Autophagy maintains Zn pools under Zn deficiency Author ...140 atg mutants exhibit accelerated Zn deficiency symptoms 141 To evaluate the relationship between Zn deficiency and autophagy

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Short title: Autophagy maintains Zn pools under Zn deficiency 1

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Author for Contact: 3

Kohki Yoshimoto 4

E-mail: [email protected] 5

Department of Life Science, School of Agriculture, Meiji University, Kawasaki, 6

Kanagawa, 214-8571, Japan 7

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Article title: Autophagy Increases Zinc Bioavailability to Avoid Light-Mediated 9

ROS Production under Zn Deficiency 10

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Daiki Shinozaki1, 2, Ekaterina A. Merkulova3, Loreto Naya3, Tetsuro Horie4, 5, Yuri 12

Kanno6, Mitsunori Seo6, Yoshinori Ohsumi5, Céline Masclaux-Daubresse3, and Kohki 13

Yoshimoto1, 2, 3, * 14

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1 Department of Life Science, School of Agriculture, Meiji University, Kawasaki, 16


2 Life Science Program, Graduate School of Agriculture, Meiji University, Kawasaki, 18


3 Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 20

RD10, F-78000 Versailles, France 21

4 Research Center for Odontology, School of Life Dentistry at Tokyo, The Nippon Dental 22

University, Chiyoda-ku, Tokyo, 102-8159, Japan 23

Plant Physiology Preview. Published on January 15, 2020, as DOI:10.1104/pp.19.01522

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5 Research Unit for Cell Biology, Institute of Innovative Research, Tokyo Institute of 24

Technology, Yokohama, Kanagawa, 226-8503, Japan 25

6 RIKEN Center for Sustainable Resource Science, Suehiro-cho, Tsurumi-ku, 26

Yokohama, Kanagawa, 230-0045, Japan. 27

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One sentence summary: Resupply of free zinc ions via autophagic degradation 29

suppresses photosynthesis-related Fenton-like reaction-induced chlorosis under zinc 30

starvation in Arabidopsis thaliana. 31

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Author Contributions: K.Y., Y.O., and C.M. designed the research. D.S., E.M., L.N., 33

T.H., M.S. and Y.K. performed the experiments. D.S. and K.Y. analyzed the data and 34

wrote the manuscript with input from all other authors. 35

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* Author for correspondence: 37

Kohki Yoshimoto 38

E-mail: [email protected] 39

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ABSTRACT 41

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Zinc (Zn) is an essential micronutrient for plant growth. Accordingly, Zn deficiency in 43

agricultural fields is a serious problem, especially in developing regions. Autophagy, a 44

major intracellular degradation system in eukaryotes, plays important roles in nutrient 45

recycling under nitrogen and carbon starvation. However, the relationship between 46

autophagy and deficiencies of other essential elements remains poorly understood, 47

especially in plants. In this study, we focused on Zn due to the property that within cells 48

most Zn is tightly bound to proteins, which can be targets of autophagy. We found that 49

autophagy plays a critical role during Zn deficiency in Arabidopsis thaliana. 50

Autophagy-defective plants (atg mutants) failed to grow and developed accelerated 51

chlorosis under Zn starvation. As expected, a shortage of Zn induced autophagy in 52

wild-type plants, whereas in atg mutants, various organelle proteins accumulated to high 53

levels. Additionally, the amount of free Zn2+ was lower in atg mutants than in control 54

plants. Interestingly, Zn-deficiency symptoms in atg mutants recovered under low-light, 55

iron-limited conditions. The levels of hydroxyl radicals in chloroplasts were elevated, and 56

the levels of superoxide were reduced in Zn-deficient atg mutants. These results imply 57

that the photosynthesis-mediated Fenton-like reaction, which is responsible for the 58

chlorotic symptom of Zn deficiency, is accelerated in atg mutants. Together, our data 59

indicate that autophagic degradation plays important functions in maintaining Zn pools to 60

increase Zn bioavailability and maintain ROS homeostasis under Zn starvation in plants. 61

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INTRODUCTION 63

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Plant essential nutrients, defined as those elements indispensable for optimal plant 65

growth, are classified as macronutrients or micronutrients according to the amounts 66

required. Thus, the macronutrients are carbon (C), hydrogen (H), oxygen (O), nitrogen 67

(N), phosphorus (P), potassium (K), sulfur (S), calcium (Ca), and magnesium (Mg), 68

whereas the micronutrients are iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), nickel 69

(Ni), molybdenum (Mo), chlorine (Cl), and boron (B). C and O can be obtained from air, 70

and H from water, but the other nutrients must be absorbed from the soil through the 71

roots. 72

In this study, we focused on Zn, a metallic element that is essential for all living 73

organisms. Most cellular Zn is tightly bound to proteins, thus levels of free Zn ions are 74

quite low within cells. Zn serves as a catalytic or structural co-factor in a large number of 75

enzymes including alcohol dehydrogenase, superoxide dismutase (SOD), and regulatory 76

proteins such as transcription factors containing zinc-finger domains (Vallee and Auld, 77

1990; Maret, 2009). Therefore, Zn deficiency disturbs cellular homeostasis. In the 78

context of agriculture, Zn deficiency is a serious problem because it dramatically 79

decreases the quality and quantity of crop commodities, especially in developing regions. 80

Previous research on Zn deficiency in plants has focused primarily on uptake of Zn by 81

transporters (Grotz et al., 1998) and gene regulation by transcription factors that function 82

under Zn starvation (Assunção et al., 2010). By contrast, relatively few studies have 83

focused on the redistribution of intracellular Zn (Eguchi et al., 2017) and the detailed 84

mechanisms of the onset of Zn starvation symptoms remains unclear. 85



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Autophagy is a major intracellular degradation mechanism that is conserved 86

throughout the eukaryotes. During autophagy, degradation targets are surrounded by an 87

isolation membrane (IM) and encapsulated in an autophagosome (AP). The outer 88

membrane of the AP fuses with the vacuolar membrane, and the inner membrane of the 89

AP and its contents (i.e., degradation targets) are released into the vacuolar lumen. This 90

single membrane–bound vesicle inside the vacuole is called the autophagic body (AB). 91

The AB is rapidly degraded by vacuolar lipases and proteases, and the contents are 92

recycled for use as nutrients. 93

Autophagic processes are driven by a number of autophagy-related (ATG) 94

proteins (Mizushima et al., 2011). The ATG genes were first discovered in yeast 95

(Saccharomyces cerevisiae), and many ATG genes are highly conserved in plants 96

(Hanaoka et al., 2002; Yoshimoto, 2012). In Arabidopsis thaliana, transfer DNA 97

(T-DNA) insertions that disrupted ATG genes were identified and the mutants were 98

shown to be defective in autophagy (Doelling et al., 2002; Hanaoka et al., 2002; 99

Yoshimoto et al., 2004; Thompson et al., 2005). These mutants, referred to as atg (e.g., 100

atg2 and atg5), exhibit accelerated senescence under normal conditions, but complete the 101

normal life cycle and form seeds for the next generation. 102

The physiological significance of autophagy in plants has been gradually 103

clarified by analyses of atg mutants. For example, it has become clear that autophagy 104

suppresses salicylic acid (SA) signaling. When NahG, a SA hydroxylase, is 105

overexpressed in an atg5 plant, the level of endogenous SA is reduced and SA signaling is 106

inhibited, resulting in suppression of senescence and immunity-related programmed cell 107

death (PCD). Additionally, a knockout (KO) mutant of SALICYLIC ACID INDUCTION 108

DEFICIENT 2 (SID2), a gene for a SA biosynthetic enzyme, suppresses senescence and 109



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PCD in the atg2 mutant. These data suggest that excessive SA signaling causes 110

accelerated PCD during senescence and immunity in atg mutants (Yoshimoto et al., 111

2009). 112

Nitrogen or C starvation induces autophagy in Arabidopsis (Thompson et al., 113

2005; Merkulova et al., 2014; Izumi et al., 2010), as in yeast and mammals. However, the 114

relationship between autophagy and deficiencies in many other essential elements 115

remains poorly understood, especially in plants. In yeast, Zn starvation induces 116

autophagy and plays important roles in adaptation to Zn deficiency. The transcription 117

factor Zap1, the master regulator of the Zn deficiency response in yeast, does not directly 118

control Zn deficiency–induced autophagy. Zn is thought to be supplied by bulk 119

degradation of cytoplasm via non-selective autophagy (Kawamata et al., 2017). The 120

association between autophagy and Zn has also been examined in cultured mammalian 121

cells (Liuzzi et al., 2014). These studies have revealed, for example, that 122

N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn-specific chelating 123

agent that can cross biological membranes, inhibits autophagy, whereas the addition of 124

Zn promotes autophagy (Liuzzi and Yoo, 2013). 125

In this study, we sought to characterize the relationship between Zn deficiency 126

and autophagy in plants. For this purpose, we established an experimental system for 127

inducing micronutrient deficiency in hydroponic culture, and grew atg mutants under 128

these conditions. Using various cell biological, biochemical, histochemical, and 129

physiological methods, we analyzed the phenotypes of atg mutants under Zn starvation. 130

Our results indicated that Zn deficiency–induced autophagy improves Zn bioavailability 131

in plants, as it does in yeast. In addition, we found that autophagy suppresses the 132

symptoms of Zn deficiency in a photosynthesis-dependent manner. Our findings 133



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regarding the mechanism by which plants respond to Zn deficiency should contribute to 134

methods aimed at preventing Zn starvation in crops and help to solve important 135

agricultural problems. 136

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RESULTS 138

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atg mutants exhibit accelerated Zn deficiency symptoms 140

To evaluate the relationship between Zn deficiency and autophagy in plants, we grew 141

wild-type Arabidopsis Columbia (Col) and two autophagy-defective mutants, atg2 and 142

atg5, under Zn deficiency (−Zn) conditions. Col was used as a control for normal 143

autophagic activity. First, we sowed seeds on solid media and grew the seedlings for 14 144

days (d). In the presence of sufficient Zn (+Zn), the atg mutants grew as rapidly as Col 145

(Fig. 1A, lower panels). On the other hand, as shown in the upper panels of Fig. 1A, the 146

atg mutants exhibited severe symptoms under −Zn: Chlorophyll contents and main root 147

length were significantly lower in the atg mutants than in Col (Fig. 1B), and the levels of 148

chloroplastic proteins, such as D1 protein of photosystem II (PSII) (PsbA) and Rubisco, 149

were much lower in the atg5 mutant than in Col 10 d after sowing on −Zn media (Fig. 150

1C). These results suggested that autophagy has an important function in adaptation to Zn 151

starvation. 152

However, these phenotypes could be explained by lower Zn contents in atg 153

mutant seeds. If autophagy is involved in remobilization of Zn ions from leaves to seeds, 154

the level of Zn could have been lower in the seeds of atg mutants than in Col seeds, 155

leading to a growth defect on media lacking Zn. In order to exclude this possibility, we 156

next conducted hydroponic transplant experiments. Plants were first grown in +Zn 157

hydroponic media for 21 d. After the roots were washed well in −Zn media, the plants 158

were transferred to −Zn and then grown for another 32 d. The results revealed that atg 159

mutants had Zn-deficiency phenotypes even in transplant experiments. Under −Zn 160

conditions, the leaves of atg mutants started to yellow at 3 d after transplanting (DAT), 161



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and at 7 DAT, many of the mutant leaves were dead (Fig. 1D). We quantified the changes 162

in leaf phenotypes under −Zn conditions. The peak percentage of damaged (yellow) 163

leaves occurred at 12 DAT in Col vs. 5 DAT in the atg mutants (Fig. 1E upper panel). In 164

addition, the percentage of completely dead leaves was elevated earlier in the atg 165

mutants, and the rate of the increase was also more rapid (Fig. 1E lower panel). These 166

data suggested that a difference in seed Zn level is not responsible for the Zn deficiency–167

sensitive phenotype of atg mutants. Indeed, neither seed weight (Fig. 1F) nor seed Zn 168

content (Fig. 1G) differed between atg mutants and Col. 169

170

Excessive SA signaling in atg mutant plants is not a main cause of Zn deficiency–171

induced chlorosis 172

SA is a phytohormone involved in defense against pathogens and senescence, and 173

autophagy negatively regulates SA signaling. We previously showed that atg mutants 174

exhibit accelerated PCD during senescence and immunity. When NahG is overexpressed, 175

leading to suppression of SA signaling, the early PCD phenotype in atg mutants is 176

suppressed (Yoshimoto et al., 2009). To determine whether excessive SA signaling is the 177

cause of Zn deficiency–induced chlorosis in atg mutants, we compared NahG and NahG 178

atg5 plants. In hydroponic transplant experiments, NahG atg5 plants exhibited more 179

severe Zn-deficiency symptoms (chlorosis) than NahG plants. In NahG atg5 plants, 180

chlorosis occurred at 5 DAT, and the progression of symptoms was also much faster than 181

in NahG plants (Fig. 2A and 2B). This suggests that the defect in autophagy is a major 182

factor in Zn deficiency–induced chlorosis, independent of SA signaling. Since the severe 183

chlorosis phenotype of atg mutants under −Zn causes difficulties for sampling enough 184



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materials, we used NahG plants as a control and NahG atg5 plants as an autophagy 185

defective plant for further experiments. 186

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Zn starvation induces bulk autophagy 188

Starvation of N or C induces autophagy in Arabidopsis. To determine whether Zn 189

starvation also induces autophagy in plants, we observed APs using plants expressing 190

GFP-fused AUTOPHAGY-RELATED PROTEIN 8a (GFP-ATG8a). ATG8 is a 191

lipid-conjugated protein localized to autophagy-related membrane structures such as the 192

AP (Kirisako et al., 1999; Kabeya et al., 2000; Yoshimoto et al., 2004). GFP-ATG8a–193

expressing plants grown on +Zn solid media for 7 d were transferred to −Zn or +Zn 194

media, and then their cotyledons and roots were observed by confocal laser scanning 195

microscopy. At 18 h after transplanting, plants under −Zn exhibited more dots labeled 196

with GFP-ATG8a in both cotyledon and root cells than plants under +Zn (Fig. 3A–C). 197

Temporal changes in the number of APs in roots under +/−Zn are shown in Supplemental 198

Fig. S1. 199

In order to further confirm the autophagy induction by Zn deficiency, we 200

checked autophagy flux using a V-ATPase inhibiter, concanamycin A (ConA). When 201

roots are treated with ConA, vacuolar acidification is inhibited, leading to accumulation 202

of ABs inside vacuolar lumens if autophagy is activated. Under −Zn, AB numbers were 203

significantly increased compared to under +Zn (Supplemental Fig. S2A and S2B). In 204

addition, we quantified autophagic degradation activity by a GFP-ATG8 processing 205

assay. In GFP-ATG8 plants, amounts of free GFP were increased inside the vacuole 206

along with the activation of autophagy. Therefore, the activity of autophagy can be 207

estimated by detecting the ratio of free GFP against GFP-ATG8 by western blot analysis 208



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using anti-GFP antibody. As expected, under −Zn, the GFP/GFP-ATG8 ratio was 209

increased compared to +Zn conditions (Supplemental Fig. S3). These data indicate that 210

autophagy activity was upregulated by Zn starvation. 211

Furthermore, we checked autophagy activity in roots using a split-root assay. 212

After splitting a root of a GFP-ATG8 plant, two roots were transplanted to a +Zn and −Zn 213

separated media plate (−Zn/+Zn plate) as shown in Supplemental Fig. S4A and incubated 214

for 18 h under continuous light conditions. Then, we quantified the number of APs using 215

a confocal microscope. The result showed that the −Zn side root had significantly higher 216

autophagy activity than that of the control (+Zn/+Zn plate). On the other hand, the +Zn 217

side of the −Zn/+Zn plate did not show an increase of APs (Supplemental Fig. S4B), 218

implying that autophagy is induced locally at the Zn deficient site, such as at organ or 219

tissue levels. 220

In yeast, it has been suggested that induction of autophagy by Zn deficiency is 221

not a selective process (Kawamata et al., 2017). To investigate the degradation targets of 222

plant autophagy under −Zn, we conducted western blot analyses. Using antibodies 223

against representative organelle marker proteins, we compared the amount of each 224

organelle protein in NahG and NahG atg5 leaves under −Zn and +Zn at 3 and 5 DAT. We 225

reasoned that if −Zn conditions induced selective autophagy, specific organelles fated for 226

degradation would accumulate to higher levels in NahG atg5 than in NahG plants under 227

−Zn. ADP-ribosylation factor 1 (ARF1), cytochrome oxidase subunit II (COXII), 228

catalase (CAT), sterol methyltransferase 1 (SMT1), and PsbA were used as markers for 229

Golgi apparatus, mitochondrion, peroxisome, endoplasmic reticulum (ER), and 230

chloroplast, respectively. Ribosomes are complexes of proteins and RNAs, and several 231

ribosomal proteins, such as RPL37, are Zn-binding proteins (Klinge et al., 2011). In 232



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addition, because ribosomes can be degraded by ribophagy, a form of selective autophagy 233

(Kraft et al., 2008; Floyd et al., 2015; Bassham and MacIntosh, 2017), we performed 234

western blot using antibodies against 40S ribosomal protein S14-1 (RPS14) and 60S 235

ribosomal protein L13-1 (RPL13), along with the other organelle markers. Under +Zn, 236

the levels of each protein were almost equal in NahG atg5 and NahG plants, with the 237

exception of CAT. Higher accumulation of CAT is consistent with our previous report 238

that peroxisomes constantly accumulate in atg mutant leaves (Yoshimoto et al., 2014). 239

However, under −Zn, many organelle markers accumulated to higher levels in NahG atg5 240

than in NahG plants (Fig. 3D). RPS14, RPL13, ARF1, COXII, and CAT were 241

significantly more abundant in NahG atg5 plants, especially at 5 DAT. Even at 3 DAT, 242

RPS14, ARF1, and CAT were present in NahG atg5 plants at higher levels than in the 243

control. These data suggest that autophagy degrades various targets (proteins and 244

organelles) as Zn starvation progresses. 245

246

Oxidative stress mediated by light and Fe is a cause of Zn deficiency–induced 247

chlorosis in atg mutant plants 248

The severity of Zn deficiency symptoms in plants depends on light intensity (Cakmak, 249

2000). As shown in Fig. 2A and 2B, atg5 and NahG atg5 plants developed –Zn-induced 250

chlorosis at 3 and 5 DAT, respectively, under normal light intensity. In order to 251

investigate the influence of light intensity on the onset of Zn-deficiency symptoms in atg 252

mutant plants, we performed the same transplant experiment under low light intensity. 253

For this experiment, plants were grown under normal light intensity for 21 d in +Zn, and 254

then transferred to low light conditions simultaneously with transplantation to −Zn or 255

+Zn media. Zn deficiency–induced chlorosis in the atg mutant plants was observed later 256



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under low light intensity than under normal light (Fig. 4A): atg5 mutants developed 257

−Zn-induced chlorosis at 7 DAT, and NahG atg5 plants did not exhibit chlorosis even at 7 258

DAT. These data indicate that severe Zn deficiency symptoms in atg5 mutants were 259

induced in a light-dependent manner, as in crops (Cakmak, 2000). 260

Because the onset of –Zn-induced chlorosis was dependent on light intensity, we 261

next investigated the mechanism underlying light-dependent chlorosis by comparing 262

oxidized protein levels in leaves. To detect oxidized proteins, we performed anti–263

2,4-dinitrophenol (DNP) western blot. When extracted proteins are treated with 264

2,4-dinitrophenylhydrazine (DNPH), DNP is added to their carbonylation sites, and 265

DNP-modified proteins can be detected by western blot with anti-DNP antibody 266

(Romero-Puertas et al., 2002). Because ATG-knockdown plants accumulate oxidized 267

proteins at a certain level even under normal conditions (Xiong et al., 2007), we 268

anticipated that it would be difficult to detect differences in oxidized protein levels 269

between +Zn and −Zn conditions. Therefore, for these experiments, we used NahG and 270

NahG atg5 plants because inhibition of SA signaling in atg mutants by NahG decreases 271

oxidative stress and prolongs life (Yoshimoto et al., 2009). Under normal light intensity, 272

Zn starvation induced the accumulation of higher levels of oxidized proteins in NahG 273

atg5 plants compared to NahG plants, but this difference was not observed under +Zn 274

(Fig. 4B). As expected, under low light intensity, the oxidized protein levels in NahG 275

atg5 plants were not high even under −Zn; this was almost the same accumulation pattern 276

observed in the control (+Zn and low light). These data indicate that the appearance of 277

Zn-deficiency symptoms depends on oxidative stress levels. 278

Next, because Fe contents are elevated in aerial tissues under Zn deficiency 279

(Cakmak, 2000), we focused on the effect of Fe under −Zn. To investigate the 280



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relationships between Zn-deficiency phenotypes in atg mutant plants and Fe nutrition, we 281

performed transplant experiments as described above, using media that were Zn-deficient 282

and Fe-limited. In media containing 1% of the normal level of iron (Fe/100), Zn 283

starvation–induced chlorosis in NahG atg5 plants was suppressed (Fig. 4C). The 284

phenotype in NahG atg5 plants in -Zn Fe/100 was similar to that in NahG plants, and Zn 285

starvation–induced chlorosis was not apparent. On Zn-deficient and Fe-sufficient media 286

(−Zn +Fe), NahG atg5 plants exhibited intense chlorosis throughout all leaves at 10 DAT. 287

Additionally, NahG atg5 plants did not exhibit an obviously different phenotype 288

compared to NahG plants under Fe starvation alone (+Zn Fe/100) (Fig. 4C). These data 289

suggest that Fe is an important determinant of Zn deficiency–induced chlorosis in atg 290

mutant plants, and that autophagy does not affect Fe starvation resistance in plants. 291

292

ROS production and homeostasis are not controlled in atg mutant plants under Zn 293

starvation 294

Because oxidative stress is enhanced under Zn deficiency, we next sought to determine 295

the levels of reactive oxygen species (ROS) in atg mutant plants under −Zn. First, we 296

detected hydroxyl radicals (•OH) in leaves by fluorescence of 3’-(p-hydroxyphenyl) 297

fluorescein (HPF). HPF produces fluorescein when it is oxidized by ROS, especially by 298

•OH, with quite high specificity. At 3 DAT in NahG atg5 plants under −Zn, we detected 299

fluorescein fluorescence co-localized with autofluorescence of chlorophyll (Fig. 5A). In 300

NahG plants or in NahG atg5 plants under +Zn, no such fluorescence was detected. 301

Additionally, there was no fluorescence in leaves treated with N,N-dimethylformamide 302

(DMF), which served as a control for the HPF solvent. Quantification of fluorescence 303



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intensity clearly showed that •OH levels were significantly elevated in NahG atg5 plants 304

under −Zn (Fig. 5B). 305

In atg5 mutants under Zn starvation, we observed marked elongation of 306

stromules (stroma-filled tubules) (Supplemental Fig. S5A and S5B). Stromules elongate 307

under various environmental stresses (Brunkard et al., 2015), suggesting that the 308

chloroplasts in atg mutant plants under −Zn were experiencing stress. This result suggests 309

that chloroplasts are responsible for stress development in the atg mutants under −Zn. 310

Next, we detected two other ROS species, superoxide (•O2−) and hydrogen 311

peroxide (H2O2), using nitro blue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) 312

staining, respectively (Doke, 1983; Thordal-Christensen et al., 1997). •O2− accumulated 313

at higher levels in NahG atg5 than in NahG plants under +Zn, whereas under −Zn, the 314

level of •O2− was dramatically lower in NahG atg5 plants (Fig. 5C). NahG atg5 plants 315

contained slightly higher levels of H2O2 than NahG plants (Fig. 5D), consistent with our 316

previous report (Yoshimoto et al., 2009). These data suggest that changes in ROS 317

production, specifically reduced •O2− and elevated •OH, are responsible for the more 318

severe Zn-deficiency symptoms in atg mutant plants. 319

320

Autophagy maintains Zn pools under Zn deficiency 321

Finally, using the Zin-Pro Capture reagent (Miki et al., 2016), we investigated how 322

autophagy affects the level of free Zn ions in individual plants. When bound to mobile Zn 323

ions, Zin-Pro Capture can react with proteins and bind fluorescein (Supplemental Fig. 324

S6). We extracted proteins from plants infiltrated with Zin-Pro Capture, subjected them 325

to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and 326

observed the resultant gels on a fluorescence scanner. The levels of mobile Zn ions in 327



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NahG and NahG atg5 plants were estimated based on the levels of fluorescently labeled 328

endogenous proteins. As shown in Fig. 6A, the level of fluorescently labeled proteins was 329

lower in NahG atg5 than in NahG plants, indicating that the level of mobile Zn ions inside 330

plants was increased by autophagy under −Zn conditions. 331

Next, we investigated the activity of SODs, as representative Zn-requiring 332

metalloenzymes. Plants have Cu/ZnSODs that require both Zn and Cu, as well as 333

FeSODs and MnSODs that require Fe and Mn, respectively, for their enzyme activities 334

and/or maintenance of enzyme structures. The activities of Cu/ZnSODs and other SODs 335

were measured separately using potassium cyanide (KCN), which is an inhibitor of 336

Cu/ZnSOD. Cu/ZnSOD activities were calculated by subtraction of MnSOD and FeSOD 337

activities, which were measured in reaction solutions with KCN, from total SOD 338

activities. Under +Zn, the SOD activity in NahG atg5 plants was similar to that in NahG 339

plants. By contrast, under −Zn, the activity of Cu/ZnSOD was dramatically lower in 340

NahG atg5 than in NahG plants (Fig. 6B). Under −Zn, the abundance of Cu/ZnSOD 341

proteins was rather higher in NahG atg5 than in NahG plants (Supplemental Fig. S7), 342

indicating that a change in protein level was not the cause of the reduction in Cu/ZnSOD 343

activity. These results indicate that an increase in Zn bioavailability, mediated by 344

autophagy, helps maintain the activity of Zn-requiring enzymes under Zn deficiency. 345



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DISCUSSION 346

347

Zn remobilization and autophagy 348

Autophagy is involved in N remobilization in Arabidopsis (Guiboileau et al., 2012), rice 349

(Oryza sativa) (Wada et al., 2015), and maize (Zea mays) (Li et al., 2015). In addition, the 350

efficiency of Fe remobilization from leaves to seeds is reduced in Arabidopsis atg5 351

mutants (Pottier et al., 2019). Those facts led us to hypothesize that autophagy also 352

contributes to Zn remobilization to seeds, as suggested previously (Pottier et al., 2019). 353

That study reported that the Zn content in seed normalized against the Zn content in the 354

whole plant is lower in atg5 mutants than in Col. By contrast, our data showed that seed 355

weight and Zn content did not differ among atg2, atg5, and Col plants (Fig. 1F and 1G). 356

The discrepancy can be explained by the fact that the number of seeds per individual plant 357

is smaller in atg mutants than in Col, in addition to that the Zn content of leaves and stems 358

is higher in atg mutants than in Col (Pottier et al., 2019). 359

360

Relationships between SA and Zn starvation–induced chlorosis 361

Our biochemical and histochemical analyses revealed that oxidative stress is enhanced by 362

the increase in ROS under Zn deficiency (Fig. 4B and Fig. 5A–D). SA induces ROS 363

production, and ROS activates SID2, a SA synthetase, through ENHANCED DISEASE 364

SUSCEPTIBILITY 1, thereby promoting SA biosynthesis during senescence and 365

pathogen-induced PCD (Yoshimoto et al., 2009; Hofius et al., 2009). This SA-ROS 366

amplification loop may function even under −Zn conditions. Indeed, as shown in Fig. 2A 367

and 2B, overexpression of NahG is effective in suppressing –Zn-induced chlorosis. The 368

symptoms of NahG atg5 plants were less severe than those of the atg5 single mutant: 369



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Chlorosis was observed starting at 3 DAT in atg5 mutants, but at 5 DAT in NahG atg5 370

plants (Fig. 2A and 2B). This implies that the expression of NahG itself plays a role in 371

suppressing chlorosis, consistent with the observation that NahG plants developed 372

chlorosis later than Col plants. Zn deficiency increases ROS levels, and SA further 373

enhances this effect. Although autophagy functions as a suppressor of SA signaling, 374

considering that there was a marked difference in –Zn-induced chlorosis between NahG 375

and NahG atg5 plants, we concluded that SA is not the main cause of Zn-deficiency 376

symptoms in atg mutant plants. When we measured SA contents in leaves and compared 377

them among Col, atg5, NahG, and NahG atg5 plants under −/+Zn conditions, the SA 378

contents of NahG atg5 plants under −Zn was much lower than those of Col under −Zn 379

and even under +Zn (Supplemental Fig. S8), supporting our conclusion that excessive SA 380

signaling is not a main cause of Zn deficiency symptoms in the atg mutant. 381

382

Autophagic defects cause damage to chloroplasts under Zn starvation 383

In plants, Zn-deficiency symptoms manifest primarily as chlorosis, i.e., a reduction in the 384

level of chlorophyll. This chlorosis was further enhanced in the atg mutants under −Zn, 385

suggesting that chloroplasts are damaged and become less abundant in atg mutants (Fig. 386

1A–C). Additionally, the levels of SMT1 and PsbA were decreased in NahG atg5 plants 387

under −Zn (Fig. 3D). There are two possible reasons why these proteins might be less 388

abundant in NahG atg5 plants. First, because Zn is important for transcriptional and 389

translational activities, the expression of these proteins might decrease under Zn 390

deficiency. Second, Zn deficiency might cause these proteins to be damaged so that they 391

are broken down by non-autophagic pathways or cannot react with the antibodies we 392

used. The second possibility is consistent with the data in Fig. 1D, which show that 393



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chloroplasts were damaged and the levels of chloroplastic proteins were reduced in atg5 394

mutants under −Zn. The localization of •OH in chloroplasts (Fig. 5A) is consistent with 395

the idea that chloroplasts are the main onset place of Zn deficiency symptoms. 396

As shown in Supplemental Fig. S5A and S5B, stromules significantly elongated 397

under −Zn in atg5 plants. There are two possible causes for this phenotype. First, the 398

chloroplasts could be experiencing stress. Consistent with this, stromule elongation is 399

induced by various environmental stresses (Brunkard et al., 2015). Alternatively, partial 400

degradation of chloroplasts may be promoted by Zn starvation. Rubisco-containing 401

bodies (RCBs), small chloroplast-derived vesicles containing stromal proteins, have been 402

proposed to be pinched off from stromules through sequestration by isolation membranes 403

(Ishida et al., 2008); consequently, defects in autophagy promotes elongation of 404

stromules. In other words, stromule elongations in atg5 mutants under −Zn mean that the 405

RCB degradation pathway is activated under Zn starvation. To investigate whether such 406

partial chloroplast degradation is induced under Zn deficiency, we examined RCB 407

accumulation inside vacuoles after treating plants expressing stroma-targeted GFP with 408

ConA. Because ConA inhibits vacuolar acidification, RCB-derived vesicles (ABs) can be 409

detected inside vacuolar lumens if autophagy is activated. Under dark, long-day, and 410

sucrose-added conditions, we detected no difference in accumulation of RCB-derived 411

vesicles, irrespective of the presence or absence of Zn (Supplemental Fig. S9). These 412

results suggest that chloroplast degradation by the RCB pathway is not enhanced under 413

Zn starvation, in contrast to C starvation (Izumi et al., 2010). Based on these 414

observations, we conclude that chloroplasts in the atg mutant plants are exposed to 415

intense stress under Zn deficiency. 416



20

Zn deficiency–induced chlorosis in the atg mutant plants was suppressed under 417

low light conditions (Fig. 4A). Additionally, oxidative stress in atg mutant plants was 418

enhanced under Zn starvation in a light intensity–dependent manner (Fig. 4B). These 419

results led us to hypothesize that Zn deficiency symptoms in atg mutant plants were 420

caused by light-dependent ROS production, which is largely a consequence of 421

photosynthesis and photorespiration. To determine whether photorespiration is 422

responsible for ROS production under Zn deficiency, we grew plants under high CO2, 423

which suppresses photorespiration (Reumann and Weber, 2006). We reasoned that if 424

photorespiration is the source of ROS under Zn starvation, chlorosis should be suppressed 425

under high CO2. However, when NahG and NahG atg5 plants were grown in +Zn media 426

under ambient CO2 conditions (400 ppm) for 21 d, and then transferred to −Zn media 427

under high CO2 conditions (3000 ppm), Zn deficiency symptoms were still observed in 428

NahG atg5 plants (Supplemental Fig. S10). Indeed, the chlorosis in NahG atg5 plants was 429

slightly accelerated relative to that observed in plants of the same genotype grown in 430

ambient CO2 (Fig. 2A). These data suggest that the main cause of Zn deficiency–induced 431

chlorosis is photosynthesis-derived ROS. This is consistent with our observation that 432

chloroplasts were significantly damaged (Fig. 1B left and 1C) and that •OH accumulated 433

exclusively in chloroplasts (Fig. 5A). 434

435

Bulk autophagy resupplies Zn in plants under Zn starvation 436

Here, we propose a model of how autophagy helps plants tolerate Zn deficiency (Fig. 7). 437

When plants sense Zn deficiency, they induce autophagy. This −Zn induced autophagy 438

seems to be bulk autophagy. Western blot analysis using antibodies against organelle 439

marker proteins (Fig. 3D) revealed that multiple organelles and proteins are targets for 440



21

autophagic degradation under Zn starvation. In yeast, bulk autophagy is induced under Zn 441

deficiency (Kawamata et al., 2017); therefore, it is reasonable that the Zn starvation–442

induced autophagy in plants is also bulk autophagy. In contrast to yeasts, however, plants 443

are multicellular organisms, and may have different mechanisms for achieving selectivity 444

in a tissue- or organ-specific manner. Therefore, future studies should investigate whether 445

selective autophagic degradation occurs under Zn deficiency. 446

Zn-binding proteins or Zn-including organelles enclosed by the autophagosomes 447

are transported to the vacuole to be degraded to release Zn ions. Since more than 5% of 448

proteins bind Zn, the degradation of these intracellular components increases the level of 449

free Zn ion (Fig. 6A), thereby alleviating Zn deficiency. In other words, bulk autophagy 450

promotes Zn bioavailability under Zn starvation. 451

Zn supplied by autophagy is redistributed where it is needed. Cu/ZnSOD, a 452

representative Zn-requiring enzyme, plays an important role in oxidative stress 453

responses. Under Zn deficiency, despite the high protein levels of Cu/ZnSOD in the atg 454

mutant plants (Supplemental Fig. S7), the activity of Cu/ZnSOD was lower in atg mutant 455

plants than in control plants (Fig. 6B), suggesting that the resupply of Zn to Zn enzymes 456

by autophagy is important for resistance to Zn starvation. 457

458

Zn deficiency symptoms are due to a Fenton-like reaction in atg mutant plants 459

Due to their reduced efficiency of Zn utilization, atg mutants exhibit more severe Zn 460

deficiency symptoms (Fig. 1A–E). Cakmak (2000) predicted that Fe-mediated •OH 461

formation reactions are responsible for the symptoms of Zn deficiency. We speculated 462

that this reaction was facilitated by a defect in autophagy. The upper part of Fig. 7 shows 463

the possible reactions in the chloroplast that could produce ROS responsible for Zn 464



22

starvation–induced chlorosis in an Fe- and light intensity–dependent manner. Under −Zn, 465

Fe ions promote the Fenton-like reaction that generates •OH from H2O2. In this reaction, 466

Fe2+ is oxidized to Fe3+. As shown in Fig. 5A, •OH is produced in chloroplasts. On the 467

other hand, Fe3+ is reduced to Fe2+ by oxidation of •O2− to oxygen. The marked reduction 468

in the level of •O2− in the atg mutant plants under −Zn (Fig. 5C) supports this model. The 469

suppression of chlorosis by Fe limitation under −Zn (Fig. 4C) suggests that the main 470

cause of Zn deficiency symptoms is •OH production in chloroplasts via this loop 471

involving Fe2+ and Fe3+. Furthermore, the suppression of chlorosis by reduced light 472

intensity (Fig. 4A) suggests that this reaction occurs during photosynthesis. Therefore, 473

we propose that Zn deficiency–induced chlorosis is caused by chloroplast damage due to 474

ROS production via this photosynthesis-dependent Fenton-like reaction. 475

476



23

CONCLUSIONS 477

In this study, we developed a hydroponic system that can precisely control nutrient 478

conditions. Since sucrose was not added in the media in this system, plants 479

photosynthesize like they do in normal field conditions, allowing us to analyze only the 480

effects of Zn deficiency on phenotypes under more natural conditions. Additionally, the 481

use of NahG atg5 plants allowed us to analyze true Zn deficiency phenotypes excluding 482

the effects of SA-dependent senescence and constitutive ROS accumulation in atg mutant 483

plants. Thus, we clearly demonstrated that intracellular self-degradation by plant 484

autophagy resupplies Zn ions from proteins and organelles to increase usable Zn under Zn 485

deficiency. Furthermore, here we also demonstrated the importance of the relationship 486

between Zn and Fe. Namely, the main cause of Zn starvation–induced chlorosis is •OH 487

production mediated by Fe (Fenton-like reaction), which occurs in chloroplasts in a 488

light-dependent manner. This was clearly demonstrated experimentally using 489

autophagy-defective plants which show reduced Zn bioavailability. Our current findings 490

would shed light on further mechanisms for plants to grow robustly under unfavorable 491

soil environments. 492

493



24

METHODS 494

495

Plant materials and growth conditions 496

Arabidopsis thaliana ecotype Columbia-0 (Col) was used in this study. Details of atg2 497

(SALK_076727), atg5 (SAIL_129_B07), NahG, and NahG atg5 plants were described in 498

our previous report (Yoshimoto et al., 2009). Plants were grown under long-day 499

conditions (16-h-light/8-h-dark) at 22°C. Light intensity was 60 and 17 mol m-2 s-1 in 500

normal and low-light conditions, respectively. For high CO2 conditions, plants were 501

grown under 3000 ppm CO2 (LPH-411SPC; NK System, Osaka, Japan). Seeds were 502

surface-sterilized, chilled at 4°C for 3 d in the dark, and then sown on solid media (for 503

plate cultures) or rockwool (for hydroponic cultures). Plant growth media were based on 504

MGRL media (Fujiwara et al., 1992). Media for Zn starvation were prepared without 505

ZnSO4. Media for Fe limitation were prepared by reducing [FeSO4] to 0.086 M and 506

[Na2-EDTA] to 0.67 M. Solid media plates were prepared by addition of 0.7% (w/v) 507

agarose (A9539; Sigma-Aldrich, St. Louis, MO, USA) and 1.0% (w/v) sucrose to MGRL 508

liquid media. In hydroponic transplant experiments, the roots of plants grown for 21 d on 509

+Zn media with aeration were well rinsed with −Zn media, and then transferred to −Zn 510

media; images were acquired every day after transplantation. Two liters of liquid media 511

were used per 12 individual plants. Hydroponic media were changed weekly. In the 512

split-root assay, plants were vertically grown for 4 days under continuous light conditions 513

on a +Zn plate gelled by 0.4% (w/v) gelrite (075-05655; Fujifilm Wako Pure Chemical, 514

Tokyo, Japan), then the main root was cut under the hypocotyl. Five days after cutting the 515

root, split roots were transplanted to −Zn/+Zn separated solid media gelled with agarose. 516

517



25

Measurements of chlorophyll contents 518

The fresh weight of shoots of plants grown for 14 d on agarose plates was measured. The 519

shoots were immersed in N,N-dimethylformamide (DMF) and placed at 4°C overnight in 520

the dark. The absorbance of the resultant DMF solutions was measured at 664, 647, and 521

750 nm on a spectrophotometer (DS-11+; DeNovix, Wilmington, DE, USA) using glass 522

cells (596492; Beckman Coulter, Brea, CA, USA), and the total amount of chlorophyll a 523

and chlorophyll b was calculated by the equation of Porra (Porra et al., 1989). Finally, the 524

amount of total chlorophyll per fresh weight (chlorophyll concentration) was calculated. 525

526

Measurements of metal contents 527

Contents of Zn in seeds were measured by inductively coupled plasma mass spectrometry 528

(ICP-MS). Five hundred seeds were counted and weighed after drying in a desiccator. 529

Sample processing and metal measurements were performed as described previously 530

(Horie et al., 2017). 531

532

Measurements of SA contents 533

Freeze dried samples (2-20 mg) were placed in 2 mL tubes and ground to powder with 3 534

mm zirconia beads using a Tissue Lyser (Qiagen, Hilden, Germany). The samples were 535

extracted with 1 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic acid and 5 ng 536

D6-SA (Isotec, Miamisburg, OH, USA) for 2 hours at 4°C in the dark. After 537

centrifugation of the samples at 15000 g for 5 min, supernatants were transferred to fresh 538

tubes and precipitates were extracted again with 1 mL 80% (v/v) acetonitrile containing 539

1% acetic acid (v/v) for 2 hours at 4°C in the dark. After centrifugation of the samples at 540

15000 g for 5 min, supernatants were combined with the first extracts and dried under a 541



26

stream of N2 gas. The dried extracts were dissolved in 1 mL water containing 1% (v/v) 542

acetic acid and then applied to Oasis WAX column (1 cc) (Waters, Milford, MA, USA), 543

which had been pre-treated with 1 mL acetonitrile, 1 mL KOH (0.1 N) and 1 mL water 544

containing 1% (v/v) acetic acid. The columns were washed with 1 mL water containing 545

1% (v/v) acetic acid and then with 1 mL 80% (v/v) acetonitrile containing 1% (v/v) acetic 546

acid. Fractions containing SA were eluted with 2 mL 80% (v/v) acetonitrile containing 547

5% (v/v) formic acid and dried under a stream of N2 gas. The dried samples were 548

dissolved in 50 l of water containing 1% (v/v) acetic acid and analyzed with liquid 549

chromatography tandem-mass spectrometry as described previously (Kanno et al., 2016). 550

551

Immunoblot analysis 552

Total protein lysates were prepared as described previously (Yoshimoto et al., 2004). 553

Western blot using anti-PsbA, -ARF1, -COXII, -CAT, and -SMT1 antibodies was 554

performed as described previously (Yoshimoto et al., 2014). Anti-RPS14 (AS12 2111), 555

anti-RPL13 (AS13 2650), and anti-Cu/ZnSOD2 (CSD2) (AS06 170) were acquired from 556

Agrisera (Vännäs, Sweden). Anti-GFP antibody (3H9-100) was acquired from 557

Chromotek (Munich, Germany). The secondary antibodies were horseradish peroxidase–558

conjugated goat anti–rabbit IgG antibody (111-035-003; Jackson ImmunoResearch, West 559

Grove, PA, USA) and anti–rat IgG antibody (112-035-003; Jackson ImmunoResearch). 560

Protein extraction, DNPH treatment, and anti-DNP western blot for detection of oxidized 561

protein were performed using the Oxidized Protein Western Blot detection kit (ab178020; 562

Abcam, Cambridge, UK). Blots were visualized by chemiluminescence using ECL 563

substrate (1705062, clarity max western ECL substrate; Bio-Rad, Hercules, CA, USA) 564

and recorded using a CCD imager (LAS-4000; Fujifilm, Tokyo, Japan). 565



27

566

Fluorescence microscopy 567

GFP fusion proteins, autofluorescence of chlorophyll, and fluorescein were visualized 568

using a confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan). Details 569

of GFP-ATG8a and stroma-targeted GFP expressed plants were described previously 570

(Yoshimoto et al., 2004; Izumi et al., 2010). To observe RCB accumulation, 10 mM 571

MES-KOH (pH 5.7) containing 1 M concanamycin A and 100 M E-64d were 572

infiltrated into leaves of 3 DAT plants with a syringe and incubated for 20 h at 22°C. In 573

sucrose-added conditions, excised leaves were incubated in MGRL media containing 3% 574

(w/v) sucrose. For HPF treatments, a 5 mM stock solution of HPF (H36004; Molecular 575

probes, Eugene, OR, USA) was diluted in 10 mM MES-KOH (pH 5.7) to prepare a 10 576

M working solution. The solution was infiltrated into leaves with a syringe and 577

incubated for 1.5 h in a growth chamber. The fluorescence intensities of HPF were 578

quantified using ImageJ (NIH; Schneider et al., 2012). 579

580

Histochemical staining 581

For NBT staining, detached leaves from 3 DAT plants were submerged in 10 mM KPO4 582

(K2HPO4 and KH2PO4) buffer (pH 7.8) containing 10 mM NaN3, and then vacuum 583

infiltrated 3 times at 0.09 MPa for 20 sec. Next, the buffer was removed, 0.1% (w/v) NBT 584

(24720-01; Nacalai Tesque, Kyoto, Japan) in 10 mM KPO4 buffer was added, and the 585

leaves were incubated at room temperature for 30 min. For DAB staining, DAB 586

(046-26861; Fujifilm Wako Pure Chemical, Tokyo, Japan) was added to water at 1 587

mg/mL and heated to 60°C, with addition of HCl to promote dissolution. After 588

dissolution, NaOH was added to adjust the pH of the DAB solution, which was then 589



28

cooled on ice. Detached leaves were vacuum infiltrated with DAB solutions. Then, the 590

solution was discarded, and the leaves were rinsed with tap water and incubated at room 591

temperature for 7 h in the dark. After NBT and DAB staining, decolorization of 592

chlorophyll was performed by boiling leaves with solutions containing 7% (v/v) lactate, 593

13% (v/v) glycerol, 7% (v/v) phenol, and 67% (v/v) ethanol for 2 min. Pictures were 594

taken with a stereomicroscope. 595

596

Detection of mobile Zn ions in plants by Zin-Pro Capture 597

A stock solution of Zin-Pro Capture (FDV-0013A; Funakoshi, Tokyo, Japan) was 598

prepared at 1 mM by dissolving in dimethyl sulfoxide (DMSO). The working solution 599

was 1 M in 3 mM MES-KOH (pH 7.0). The same amount of DMSO was used as a 600

control. The solution was vacuum-infiltrated into shoots of plants grown for 2 d under 601

−Zn, which were then incubated at 22°C in the dark for 24 h. The samples were frozen in 602

liquid nitrogen, homogenized in 50 mM Tris-HCl (pH 7.5), and then centrifuged at 21900 603

g for 5 min at 4°C to recover the supernatant. Protein concentration was measured using 604

the BCA protein assay kit (23227; Thermo Fisher Scientific, Waltham, MA, USA), and 605

equal quantities of proteins were subjected to SDS-PAGE. After electrophoresis, gels 606

were scanned at 488 nm on a fluorescence imager (Fluorimager 595; Molecular 607

Dynamics, Chicago, IL, USA). Subsequently, CBB-R250 staining was performed, and a 608

transmitted light image was acquired to confirm equal protein loading. 609

610

Measurements of SOD activities 611

Leaves of 5 DAT plants were frozen in liquid nitrogen and homogenized in 50 mM 612

Tris-HCl (pH 7.5) containing 2% (w/v) polyvinylpolypyrrolidone, and then centrifuged 613



29

at 14000 g for 10 min at 4°C. The supernatant was used for measurement of SOD 614

activities using the SOD Assay Kit-WST (S311; Dojindo, Kumamoto, Japan). KCN was 615

added to the samples at 10 mM to inhibit Cu/ZnSOD activities. After samples were 616

aliquoted into a 96-well plate and reagents were added, they were incubated at 22°C for 617

20 min, and then absorbance was measured at 450 nm on a microplate reader (Epoch 2; 618

BioTek, Winooski, VT, USA). The inhibition rate was calculated from absorbance and 619

used as an index of SOD activities (arbitrary unit). The inhibition rate is a measure of 620

SOD activity because SOD inhibits the color reaction facilitated by •O2−. 621

622

Statistical Analyses 623

Statistical analysis was performed with Microsoft Office Excel 2016 software. Student’s 624

t-test was used to compare samples as indicated in the figure legends. 625

626

Accession Numbers 627

Genetic information in this article can be obtained from the Arabidopsis Information 628

Resource (TAIR) under following accession numbers: ARF1, AT1G23490; ATG2, 629

AT3G19190; ATG5, AT3G51830; ATG8a, At4g21980; CAT, AT4G35090; COXII, 630

ATMG00160; CSD2, AT2G28190; PsbA, ATCG00020; RPL13, AT3G49010; RPS14, 631

AT2G36160; SMT1, AT5G13710. 632



30

SUPPLEMENTAL DATA 633

The following supplemental information is available. 634

Supplemental Figure S1. Temporal changes in the number of APs under Zn starvation. 635

Supplemental Figure S2. Accumulation of ABs under Zn starvation. 636

Supplemental Figure S3. Increase of autophagic degradation activity under Zn 637

starvation. 638

Supplemental Figure S4. Autophagy is induced in the −Zn side but not in the +Zn side 639

in the split-root assay. 640

Supplemental Figure S5. Marked elongation of stromules in atg5 mutants under Zn 641

starvation. 642

Supplemental Figure S6. Visualization of mobile Zn ion contents in plants by Zin-Pro 643

Capture. 644

Supplemental Figure S7. Cu/ZnSOD protein levels in NahG and NahG atg5 leaves. 645

Supplemental Figure S8. SA levels of Col, atg5, NahG, and NahG atg5 leaves under 646

−/+Zn conditions. 647

Supplemental Figure S9. Zn deficiency does not affect RCB accumulation. 648

Supplemental Figure S10. Zn deficiency induces chlorosis even under high CO2 649

conditions. 650

651

ACKNOWLEDGMENTS 652

We thank all members of Yoshimoto Laboratory for providing a great deal of useful 653

advice for experiments, as well as valuable discussion. We also thank Suzukakedai 654

Materials Analysis Division, Technical Department, Tokyo Institute of Technology, for 655

ICP-MS analysis. This work was partly supported by Grant-in-Aid for Research Activity 656



31

Start-up [grant number 16H07255 to K.Y.], Grant-in-Aid for Scientific Research on 657

Innovative Areas (Research in a Proposed Research Area) [grant number 19H05713 to 658

K.Y.], Research Project Grant A by Institute of Science and Technology, Meiji 659

University, and the Program for the Strategic Research Foundation at Private 660

Universities, 2014-2018 of MEXT, Japan. L.N. and K.Y. were supported by the INRA 661

Package program (2012-2015). Additional funding was provided by the 662

ANR-12-ADAPT-001-01-AUTOADAPT program. 663

664

Figure Legends 665

Figure 1. Autophagy-defective plants exhibit accelerated Zn deficiency symptoms 666

(A) Phenotypes of Col, atg2, and atg5 plants on solid media, grown vertically for 14 d 667

after sowing under long-day conditions. −Zn: Zn deficiency (0 M); +Zn: normal media 668

containing a sufficient amount of Zn (1 M). Scale bars, 1 cm. (B) Values of −Zn/+Zn 669

ratios of chlorophyll concentration (left) and main root length (right) shown in (A). n = 3, 670

*: p < 0.05 Student’s t-test, error bars show standard deviation (SD). (C) Protein levels in 671

Col and atg5 plants under +Zn and −Zn conditions. PsbA was detected by western blot. 672

Total proteins were separated by SDS-PAGE and stained with Coomassie Brilliant Blue 673

(CBB). Approximate molecular mass (kDa) is displayed on the left. Equal amount of total 674

protein was loaded in each lane. (D) Phenotypes of Col, atg5, and atg2 plants in 675

hydroponic transplant experiments. Twenty-one days after sowing on +Zn media, plants 676

were transplanted to −Zn or +Zn media. Images were acquired at 0, 3, and 7 d after 677

transplanting (DAT). Scale bar, 2 cm. (E) Quantified data of (D). Percentages of 678

damaged (chlorotic) leaves (upper panel) and dead (completely dried) leaves (lower 679

panel) in a time course manner. n = 4. Error bars show SD. (F) Seed weight of Col, atg2, 680



32

and atg5 plants. Seeds were harvested from soil-grown plants and 500 seeds were 681

counted and weighted. Individual seed weight was calculated by dividing the total weight 682

by 500. n = 4. Error bars show SD. No significant difference was detected among the 683

three samples in Student’s t-test (p > 0.05) (N.S.). (G) Zn contents per 1 mg of seeds of 684

Col, atg2, and atg5 plants. Quantification was performed by inductively coupled plasma 685

mass spectrometry (ICP-MS). n = 4. Error bars show SD. No significant difference was 686

detected among the three samples in Student’s t-test (p > 0.05) (N.S.). 687

688

689

Figure 2. Phenotypes of NahG and NahG atg5 plants under Zn deficiency 690

(A) Phenotypes of Col, atg5, NahG, and NahG atg5 plants in hydroponic transplant 691

experiments. Twenty-one days after sowing on +Zn media, plants were transplanted to 692

−Zn media. Images were acquired at 0, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 DAT. Scale 693

bar, 2 cm. (B) Quantified data of (A). Temporal changes in the percentages of damaged 694

leaves and dead leaves were shown in the same manner as in Fig. 1E. n = 3. Error bars 695

show SD. 696

697

698

Figure 3. Zn starvation induces autophagic degradation of many targets in plants 699

(A) GFP-ATG8a–labeled dots indicate APs. Images of main root cells (left panels) and 700

cotyledon cells (right panels) were acquired at 18 h after transplanting to +/−Zn 701

conditions. Under +Zn, APs were barely observed (lower panels), but under −Zn, many 702

APs were observed in both roots and cotyledons (upper panels). Representative APs are 703

indicated by white arrowheads. Scale bars, 20 m. (B) and (C) Quantified data of (A). 704



33

Number of APs per image in roots (B) and cotyledons (C) at 18 h after transplant to +/−Zn 705

media. n = 4, **: p < 0.01 Student’s t-test, error bars show SD. (D) Various organelle 706

proteins were compared by western blot under +/−Zn. Proteins were extracted from 707

leaves of NahG and NahG atg5 plants at 3 and 5 DAT under +/−Zn. Antibodies used in 708

this experiment are indicated on the right. The lower panel shows a CBB-stained gel 709

confirming equal protein loading. Approximate molecular mass (kDa) is displayed on the 710

right. 711

712



34

713

Figure 4. Zn deficiency symptoms are promoted by light and Fe ions 714

(A) Phenotypes of Col, atg5, NahG, and NahG atg5 plants at 0, 3, and 7 DAT in 715

hydroponic transplant experiments under low light conditions (17 mol m-2 s-1). Plants 716

grown for 21 d on +Zn under normal light intensity were transplanted to −Zn or +Zn 717

media and grown under low light intensity. Scale bar, 2 cm. (B) Detection of oxidized 718

protein levels by western blot with anti-DNP antibody. Proteins were extracted from 719

leaves at 3 DAT under normal or low light intensity. Upper panel shows the result of 720

western blot. Lower panel shows a CBB stained gel (loading control). (C) Phenotypes of 721

NahG and NahG atg5 plants in hydroponic transplant experiments under Fe-limited 722

conditions. Plants grown for 21 d on +Zn media were transplanted to Zn-deficient, 723

Fe-limited (−Zn Fe/100), Zn-deficient, Fe-sufficient (−Zn +Fe), Zn-sufficient, Fe-limited 724

(+Zn Fe/100), or Zn-sufficient, Fe-sufficient (+Zn +Fe) media. Photographs were taken at 725

0, 3, and 10 DAT. Scale bar, 2 cm. 726

727

728

Figure 5. Changes of ROS under Zn starvation in autophagy-defective plants 729

(A) Detection of •OH was performed using HPF. Fluorescence of fluorescein, generated 730

from HPF by reaction with •OH, is shown in green; autofluorescence of chlorophylls is 731

shown in magenta. Merged images are shown in the right panels. Fluorescein 732

fluorescence was increased in NahG atg5 plants at 3 DAT (from +Zn to −Zn), and was 733

colocalized with chloroplasts. DMF, the solvent for HPF, was used as the negative 734

control. Scale bar, 50 m. (B) Quantified data from (A). Relative intensity of green 735

fluorescence was measured using ImageJ. Columns with the same letter are not 736



35

significantly different (p < 0.01, Student’s t-test). n = 4. Error bars show SD. (C) and (D) 737

Results of NBT (C) and DAB (D) staining of NahG and NahG atg5 leaves at 3 DAT 738

under −/+Zn. Left panels show whole leaves digitally extracted from the images taken by 739

stereo microscope and shown on a white background. Right panels show higher 740

magnifications of the left panels. For each magnification, the left column shows the lower 741

leaves, and the right column shows the upper leaves. Scale bars: 5 mm in left panels 742

showing whole leaves, and 1 mm in right panels. 743

744

745

Figure 6. Decrease of mobile Zn levels in atg mutant plants causes decay of 746

Zn-requiring enzyme activity 747

(A) Proteins were extracted from Zin-Pro Capture (ZPC)-treated plants (NahG and NahG 748

atg5, 3 DAT to −Zn), and subjected to SDS-PAGE; fluorescence was detected in-gel. The 749

lower panel shows a CBB-stained gel, confirming equal protein loading. Approximate 750

molecular mass (kDa) is shown on the right. (B) SOD activities of NahG and NahG atg5 751

plants at 5 DAT to −/+Zn. Gray parts indicate Cu/ZnSOD activities, and white parts 752

indicate MnSOD and FeSOD activities. n = 4. Error bars show SD. 753

754

755

Figure 7. Model of autophagy-mediated Zn deficiency tolerance mechanism in 756

plants 757

During Zn deficiency, autophagy is induced and supplies mobile Zn ions by degrading 758

various proteins and organelles. This increases Zn bioavailability and alleviates the Zn 759

deficiency. •OH is generated in a light-dependent manner via the Fe-mediated 760



36

Fenton-like reaction in chloroplasts. Production of •OH is responsible for Zn deficiency–761

induced chlorosis. IM: isolation membrane; AB: autophagic body. 762

763



37

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Eguchi M, Kimura K, Makino A, Ishida H (2017) Autophagy is induced under Zn limitation and contributes to Zn-limited stress tolerancein Arabidopsis (Arabidopsis thaliana). Soil Sci. Plant Nutr. 63: 342-350.


Floyd BE, Morriss SC, MacIntosh GC, Bassham DC (2015) Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis. Autophagy. 11: 2199-2212.


Fujiwara T, Hirai MY, Chino M, Komeda Y, Naito S (1992) Effects of sulfur nutrition on expression of the soybean seed storage proteingenes in transgenic petunia. Plant Physiol. 99: 263-268.


Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D (1998) Identification of a family of zinc transporter genes from Arabidopsisthat respond to zinc deficiency. Proc Natl Acad Sci. 95: 7220-7224.


Guiboileau A, Yoshimoto K, Soulay F, Bataille MP, Avice JC, Masclaux-Daubresse C (2012) Autophagy machinery controls nitrogenremobilization at the whole-plant level under both limiting and ample nitrate conditions in Arabidopsis. New Phytol. 194: 732-740.


Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y (2002) Leaf senescence and starvation-inducedchlorosis are accelerated by the disruption of an Arabidopsis autophagy gene. Plant Physiol. 129: 1181-1193.


Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI, Petersen NH, Mattsson O, Jørgensen LB, Jones JD, Mundy J, Petersen M(2009) Autophagic components contribute to hypersensitive cell death in Arabidopsis. Cell. 137: 773-783.


Horie T, Kawamata T, Matsunami M, Ohsumi Y (2017) Recycling of iron via autophagy is critical for the transition from glycolytic torespiratory growth. J. Biol. Chem. 292: 8533-8543.


Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, Mae T (2008) Mobilization of rubisco and stroma- www.plantphysiol.orgon April 3, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.

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localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant physiol. 148: 142-155.


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https://scholar.google.com/scholar?as_q=NIH Image to ImageJ: 25 years of image analysis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Schneider&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Thompson AR%2C Doelling JH%2C Suttangkakul A%2C Vierstra RD %282005%29 Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways%2E Plant Physiol&dopt=abstract

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https://scholar.google.com/scholar?as_q=Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Autophagic nutrient recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Thompson&as_ylo=2005&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Thordal%2DChristensen H%2C Zhang Z%2C Wei Y%2C Colloge DB %281997%29 Subcellular localization of H2O2 in plants%2E H2O2 accumulation in papillae and hypersensitive response during the barley%2Dpowdery mildew interaction%2E Plant J&dopt=abstract

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https://scholar.google.com/scholar?as_q=Subcellular localization of H2O2 in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Subcellular localization of H2O2 in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Thordal-Christensen&as_ylo=1997&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Vallee BL%2C Auld DS %281990%29 Zinc coordination%2C function%2C and structure of zinc enzymes and other proteins%2E Biochemistry&dopt=abstract

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https://scholar.google.com/scholar?as_q=Zinc coordination, function, and structure of zinc enzymes and other proteins&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Zinc coordination, function, and structure of zinc enzymes and other proteins&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Vallee&as_ylo=1990&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Wada S%2C Hayashida Y%2C Izumi M%2C Kurusu T%2C Hanamata S%2C Kanno K%2C Kojima S%2C Yamaya T%2C Kuchitsu K%2C Makino A%2C Ishida H %282015%29 Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice%2E Plant physiol&dopt=abstract

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https://scholar.google.com/scholar?as_q=Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Autophagy supports biomass production and nitrogen use efficiency at the vegetative stage in rice&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Wada&as_ylo=2015&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Xiong Y%2C Contento AL%2C Nguyen PQ%2C Bassham DC %282007%29 Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis%2E Plant Physiol&dopt=abstract

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https://scholar.google.com/scholar?as_q=Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Xiong&as_ylo=2007&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshimoto K%2C Hanaoka H%2C Sato S%2C Kato T%2C Tabata S%2C Noda T%2C Ohsumi Y %282004%29 Processing of ATG8s%2C ubiquitin%2Dlike proteins%2C and their deconjugation by ATG4s are essential for plant autophagy%2E Plant Cell&dopt=abstract

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https://scholar.google.com/scholar?as_q=Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are essential for plant autophagy&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshimoto&as_ylo=2004&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshimoto K%2C Jikumaru Y%2C Kamiya Y%2C Kusano M%2C Consonni C%2C Panstruga R%2C Ohsumi Y%2C Shirasu K %282009%29 Autophagy negatively regulates cell death by controlling NPR1%2Ddependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis%2E Plant Cell&dopt=abstract


https://scholar.google.com/scholar?as_q=Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshimoto&as_ylo=2009&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshimoto K %282012%29 Beginning to understand autophagy%2C an intracellular self%2Ddegradation system in plants%2E Plant Cell Physiol&dopt=abstract


https://scholar.google.com/scholar?as_q=Beginning to understand autophagy, an intracellular self-degradation system in plants&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Beginning to understand autophagy, an intracellular self-degradation system in plants&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshimoto&as_ylo=2012&as_allsubj=all&hl=en&c2coff=1

https://www.ncbi.nlm.nih.gov/pubmed?term=Yoshimoto K%2C Shibata M%2C Kondo M%2C Oikawa K%2C Sato M%2C Toyooka K%2C Shirasu K%2C Nishimura M%2C Ohsumi Y %282014%29 Organ%2Dspecific quality control of plant peroxisomes is mediated by autophagy%2E J%2E Cell Sci&dopt=abstract


https://scholar.google.com/scholar?as_q=Organ-specific quality control of plant peroxisomes is mediated by autophagy&num=10&btnG=Search+Scholar&as_epq=&as_oq=&as_eq=&as_occt=any&as_publication=&as_yhi=&as_allsubj=all&hl=en&lr=&c2coff=1

https://scholar.google.com/scholar?as_q=Organ-specific quality control of plant peroxisomes is mediated by autophagy&num=10&btnG=Search+Scholar&as_occt=any&as_sauthors=Yoshimoto&as_ylo=2014&as_allsubj=all&hl=en&c2coff=1


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Autophagy maintains Zn pools under Zn deficiency Author ...140 atg mutants exhibit accelerated Zn deficiency symptoms 141 To evaluate the relationship between Zn deficiency and autophagy