 determined. When a high concentration of Co2+ and Ni2+ were provided to seedlings grown

378 on ½ MS solid media, arg1 seedlings were entirely albino, closely resembling the albino

379 phenotype of arg1 mutant grown in the paddy field (Fig. 3c and Fig. S5a). In view of the

380 fact that Co and Ni have chemical properties analogous to that of Fe, the influence of Fe

381 on the phenotypes of WT and arg1 mutants was also tested. In contrast with the results

382 seen with Co and Ni, when excess Fe2+ or Fe3+ was provided to arg1 seedlings, arg1

383 seedlings were not albino (Fig. S5b). In addition, when arg1 mutants were grown on

384 Hoagland’s hydroponic medium lacking Co2+ and Ni2+, arg1 plants remained green after

385 the one-leaf stage until the heading stage (Fig. S5c–e). Based on these results, we

386 deduced that the excess accumulation of Co and Ni in arg1 may be closely related to the

387 albino phenotype.

388 We further expressed ARG1 in the Salmonella strain MM281 to test the ability of ARG1

389 to transport Co and Ni. The results showed that strains expressing ARG1 showed

390 hypersensitivity to Co2+ and Ni2+ and higher contents of Co2+ and Ni2+, compared with

391 strains expressing empty vector pET32 and MM281 alone (Fig. 3d,e and Fig. S6a,b).

392 However, no obvious difference was detected in growth and/or contents between strains

393 expressing ARG1 and strains expressing pET32, when grown on media supplemented

394 with Zn, Cu, Fe, Fe, or Mn ion (Fig. S6c–j and Fig. S7). These findings demonstrated that

395 ARG1 transports Co and Ni specifically rather than any other metal tested.

396

397 ARG1-OsABCI7/OsABCI8 transporter complex

398 ARG1, as a transmembrane protein, was presumed to assemble with other ATP-binding

399 components to form a functional heterologous complex to transport substrates. Two ABCI

400 proteins potentially interacting with ARG1, OsABCI7 (Os01g0770500) and OsABCI8, Acc

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401 were identified in rice based on the protein interaction database (https://string-db.org),

402 both of which have ABC signature motifs and ATP binding domains. Transient transfection

403 assays in protoplasts revealed that both OsABCI7 and OsABCI8 proteins are localized to

404 chloroplasts (Fig. 3f). Moreover, split ubiquitin-based yeast two-hybrid assays confirmed

405 that there are heterologous interactions between ARG1-OsABCI7 and ARG1-OsABCI8

406 (Fig. 3g). Therefore, we concluded that an ARG1-OsABCI7/OsABCI8 transporter complex

407 may be responsible for functional transmembrane transport of Co and Ni in rice.

408

409 The influence of the ARG1 mutation on ion-binding and photosynthesis

410 We performed gene expression analyses of arg1 and WT plants using RNA-seq to further

411 investigate the consequences of the loss of ARG1 function. The RNA-seq results

412 indicated that 7,133 genes were differentially expressed in arg1 compared with the WT

413 libraries, and the expression of plenty of genes encoding ion binding proteins were

414 significantly down-regulated (Fig. 4a and Table S6, see later). Furthermore, for

415 down-regulated genes, ion-binding and photosynthesis were significantly enriched in GO

416 terms and KEGG pathways, respectively (Fig. 4b and Table S6, see later). Subsequently,

417 10 genes involved in Photosystem I, Photosystem II, and the electron transport chain of

418 photosynthesis were selected for qRT-PCR analysis (Fig. 4c). All selected genes showed

419 significant differences in expression between arg1 mutant and WT plants, consistent with

420 the RNA-seq data. These findings confirmed the reliability of the RNA-seq analysis and

421 supported our view that many genes encoding ion binding and photosynthesis-related

422 proteins show reduced transcript levels in the arg1 mutant compared to WT.

423 We prepared polyclonal antibodies against Os02g0103800 (Ferredoxin-NADP

424 reductase) and Os07g0556200 (Rieske iron-sulfur protein), which are involved in the

425 photosynthetic electron transport, and employed western blot to detect their protein levels.

426 Our results showed that their protein levels were markedly down-regulated in the arg1

427 mutant compared to WT (Fig. 4d).

428 Considering that the photosynthetic electron transport chain is a major Acc

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429 ROS-producing center and that abundant Co and Ni can induce excessive accumulation

430 of ROS in plants (Anjum et al., 2015), ROS levels in arg1 and WT plants were measured.

431 The ROS content in arg1 protoplasts and chloroplasts was significantly higher than in WT

432 (Fig. 5a–f). RNA-seq showed the expression levels of ROS scavenging genes were higher

433 in arg1 than in WT (Fig. S8), indicating oxidative stress was induced in arg1. Because we

434 performed experiments with Co and Ni stress under low light (Fig. 3c and Fig. S5a–e), we

435 also exclude ROS over-accumulation caused by high light. Altogether, we concluded

436 that excess Co and Ni accumulation in chloroplasts, as a consequence of the ARG1

437 mutation, has a negative impact on the expression levels of ion-binding and

438 photosynthetic proteins, and results in ROS accumulation.

439

440 Natural allelic variation of ARG1

441 Sequence analysis revealed six non-synonymous single-nucleotide polymorphisms

442 (SNPs) in the coding DNA sequence of ARG1 between temperate japonica Nipponbare

443 and Indica 93-11 cultivars (Fig. 6a). Among the six SNPs, four SNPs occurred in the

444 predicted transit peptide and did not alter the subcellular localization of ARG1 in

445 chloroplasts (Fig. S9a). One SNP with a hydrophobic isoleucine (I) in Nipponbare and a

446 hydrophobic valine (V) in 93-11 at amino acid 156 occurred in the predicted

447 transmembrane domain and most likely had no effect on the structure of

448 ARG1-OsABCI7/OsABCI8 complex. However, the polymorphic c.922G>A difference

449 resulted in a missense mutation, with an acidic glutamic acid (E) in Nipponbare

450 corresponding to a basic lysine (K) in 93-11 (a p.E308K substitution). The amino acid

451 E308K substitution occurred in the R1 loop, which is predicted to be the CbiQ-CbiO

452 interaction region and likely crucial for the energy coupling and transport activity of this

453 complex (Xu et al., 2013; Bao et al., 2017).

454 To investigate the genetic variation in ARG1 across the O. sativa species, we examined

455 the sequence of ARG1 in the genomes of 4,726 diverse accessions of cultivated rice

456 (Zhao et al., 2015). This analysis revealed that the frequency of the c.922A (p.K308) allele Acc

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457 of ARG1 was rare in the temperate japonica (tej) subpopulation (5.90%), but was very

458 high in indica rice subspecies accessions (99.40%) (Table 1). Moreover, the genetic

459 variation of ARG1 in 301 accessions of wild rice Oryza rufipogon (Huang et al., 2012), the

460 immediate ancestral progenitor of Asian cultivated rice Oryza sativa, was analysed further.

461 Results indicated that the frequency of the c.922A (p.K308) allele of ARG1 in Oryza

462 rufipogon-I (Or-I), Oryza rufipogon-II (Or-II) and Oryza rufipogon-III (Or-III) subpopulations

463 were 90.38%, 79.57% and 54.81%, respectively (Table S4).

464 To test potential associations between genetic variation in ARG1 and the content of Co

465 and Ni in rice, we re-sequenced and investigated the allelic state of c.922G>A of ARG1 in

466 8 indica and 8 temperate japonica varieties, and subsequently examined the

467 concentration of Co and Ni in chloroplasts of these indica and temperate japonica

468 accessions. The results indicated that the accessions with the indica allele ARG1ind

469 (c.922A) generally accumulated more Co in chloroplasts, compared with the temperate

470 japonica allele ARG1tej (c.922G) (Fig. 6b and Table S5). However, there were no

471 significant differences in the chloroplast Ni concentrations between indica and temperate

472 japonica varieties (Fig. S9b). The expression level of ARG1 did not differ significantly

473 between the temperate japonica and indica accessions (Fig. S9c), suggesting that the

474 difference in chloroplast Co accumulation was due most likely to the genetic variation in

475 the coding region.

476 To test the effects of natural variation of ARG1 on transporting activity, we observed the

477 growth conditions of Salmonella MM281 expressing ARG1tej and ARG1ind grown on

478 medium supplemented with Co2+ and Ni2+. Results demonstrated that compared with

479 strains expressing the indica allele, strains expressing the temperate japonica ARG1 allele

480 showed hypersensitivity to Co2+ but not to Ni2+ (Fig. 6c,d and Fig. S9d,e), suggesting

481 ARG1tej had stronger Co2+ transporting ability compared to ARG1ind. Site directed

482 mutagenesis further revealed that the E308K variation exerted greater influence on

483 transporting activity than I156V (Fig. S9f,g), suggesting that the E/K substitution largely

484 explained the differences in chloroplast Co content between temperate japonica and Acc

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485 indica varieties.

486 To determine the biological significance of natural allelic variation in ARG1, we

487 generated transgenic lines expressing the gene from indica (gARG1ind) and temperate

488 japonica (gARG1tej) into the arg1 mutant and selected lines with similar expression levels

489 (Fig. 7a). The gARG1tej transgenic plants exhibited stronger tolerance compared to

490 gARG1ind transgenic plants under Co and Ni stress (Fig. 7b). As expected, the gARG1tej

491 transgenic lines accumulated less Co in their chloroplasts (Fig. 7c). Consistent with this

492 observation, the chromosome single-segment substitution line (CSSSL) N9 5-1, which

493 was identified to include the temperate japonica ARG1 allele genomic segment in the

494 93-11 (indica) background, displayed stronger tolerance and accumulated less Co in

495 chloroplasts than indica 93-11 haboring the indica ARG1 allele (Fig. S10a–c).

496 Given that the severity of photosynthesis and chlorophyll content are affected by Co

497 dependent on its concentration (Palit et al., 1994; Jayakumar et al., 2009; Anjum et al.,

498 2015), we measured photosynthetic parameters and chlorophyll content of gARG1tej and

499 gARG1ind transgenic plants. Our results showed that, compared to gARG1ind transgenic

500 plants, gARG1tej transgenic lines produced a higher PSI quantum yield, higher electron

501 transport rate of PSI, and had higher chlorophyll a and chlorophyll b contents (Fig. 7d–f

502 and Fig. S11a,b). In accordance with this, CSSSL analyses confirmed the improved

503 photosynthetic properties and higher chlorophyll content in lines haboring the gARG1tej

504 allele compared to plants bearing the gARG1ind allele (Fig. S10d–f).

505

506 Discussion

507 ARG1 is a chloroplast-localized Co and Ni transporter

508 The prokaryotic ECF transport complex, composed of EcfT-EcfS-EcfA/A’ modular

509 components, transports vitamins, transition metals, and other micronutrients (Slotboom,

510 2014). The CbiMNQO complex, widely distributed in bacteria, is an ECF transporter and

511 functions in the transport of Co and Ni in bacterial cells (Rodionov et al., 2006; Kirsch &

512 Eitinger, 2014; Bao et al., 2017). Our results indicated that ARG1, localized in the Acc

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513 chloroplast, had the ability to transport directly Co and Ni (Fig. 2b–d, Fig. 3d,e and Fig.

514 S6a,b).

515 Phylogenetic analysis revealed that the cyanobacterial genes encoded proteins

516 homologous to ARG1 are the evolutionarily closest to those of plants, consistent with the

517 hypothesis that the ARG1 genes entered the plant lineage via endosymbiotic plastids from

518 ancient cyanobacteria (Tomitani et al., 1999). OsABCI7 and OsABCI8 are both localized

519 to the chloroplasts (Fig. 3f) and physically interact with ARG1 (Fig. 3g). Knock-out of

520 OsABCI8 was recently shown to result in an albino-revertible phenotype under continuous

521 rainy day conditions and after transplantation (Zeng et al., 2017; Shi et al., 2018). Thus,

522 the subcellular localization, protein interaction, and the albino phenotype of these

523 knock-out mutants support the view that OsABCI7 and/or OsABCI8 likely function as CbiO

524 components in rice. However, the substrate recognition component of CbiM/N in rice

525 remains to be investigated. A transporter complex consisting of ARG1-OsABCI7/OsABCI8

526 in rice implies that the metal transport mechanism of the CbiMNQO complex has been

527 conserved between bacteria and plants.

528

529 A key role for ARG1 in maintaining the Co- and Ni-homeostasis in rice chloroplasts

530 To balance the need for metals with their toxicity, plants have evolved transport systems

531 to maintain metal homeostasis (Anjum et al., 2015), and metal transporters are closely

532 associated with metal homeostasis in plants.

533 It has been inferred that Co and Ni can compete with and/or substitute for other metal

534 cofactors in many metal-binding proteins, and that the transcription and translation of

535 proteins that do not associate with appropriate metal cofactors will typically be inhibited,

536 and their mRNAs and proteins degraded (Wildner & Henkel, 1979; Morel & Price, 2003;

537 Ranquet et al., 2007; Thorgersen & Downs, 2007; Chen et al., 2009; Blaby et al., 2013;

538 Anjum et al., 2015). Our results showed that in arg1, Co and Ni are overenriched (Fig.

539 3a,b) and the expression of many genes encoding ion binding proteins was reduced at the

540 level of transcription and translation (Fig. 4a–d and Table S6). As a consequence, the Acc

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541 physiological and biochemical functions of these proteins were greatly perturbed in the

542 mutant.

543 It has been shown that excess Co and Ni can induce over-accumulation of ROS in

544 plants, thus resulting in membrane lipid peroxidation and oxidative stress (Chen et al.,

545 2009; Anjum et al., 2015). Production of ROS in leaf cells was mainly concentrated in

546 chloroplasts in this study (Fig. 5a–f), consistent with photosynthesis being the main source

547 of ROS production (Edreva, 2005; Asada, 2006). The elevated level of ROS in the arg1

548 chloroplasts may arise from the competition and substitution of Co and Ni for other

549 biologically essential metals: the displacement of the endogenous metals by Co or Ni

550 inhibits the proper function of these metal cofactor-associated proteins. For example, Fe-S

551 proteins were characterized as primary targets of Co, which competes with Fe in the

552 biogenesis of Fe-S clusters of metabolically essential proteins (Ranquet et al., 2007;

553 Thorgersen & Downs, 2007). We infer that due to the ARG1 defect, Co and Ni in the

554 chloroplasts compete with and replaces Fe in the Fe-S proteins, such as ferredoxin, in the

555 photosynthetic electron-transport chain. This could result in electron transfer to O2 instead

556 of the Fe-S clusters, ferredoxin and the NADP+ receptors to increase ROS production in

557 the PSI reaction center for a short periods (Edreva, 2005; Asada, 2006). Also, ROS was

558 possibly generated by Co/Ni-mediated Fenton reactions (Valko et al., 2005; Lange et al.,

559 2017).The induction of oxidative stress from misdirected electron transfer and Fenton

560 chemistry could ultimately cause the observed distortion of chloroplast development

561 and/or function.

562 The ICP-MS results of chloroplast metals showed that the concentrations of Co and Ni

563 in chloroplasts of the arg1 mutant were significantly higher than those in WT (Fig. 3a,b),

564 which suggests strongly that ARG1 on the chloroplast membranes, likely transports Co

565 and Ni from the chloroplast to the cytoplasm. A chloroplast envelope protein NiCo, as a

566 member of the Ni/Co transporter family, accumulates increasingly following leaf

567 development and has been suggested to be involved in Co and Ni uptake into chloroplasts

568 (Duy et al., 2011; Pham et al., 2020). Hence, NiCo and ARG1 may be responsible for the Acc

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569 transport of Co and Ni into and out of chloroplasts, respectively, so as to maintain together

570 Co and Ni homeostasis in chloroplasts.

571 Considering that Co and Ni are regarded as remnants of early life (Frausto da Silva &

572 Williams, 2001, Küpper & Kroneck, 2007), it is intersting about the evolutionary

573 significance of the loss of ARG1 homologous genes in fungi and animals while retained in

574 plants. Chloroplasts are the main location of many metal binding proteins, and the

575 photosynthetic pigments, PSI and PSII reaction centers and the electron transfer chain

576 require Mg, Fe, Mn, Cu and other essential metal cofactors. Therefore, our results

577 together with previous findings, emphasize that the retention of ARG1 in plants was to

578 transport Co and Ni from the chloroplasts to restrict excessive Co and Ni from competing

579 with and replacing the necessary metal cofactors in the photosynthetic processes, thereby

580 ensuring the normal photosynthesis in plants.

581

582 Allelic variation of ARG1 may contribute to the divergence in the Co content and

583 photosynthetic capacity between indica and temperate japonica rice subspecies

584 Previous studies have revealed single SNP specifically correlated with differences in the

585 physiological traits between indica and japonica rice subspecies, including COLD1 SNP2

586 (A>T/C) for cold tolerance and NRT1.1B SNP1 (C>T) for nitrate uptake (Hu et al., 2015;

587 Ma et al., 2015). In this study, a single amino acid change (p.E308K) due to a SNP

588 (c.922G>A) in ARG1 exists between the temperate japonica and indica cultivars at

589 position 308 within the R1 loop (Fig. 6a), which is predicted to be the

590 ARG1-OsABCI7/OsABCI8 interaction site (Fig. 2a in this study;Xu et al., 2013; Bao et al.,

591 2017). The E308K variant likely influences the metal transporting activity of the

592 ARG1-OsABCI8/OsABCI7 complex, as supported by the expression of ARG1 with I/V and

593 E/K point mutations in Salmonella MM281 (Fig. S9f,g). Allele frequency analyses revealed

594 that c.922G (p.E308) of the temperate japonica ARG1 allele possibly originated from

595 Oryza rufipogon-III and c.922A (p.K308) of the indica ARG1 allele may have arisen from

596 Oryza rufipogon-I (Table 1 and Table S4), in accordance with the fact that japonica was Acc

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597 domesticated from Oryza rufipogon-III and O. sativa indica was descended from Oryza

598 rufipogon-I (Huang et al., 2012).

599 Natural variation in metal ion transporters has been shown to be tightly associated with

600 differences in metal content across the O. sativa accessions (Huang et al., 2016;

601 Campbell et al., 2017). In the present work, we confirmed that indica cultivars generally

602 accumulate more Co in chloroplasts compared to the temperate japonica cultivars (Fig.

603 6b). In accordance with this observation, the ARG1tej isoform from temperate japonica

604 showed stronger transporting ability for Co than ARG1ind from the indica subspecies (Fig.

605 6c,d). Furthermore, the introduction of ARG1tej from temperate japonica in transgenetic

606 and CSSSL plants conferred an increased tolerance and decreased Co accumulation in

607 chloroplasts than ARG1ind from indica (Fig. 7b,c and Fig. S10a–c). In contrast, no

608 significant differences were found in the Ni concentration in chloroplasts between indica

609 and temperate japonica varieties (Fig. S9b). This might be due to the higher transporting

610 activity of the ARG1-OsABCI7/OsABCI8 complex for Co than Ni in rice. In agreement with

611 this observation, ECF transporter complex CbiMNQO showed high Co transport activity

612 but low Ni transport activity (about 8%) in bacteria (Bao et al., 2017). Based on our results

613 we conclude that variations in ARG1, specifically the E308K variant, might account for the

614 differences in chloroplasts Co content between indica and temperate japonica.

615 Divergence in foliage color and photosynthetic capacity have previously been

616 demonstrated between indica and japonica cultivars, but the molecular mechanism

617 underlying this variation remains poorly understood (Chang & Bardenas, 1965; Cook &

618 Evans, 1983; Kasajima et al., 2011). Our CSSSL line and transgenic analyses confirmed

619 that the plants carrying the ARG1-temperate japonica allele achieved higher PSI quantum

620 yield, higher electron transport rates of PSI, and higher chlorophyll contents compared to

621 plants bearing the ARG1-indica allele (Fig. 7d–f and Fig. S10d–f and Fig. S11a,b), and

622 notably exhibited improved photosynthesis efficiency. We conclude that the differences in

623 photosynthesis capacity might be due to the divergence in chloroplasts Co accumulation

624 in the CSSSL line and transgenic lines expressing the ARG1tej and ARG1ind alleles (Fig. Acc

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625 7c and Fig. S10c). Our findings imply that natural allelic variation of ARG1 contribute to

626 differences in photosynthetic capacity between indica and temperate japonica, and that

627 the ARG1-temperate japonica allele potentially could improve the photosynthetic

628 efficiency of indica rice. The SNPs identified here will facilitate selection of ARG1 alleles

629 for breeding rice varieties with finely tuned Co homeostasis and improved photosynthetic

630 performance via molecular breeding.

631

632 Acknowledgements

633 We thank Hongbin Wang (Sun Yat-sen University), Nakao Kubo (Kyoto Prefectural

634 University), and Legong Li (Capital Normal University) for providing OsTRX m5-GFP,

635 OsRpl6-2-GFP, and Salmonella MM281, respectively. We thank Jia Wang and Lin Liu

636 (Chinese Academy of Sciences), and Elaine Barclay (John Innes Centre) for assistance

637 with protein structure prediction and immunogold experiments, respectively. We also

638 thank Cathie Martin (John Innes Centre), Günter Theißen (Friedrich Schiller University

639 Jena), Yuxin Hu, Song Ge and Congming Lu (Chinese Academy of Sciences) for their

640 valuable comments and suggestions. This work was supported by grants from the

641 National Natural Science Foundation of China (31771364) and the Strategic Priority

642 Research Program of Chinese Academy of Sciences (XDA08020303-2).

643

644 Competing interests

645 The authors declare no competing interests.

646

647 Author Contributions

648 HL, YL, and ZM designed research. HL, YL, HQ, XL, DT, ZW, FD, YW, TF, LW, LL, DC

649 and YZ performed research. HL, YL, and ZM analyzed data. DT, XL, WL, YS, DC, KC,

650 and ZC contributed reagents/materials/analysis tools. YL, HL, JDM, ZC, and ZM wrote

651 manuscript drafts. HL and YL contributed equally to this work.

652 Acc

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653 ORCID

654 Haixiu Li ID: https://orcid.org/0000-0003-2517-1917

655 Yuan Liu ID: https://orcid.org/0000-0003-0046-000X

656 Jeremy D. Murray ID: https://orcid.org/0000-0003-3000-9199

657 Kang Chong ID: https://orcid.org/0000-0003-4364-778X

658 Zhukuan Cheng ID: https://orcid.org/0000-0001-8428-8010

659 Zheng Meng ID: https://orcid.org/0000-0002-9372-5179

660

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Supporting Information

Fig. S1 Ultrastructure in chloroplasts and thylakoids from seedlings of WT and the arg1

mutant at the early one-leaf stage, the late one-leaf stage, the two-leaf stage, and the

five-leaf stage.

Fig. S2 Map-based cloning and functional complementation of the arg1 mutant and

expression pattern of ARG1.

Fig. S3 Phylogenetic tree of ARG1 homologous proteins.

Fig. S4 Element concentrations of WT and arg1 chloroplasts separated from the leaves of

WT and the arg1 mutant at the six-leaf stage (a and b).

Fig. S5 Effects of metal ion on the phenotype of WT and the arg1 mutant.

Fig. S6 ARG1 is involved in transporting Co and Ni but not Fe, Mg, Cu, Zn.

Fig. S7 Effect of ARG1 on metal transporting.

Fig. S8 Expression levels of ROS scavenging genes in WT and the arg1 mutant.

Fig. S9 Impact of natural variation of ARG1 on subcellular localization, chloroplasts Ni

accumulation, and Ni/Co transporting activity.

Fig. S10 Stress phenotype, Co concentration, and photosynthetic parameters of

Nipponbare, CSSSL line N9 5-1, and 93-11 plants.

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Fig. S11 Chlorophyll a (a) and chlorophyll b (b) content of Nipponbare, 93-11, gARG1tej,

and gARG1ind transgenic lines at the six-leaf stage grown under normal soil conditions.

Table S1 Gene-specific primers used in this study.

Table S2 Detailed description of ARG1 homologous proteins used for phylogenetic

analysis.

Table S3 The intactness assessment of chloroplasts isolated from rice leaves.

Table S4 Allele frequency of c.922G>A (p.E308K) of ARG1 in the Oryza rufipogon

populations.

Table S5 Allele state of c.922G>A (p.E308K) of ARG1 in the Oryza sativa indica and

temperate japonica varieties.

Table S6 Detailed information for DEGs and GO/KEGG enrichment analyses of

up-regulated and down-regulated DEGs between WT and arg1 mutant in RNA-seq.

Figure legend：

Fig. 1 The phenotype of the arg1 mutant and phylogenetic analyses of ARG1.

(a–d) The phenotype of WT and arg1 mutant at the early one-leaf stage (a), two-leaf stage

(b), six to eight-leaf stage (c), and after the eight-leaf stage (d). Insets ([c]) show a

magnified leaf at the corresponding stage. Bars = 2.5 cm. (e, f) Electron ultrastructure in

chloroplasts, thylakoids and envelopes from seedlings of the WT (e) and arg1 (f) at the six Acc

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to eight-leaf stage. Magnified chloroplasts are indicated in dark red and blue frames. Bars

= 200 nm. (g) Gene structure of ARG1 and the mutation site of arg1. Black boxes indicate

exons, black lines represent introns, and grey boxes represent the untranslated regions.

(h) The phylogenetic analyses of ARG1 homologous proteins.

Fig. 2 Subcellular location of ARG1. (a) Structural depiction of ARG1. TM:

transmembrane domain, CH: cytoplasmic helix/coupling helix. (b) Subcellular localization

of P2FGW (control), OsTRX m5-GFP (chloroplast marker), OsRpl6-2-GFP (mitochondrion

marker) and ARG1-eGFP fusion proteins in rice leaf protoplasts. Bars = 5 μm. (c, d)

Immunogold-labeling of ARG1 in the chloroplasts of WT (c) and ARG1-Myc transgenic

plants (d). Localization of ARG1 visualized by immunogold-labelling in chloroplast

envelope membranes and thylakoid membranes (d). The dark red arrowheads indicate

gold signal in chloroplast envelope membranes, and the blue arrowheads indicate gold

signal in thylakoid membranes. Bars = 0.5 μm. S, starch grain.

Fig. 3 ARG1 functions in Co and Ni transport in the form of a complex with OsABCI7

and/or OsABCI8. (a, b) The Co (a) and Ni (b) concentration of WT and arg1 chloroplasts

separated from the leaves of WT and arg1 mutant at the six-leaf stage. Data are

presented as mean ± SD (n = 3), and the significance of difference was assessed by

Student’s t test (** corresponding to P < 0.01). (c) Effects of Co and Ni on the phenotype of

WT and arg1 mutant. WT and arg1 mutant seedlings were grown on ½ MS solid medium

supplemented with indicated Co2+ and Ni2+ concentrations. Bar = 5 cm. (d, e) Function of

ARG1 on Co and Ni transport. Time course of Salmonella enterica sv. typhimurium strain

MM281 expressing empty pET32 and ARG1 cultured on media supplemented with 2.5

mM Co2+ (upper panels) and 6 mM Ni2+ (lowers panels) (d). Data are mean ± SD (n ≥ 3),

and different lowercase letters indicate significant differences by Student’s t test.

Salmonella MM281, MM281 expressing empty pET32, and MM281 expressing ARG1 Acc

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grown on medium supplemented with 1 mM and 1.5 mM Co2+, and with 2 mM and 3 mM

Ni2+ (e). (f) Subcellular localization of OsABCI7-eGFP and OsABCI8-eGFP fusion proteins

in Arabidopsis leaf protoplasts. Bars = 5 μm. (g) Split ubiquitin-based yeast two-hybrid

showing ARG1-OsABCI7 and ARG1-OsABCI8 protein interactions. Serial dilutions of 10

to 1 yeast transformant cells carrying indicated combinations were grown on the synthetic

dropout media.

Fig. 4 The mRNAs and proteins levels change between WT and arg1 mutant. (a) The up-

and down-regulated differential expressed genes (DEGs) between WT and arg1 mutant in

the RNA-seq analyses. (b) Gene Ontology enrichment analysis of down-regulated DEGs.

Asterisks represent the significantly enriched GO terms (Benjamini-Hochberg corrected P

< 0.05). (c) Verification of DEGs using qRT-PCR (real-time quantitative PCR). Data were

presented as mean ± SD (n = 3). The functional annotation was shown in parentheses for

each gene as Os01g0501800 (33kDa oxygen evolving protein of photosystem II),

Os01g0869800 (photosystem II subunit PsbS), Os02g0103800 (ferredoxin-NADP

reductase), Os03g0778100 (Photosystem I reaction center subunit III), Os04g0414700

(Photosystem I reaction center subunit PsaO), Os06g0101600 (Plastocyanin),

Os07g0141400 (Photosystem II oxygen-evolving complex protein 2), Os07g0544800

(Oxygen-evolving enhancer protein 3), Os07g0556200 (Rieske iron-sulfur protein), and

Os08g0104600 (ferredoxin). (d) Proteins levels change of ferredoxin-NADP reductase

(Os02g0103800) and Rieske iron-sulfur protein (Os07g0556200) on photosynthetic

electron transport chains between WT and arg1 mutant.

Fig. 5 The ROS levels change between WT and arg1 mutant. Rice protoplasts and

chloroplasts were stained by H2DCFDA separated from WT (a, c) and arg1 (b, d) at the

six-leaf stage, and the fluorescence was measured using a high-speed flow cytometer (a–

d). (e, f) Quantification of fluorescence intensities in rice protoplasts (e) and chloroplasts Acc

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(f). Fluorescence intensity (arbitrary units) was determined to assess the ROS content.

Data are mean ± SD (n = 3). * indicates significant difference at P < 0.05 by Student’s t

test, ** indicates significant difference at P < 0.01 by Student’s t test (n ≥ 1.5×105). SSC,

side scatter. R5 region represents the intact protoplasts (a, b) selected from all the

protoplasts, the chloroplasts used for H2DCFDA fluorescence detection (c, d) are from the

intact protoplasts in R5 region.

Fig. 6 The divergence in Co-transporting ability of ARG1 from rice indica and temperate

japonica (tej). (a) SNPs in the coding region of ARG1 between rice indica 93-11 and

temperate japonica Nipponbare. Dark blue boxes indicate the coding sequence, white

boxes indicate the 5’ and 3’ untranslated regions, and lines between the boxes indicate

introns. (b) The Co concentration of chloroplasts separated from the leaves at the six-leaf

stage in rice indica and temperate japonica accessions. Data are mean ± SD (n = 8 for

indica, 8 for temperate japonica), and the P value was generated by Student’s t test. (c, d)

Effect of ARG1tej and ARG1ind on Co transport. Salmonella enterica sv. typhimurium strain

MM281, MM281 expressing empty vector pET32, and MM281 expressing ARG1tej and

ARG1ind grown on medium supplemented with 1.2 mM and 1.7 mM Co2+ (c). Time course

of Salmonella MM281 expressing empty pET32, ARG1tej and ARG1ind cultured on medium

supplemented with 2.5 mM Co2+ (d). Data are presented as mean ± SD (n = 3), and

different lowercase letters indicate significant difference by Student’s t test.

Fig. 7 The association of natural ARG1 variation with the divergence in chloroplasts Co

content and photosynthetic capability between rice indica and temperate japonica (tej). (a)

The expression level of ARG1 in Nipponbare, gARG1tej, and gARG1ind transgenic plants.

Data represent mean ± SD (n = 3) and different lowercase letters indicate significant Acc

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differences by Duncan’s multiple range test (one-way ANOVA; P < 0.05). (b) The

phenotype of Nipponbare, gARG1tej, and gARG1ind transgenic plants under Co and Ni

stress. The inset shows a magnified leaf form the corresponding seedling. Bars = 2.5 cm.

(c–f) The Co concentration in chloroplasts (c), PSI quantum yield (d), electron transport

rate of PSI (e), and total chlorophyll content (f) of Nipponbare, 93-11, gARG1tej, and

gARG1ind transgenic lines at the six-leaf stage grown under normal soil conditions. Data

represent mean ± SD (n = 3 to 8) and different lowercase letters indicate significant

differences according to Duncan’s multiple range test (one-way ANOVA; P < 0.05).

Table 1 Allele frequency of c.922G>A (p.E308K) of ARG1 in the Oryza sativa populations.

Population (O. sativa) Population size Allele frequency of G Allele frequency of A

indica 2759 0.60% 99.40%

intermediate 90 16.70% 83.30%

temperate japonica (tej) 767 94.10% 5.90%

japonica intermediate 241 76.80% 23.20%

tropical japonica (trj) 504 8.10% 91.90%

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a rice chloroplast‐localized abc transporter arg1 ...2020/06/19 · 5 6 7 article type : regular...

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