autophagy maintains zn pools under zn deficiency author ...140 atg mutants exhibit accelerated zn...
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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
Kanagawa, 214-8571, Japan 17
2 Life Science Program, Graduate School of Agriculture, Meiji University, Kawasaki, 18
Kanagawa, 214-8571, Japan 19
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
Copyright 2020 by the American Society of Plant Biologists
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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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19
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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
www.plantphysiol.orgon April 3, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
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
www.plantphysiol.orgon April 3, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
37
LITERATURE CITED 764
765
Assunção AG, Herrero E, Lin YF, Huettel B, Talukdar S, Smaczniak C, Immink RG, van 766
Eldik M, Fiers M, Schat H, Aarts MG (2010) Arabidopsis thaliana transcription factors 767
bZIP19 and bZIP23 regulate the adaptation to zinc deficiency. Proc Natl Acad Sci. 768
107: 10296-10301. 769
Bassham DC, MacIntosh GC (2017) Degradation of cytosolic ribosomes by 770
autophagy-related pathways. Plant Sci. 262: 169-174. 771
Brunkard JO, Runkel AM, Zambryski PC (2015) Chloroplasts extend stromules 772
independently and in response to internal redox signals. Proc. Natl. Acad. Sci. 112: 773
10044-10049. 774
Cakmak I (2000) Tansley review No. 111 Possible roles of zinc in protecting plant cells 775
from damage by reactive oxygen species. New Phytol. 146: 185-205. 776
Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD (2002) The 777
APG8/12-activating enzyme APG7 is required for proper nutrient recycling and 778
senescence in Arabidopsis thaliana. J Biol Chem. 277: 33105-33114. 779
Doke N (1983) Generation of superoxide anion by potato tuber protoplasts during the 780
hypersensitive response to hyphal wall components of Phytopthora infestans and 781
specific inhibition of the reaction by suppressors of hypersensitivity. Physiol Plant 782
Pathol. 23: 359-367. 783
Eguchi M, Kimura K, Makino A, Ishida H (2017) Autophagy is induced under Zn 784
limitation and contributes to Zn-limited stress tolerance in Arabidopsis (Arabidopsis 785
thaliana). Soil Sci. Plant Nutr. 63: 342-350. 786
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Floyd BE, Morriss SC, MacIntosh GC, Bassham DC (2015) Evidence for 787
autophagy-dependent pathways of rRNA turnover in Arabidopsis. Autophagy. 11: 788
2199-2212. 789
Fujiwara T, Hirai MY, Chino M, Komeda Y, Naito S (1992) Effects of sulfur nutrition on 790
expression of the soybean seed storage protein genes in transgenic petunia. Plant 791
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Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D (1998) Identification of a 793
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Guiboileau A, Yoshimoto K, Soulay F, Bataille MP, Avice JC, Masclaux-Daubresse C 796
(2012) Autophagy machinery controls nitrogen remobilization at the whole-plant level 797
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732-740. 799
Hanaoka H, Noda T, Shirano Y, Kato T, Hayashi H, Shibata D, Tabata S, Ohsumi Y 800
(2002) Leaf senescence and starvation-induced chlorosis are accelerated by the 801
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Hofius D, Schultz-Larsen T, Joensen J, Tsitsigiannis DI, Petersen NH, Mattsson O, 803
Jørgensen LB, Jones JD, Mundy J, Petersen M (2009) Autophagic components 804
contribute to hypersensitive cell death in Arabidopsis. Cell. 137: 773-783. 805
Horie T, Kawamata T, Matsunami M, Ohsumi Y (2017) Recycling of iron via autophagy 806
is critical for the transition from glycolytic to respiratory growth. J. Biol. Chem. 292: 807
8533-8543. 808
Ishida H, Yoshimoto K, Izumi M, Reisen D, Yano Y, Makino A, Ohsumi Y, Hanson MR, 809
Mae T (2008) Mobilization of rubisco and stroma-localized fluorescent proteins of 810
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39
chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant 811
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Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, 816
Ohsumi Y, Yoshimori T (2000) LC3, a mammalian homologue of yeast Apg8p is 817
processed and localized in autophagosome membranes. EMBO J. 19: 5720-5728. 818
Kanno Y, Oikawa T, Chiba Y, Ishimaru Y, Shimizu T, Sano N, Koshiba T, Kamiya Y, 819
Ueda M, Seo M (2016) AtSWEET13 and AtSWEET14 regulate gibberellin-mediated 820
physiological processes. Nat Commun. 7: 13245. 821
Kawamata T, Horie T, Matsunami M, Sasaki M, Ohsumi Y (2017) Zinc starvation 822
induces autophagy in yeast. J. Biol. Chem. 292: 8520-8530. 823
Kirisako T, Baba M, Ishihara N, Miyazawa K, Ohsumi M, Yoshimori T, Noda T, Ohsumi 824
Y (1999) Formation process of autophagosome is traced withApg8/Aut7p in yeast. J. 825
Cell Biol. 147:435-446. 826
Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N (2011) Crystal 827
structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6. 828
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Kraft C, Deplazes A, Sohrmann M, Peter M (2008) Mature ribosomes are selectively 830
degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p 831
ubiquitin protease. Nat Cell Biol. 10: 602-610. 832
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40
Li F, Chung T, Pennington JG, Federico ML, Kaeppler HF, Kaeppler SM, Otegui MS, 833
Vierstra RD (2015) Autophagic recycling plays a central role in maize nitrogen 834
remobilization. Plant Cell. 27: 1389-1408. 835
Liuzzi JP, Yoo C (2013) Role of zinc in the regulation of autophagy during ethanol 836
exposure in human hepatoma cells. Biol Trace Elem Res. 156: 350-356. 837
Liuzzi JP, Guo L, Yoo C, Stewart TS (2014) Zinc and autophagy. Biometals. 27: 838
1087-1096. 839
Maret M (2009) Molecular aspects of human cellular zinc homeostasis: redox control of 840
zinc potentials and zinc signals. Biometals. 22: 149-157 841
Merkulova EA, Guiboileau A, Naya L, Masclaux-Daubresse C, Yoshimoto K (2014) 842
Assessment and optimization of autophagy monitoring methods in Arabidopsis roots 843
indicate direct fusion of autophagosomes with vacuoles. Plant Cell Physiol. 55: 844
715-726. 845
Miki T, Awa M, Nishikawa Y, Kiyonaka S, Wakabayashi M, Ishihama Y, Hamachi I 846
(2016) A conditional proteomics approach to identify proteins involved in zinc 847
homeostasis. Nat. Methods. 13: 931-937. 848
Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in 849
autophagosome formation. Annu. Rev. Cell Dev. Biol. 27: 107-132. 850
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction 851
coefficients and simultaneous equations for assaying chlorophylls a and b extracted 852
with four different solvents: verification of the concentration of chlorophyll standards 853
by atomic absorption spectroscopy. Biochim Biophys Acta Bioenerg. 975: 384-394. 854
Pottier M, Dumont J, Masclaux-Daubresse C, Thomine S (2019) Autophagy is essential 855
for optimal translocation of iron to seeds in Arabidopsis. J Exp Bot. 70: 859-869. 856
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41
Reumann S, Weber AP (2006) Plant peroxisomes respire in the light: some gaps of the 857
photorespiratory C2 cycle have become filled—others remain. Biochim. Biophys. 858
Acta. 1763: 1496-1510. 859
Romero-Puertas MC, Palma JM, Gomez M, Del Rio LA. Sandalio LM (2002) Cadmium 860
causes the oxidative modification of proteins in pea plants. Plant Cell Environ. 25: 861
677-686. 862
Schneider CA, Rasband, WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of 863
image analysis. Nat Methods. 9: 671-675. 864
Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient 865
recycling in Arabidopsis directed by the ATG8 and ATG12 conjugation pathways. 866
Plant Physiol. 138: 2097-2110. 867
Thordal-Christensen H, Zhang Z, Wei Y, Colloge DB (1997) Subcellular localization of 868
H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the 869
barley-powdery mildew interaction. Plant J. 11: 1187-1194. 870
Vallee BL, Auld DS (1990) Zinc coordination, function, and structure of zinc enzymes 871
and other proteins. Biochemistry. 29: 5647-5659. 872
Wada S, Hayashida Y, Izumi M, Kurusu T, Hanamata S, Kanno K, Kojima S, Yamaya T, 873
Kuchitsu K, Makino A, Ishida H (2015) Autophagy supports biomass production and 874
nitrogen use efficiency at the vegetative stage in rice. Plant physiol. 168: 60-73. 875
Xiong Y, Contento AL, Nguyen PQ, Bassham DC (2007) Degradation of oxidized 876
proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiol. 143: 877
291-299. 878
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42
Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) 879
Processing of ATG8s, ubiquitin-like proteins, and their deconjugation by ATG4s are 880
essential for plant autophagy. Plant Cell. 16: 2967-2983. 881
Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, Ohsumi Y, 882
Shirasu K (2009) Autophagy negatively regulates cell death by controlling 883
NPR1-dependent salicylic acid signaling during senescence and the innate immune 884
response in Arabidopsis. Plant Cell. 21: 2914-2927. 885
Yoshimoto K (2012) Beginning to understand autophagy, an intracellular 886
self-degradation system in plants. Plant Cell Physiol. 53: 1355-1365. 887
Yoshimoto K, Shibata M, Kondo M, Oikawa K, Sato M, Toyooka K, Shirasu K, 888
Nishimura M, Ohsumi Y (2014) Organ-specific quality control of plant peroxisomes is 889
mediated by autophagy. J. Cell Sci. 127: 1161-1168. 890
891
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Parsed CitationsAssunção AG, Herrero E, Lin YF, Huettel B, Talukdar S, Smaczniak C, Immink RG, van Eldik M, Fiers M, Schat H, Aarts MG (2010)Arabidopsis thaliana transcription factors bZIP19 and bZIP23 regulate the adaptation to zinc deficiency. Proc Natl Acad Sci. 107: 10296-10301.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Bassham DC, MacIntosh GC (2017) Degradation of cytosolic ribosomes by autophagy-related pathways. Plant Sci. 262: 169-174.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Brunkard JO, Runkel AM, Zambryski PC (2015) Chloroplasts extend stromules independently and in response to internal redox signals.Proc. Natl. Acad. Sci. 112: 10044-10049.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Cakmak I (2000) Tansley review No. 111 Possible roles of zinc in protecting plant cells from damage by reactive oxygen species. NewPhytol. 146: 185-205.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Doelling JH, Walker JM, Friedman EM, Thompson AR, Vierstra RD (2002) The APG8/12-activating enzyme APG7 is required for propernutrient recycling and senescence in Arabidopsis thaliana. J Biol Chem. 277: 33105-33114.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Doke N (1983) Generation of superoxide anion by potato tuber protoplasts during the hypersensitive response to hyphal wallcomponents of Phytopthora infestans and specific inhibition of the reaction by suppressors of hypersensitivity. Physiol Plant Pathol.23: 359-367.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Floyd BE, Morriss SC, MacIntosh GC, Bassham DC (2015) Evidence for autophagy-dependent pathways of rRNA turnover inArabidopsis. Autophagy. 11: 2199-2212.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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.
localized fluorescent proteins of chloroplasts to the vacuole by an ATG gene-dependent autophagic process. Plant physiol. 148: 142-155.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Izumi M, Wada S, Makino A, Ishida H (2010) The autophagic degradation of chloroplasts via rubisco-containing bodies is specificallylinked to leaf carbon status but not nitrogen status in Arabidopsis. Plant Physiol. 154: 1196-1209.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T (2000) LC3, a mammalianhomologue of yeast Apg8p is processed and localized in autophagosome membranes. EMBO J. 19: 5720-5728.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kanno Y, Oikawa T, Chiba Y, Ishimaru Y, Shimizu T, Sano N, Koshiba T, Kamiya Y, Ueda M, Seo M (2016) AtSWEET13 and AtSWEET14regulate gibberellin-mediated physiological processes. Nat Commun. 7: 13245.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kawamata T, Horie T, Matsunami M, Sasaki M, Ohsumi Y (2017) Zinc starvation induces autophagy in yeast. J. Biol. Chem. 292: 8520-8530.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kirisako T, Baba M, Ishihara N, Miyazawa K, Ohsumi M, Yoshimori T, Noda T, Ohsumi Y (1999) Formation process of autophagosome istraced withApg8/Aut7p in yeast. J. Cell Biol. 147:435-446.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Klinge S, Voigts-Hoffmann F, Leibundgut M, Arpagaus S, Ban N (2011) Crystal structure of the eukaryotic 60S ribosomal subunit incomplex with initiation factor 6. Science. 334: 941-948.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kraft C, Deplazes A, Sohrmann M, Peter M (2008) Mature ribosomes are selectively degraded upon starvation by an autophagypathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat Cell Biol. 10: 602-610.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Li F, Chung T, Pennington JG, Federico ML, Kaeppler HF, Kaeppler SM, Otegui MS, Vierstra RD (2015) Autophagic recycling plays acentral role in maize nitrogen remobilization. Plant Cell. 27: 1389-1408.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liuzzi JP, Yoo C (2013) Role of zinc in the regulation of autophagy during ethanol exposure in human hepatoma cells. Biol Trace ElemRes. 156: 350-356.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Liuzzi JP, Guo L, Yoo C, Stewart TS (2014) Zinc and autophagy. Biometals. 27: 1087-1096.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Maret M (2009) Molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals. Biometals. 22:149-157
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Merkulova EA, Guiboileau A, Naya L, Masclaux-Daubresse C, Yoshimoto K (2014) Assessment and optimization of autophagymonitoring methods in Arabidopsis roots indicate direct fusion of autophagosomes with vacuoles. Plant Cell Physiol. 55: 715-726.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Miki T, Awa M, Nishikawa Y, Kiyonaka S, Wakabayashi M, Ishihama Y, Hamachi I (2016) A conditional proteomics approach to identifyproteins involved in zinc homeostasis. Nat. Methods. 13: 931-937.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Mizushima N, Yoshimori T, Ohsumi Y (2011) The role of Atg proteins in autophagosome formation. Annu. Rev. Cell Dev. Biol. 27: 107-132.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for www.plantphysiol.orgon April 3, 2020 - Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.
assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards byatomic absorption spectroscopy. Biochim Biophys Acta Bioenerg. 975: 384-394.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Pottier M, Dumont J, Masclaux-Daubresse C, Thomine S (2019) Autophagy is essential for optimal translocation of iron to seeds inArabidopsis. J Exp Bot. 70: 859-869.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Reumann S, Weber AP (2006) Plant peroxisomes respire in the light: some gaps of the photorespiratory C2 cycle have become filled-others remain. Biochim. Biophys. Acta. 1763: 1496-1510.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Romero-Puertas MC, Palma JM, Gomez M, Del Rio LA. Sandalio LM (2002) Cadmium causes the oxidative modification of proteins inpea plants. Plant Cell Environ. 25: 677-686.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Schneider CA, Rasband, WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 9: 671-675.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Thompson AR, Doelling JH, Suttangkakul A, Vierstra RD (2005) Autophagic nutrient recycling in Arabidopsis directed by the ATG8 andATG12 conjugation pathways. Plant Physiol. 138: 2097-2110.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Thordal-Christensen H, Zhang Z, Wei Y, Colloge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillaeand hypersensitive response during the barley-powdery mildew interaction. Plant J. 11: 1187-1194.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Vallee BL, Auld DS (1990) Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry. 29: 5647-5659.Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Wada S, Hayashida Y, Izumi M, Kurusu T, Hanamata S, Kanno K, Kojima S, Yamaya T, Kuchitsu K, Makino A, Ishida H (2015) Autophagysupports biomass production and nitrogen use efficiency at the vegetative stage in rice. Plant physiol. 168: 60-73.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Xiong Y, Contento AL, Nguyen PQ, Bassham DC (2007) Degradation of oxidized proteins by autophagy during oxidative stress inArabidopsis. Plant Physiol. 143: 291-299.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshimoto K, Hanaoka H, Sato S, Kato T, Tabata S, Noda T, Ohsumi Y (2004) Processing of ATG8s, ubiquitin-like proteins, and theirdeconjugation by ATG4s are essential for plant autophagy. Plant Cell. 16: 2967-2983.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshimoto K, Jikumaru Y, Kamiya Y, Kusano M, Consonni C, Panstruga R, Ohsumi Y, Shirasu K (2009) Autophagy negatively regulatescell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis.Plant Cell. 21: 2914-2927.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshimoto K (2012) Beginning to understand autophagy, an intracellular self-degradation system in plants. Plant Cell Physiol. 53: 1355-1365.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Yoshimoto K, Shibata M, Kondo M, Oikawa K, Sato M, Toyooka K, Shirasu K, Nishimura M, Ohsumi Y (2014) Organ-specific qualitycontrol of plant peroxisomes is mediated by autophagy. J. Cell Sci. 127: 1161-1168.
Pubmed: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
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