1

1 2 3 4 5

THE PROTEASOME STRESS REGULON IS CONTROLLED BY A PAIR OF 6

NAC TRANSCRIPTION FACTORS IN ARABIDOPSIS 7

8

9

10

11

Running Title: Transcriptional Regulation of Proteotoxic Stress 12

13

14

15

By Nicholas P. Gladman1, Richard S. Marshall1,2, Kwang-Hee Lee1, and Richard D. 16

Vierstra1,2 17

18

19

20 1Department of Genetics, University of Wisconsin, Madison, WI 53706 USA 21

2Department of Biology, Washington University in St. Louis, St. Louis, MO 63130 USA 22

23

24

Correspondences should be addressed to: 25

Dr. Richard D. Vierstra 26

Department of Biology, Campus Box 1137 27

Washington University in St. Louis 28

One Brookings Drive 29

St. Louis, MO 63130 USA 30

314-935-5058 office 31

314-935-4432 fax 32

rdvierstra@wustl.edu 33

34

35

Plant Cell Advance Publication. Published on May 18, 2016, doi:10.1105/tpc.1501022

©2016 American Society of Plant Biologists. All Rights Reserved.

2

36

37

SYNOPSIS 38

Proteotoxic stress in Arabidopsis is attenuated by a pair of NAC transcription factors that up-39

regulate the synthesis of the 26S proteasome and other factors that promote protein 40

homeostasis. 41

42

ABSTRACT 43

Proteotoxic stress, which is generated by the accumulation of unfolded or aberrant proteins due 44

to environmental or cellular conditions, can be mitigated by several mechanisms, including 45

activation of the unfolded protein response and coordinated increases in protein chaperones 46

and activities that direct proteolysis, such as the 26S proteasome. Using RNA-seq analyses 47

combined with chemical inhibitors or mutants that induce proteotoxic stress by impairing 26S 48

proteasome capacity, we defined the transcriptional network that responds to this stress in 49

Arabidopsis thaliana. This network includes genes encoding core and assembly factors needed 50

to build the complete 26S particle, alternative proteasome capping factors, enzymes involved in 51

protein ubiquitylation/deubiquitylation and cellular detoxification, protein chaperones, autophagy 52

components, and various transcriptional regulators. Many loci in this proteasome-stress regulon 53

contain a consensus cis element upstream of the transcription start site, which was previously 54

identified as a binding site for the NAM/ATAF1/CUC2 (NAC)-78 transcription factor. Double 55

mutants disrupting NAC78 and its closest relative NAC53 are compromised in the activation of 56

this regulon, and notably are strongly hypersensitive to the proteasome inhibitors MG132 and 57

bortezomib. Given that NAC53 and NAC78 homo- and hetero-dimerize, we propose that they 58

work as a pair in activating the expression of numerous factors that help plants survive 59

proteotoxic stress, and thus play a central regulatory role in maintaining protein homeostasis. 60

61

3

INTRODUCTION 62

All cellular organisms require mechanisms to dampen the accumulation of aberrant proteins 63

caused by transcription/translation errors, misfolding, cleavage, chemical modification, and 64

environmental conditions such as heat that perturb tertiary/quaternary structures. If allowed to 65

hyper-accumulate, this detritus can negatively impact a host of intracellular activities through an 66

imbalance in protein homeostasis and excessive protein aggregation (Morimoto, 2008; Hipp et 67

al., 2014). The resulting proteotoxic stress has strong physiological consequences, including 68

genome instability, arrest of the cell cycle, inhibition of translation through a reduction in 69

ribosomes, lost membrane integrity, inhibition of metabolism, and accelerated senescence, as 70

well as amino acid starvation if protein recycling becomes severely compromised. In fact, the 71

accumulation of misfolded, aggregation prone proteins is the hallmark of a broad range of 72

human diseases (Cuanalo-Contreras et al., 2013). 73

To alleviate this stress, plants, fungi, and animals elicit a number of cytoprotective 74

responses, including expression of protein chaperones and activation of the unfolded protein 75

response designed to promote refolding and sequester protein aggregates, maintenance of 76

chromatin integrity through SUMOylation, and up-regulation of several proteolytic pathways that 77

remove these aberrant polypeptides before they become cytotoxic (Hetz, 2012; Howell, 2013; 78

Kim et al., 2013; Amm et al., 2014; Seifert et al., 2015). Excessive protein aggregation and the 79

accumulation of defective protein complexes (e.g., ribosomes and proteasomes) and organelles 80

(e.g., chloroplasts and mitochondria) often engage autophagy (Li and Vierstra, 2012; Khaminets 81

et al., 2015; Marshall et al., 2015). These damaged structures are encapsulated into 82

cytoplasmic vesicles and delivered to the vacuole/lysosome for breakdown, in many cases 83

using ubiquitylation as a signal. 84

Arguably, the most important protease up-regulated during proteotoxic stress is the 26S 85

proteasome (Amm et al., 2014; Hanssum et al., 2014; Livnat-Levanon et al., 2014). This 2.5-86

MDa, ATP-dependent proteolytic machine works in tandem with ubiquitin (Ub) to direct the 87

selective breakdown of aberrant polypeptides and normal short-lived proteins in the cytoplasm 88

and nucleus. Targets are first covalently modified with multiple Ubs; the resulting ubiquitylated 89

species dock with the 26S proteasome, which degrades the modified protein concomitant with 90

release of the Ub moieties for reuse. The 26S proteasome consists of two subcomplexes: the 91

20S core protease (CP) and the 19S regulatory particle (RP) (Finley, 2009; Bhattacharyya et al., 92

2014). The CP consists of four stacked heteroheptameric rings of distinct α and β subunits in 93

an α1-7/β1-7/β1-7/α1-7 configuration, which houses in a central chamber the proteolytic active sites 94

provided by the PBA (β1), PBB (β2) and PBE (β5) subunits. The RP has 18 or more subunits; it 95

caps one or both ends of the CP barrel and contains receptors for ubiquitylated targets, and 96

4

activities that remove the Ub moieties and unfold and translocate the target polypeptides into 97

the CP lumen before breakdown. In addition, a host of accessory factors associate 98

substoichiometrically, including dedicated chaperones that promote the sequential assembly of 99

the CP and RP complexes and final construction of the 26S particle, alternative capping factors 100

(PA200 and CDC48), shuttle proteins that help deliver ubiquitylated substrates, and ubiquitin-101

protein ligases (or E3s) and deubiquitylating enzymes (Finley, 2009; Hanssum et al., 2014). 102

Given its role(s) in maintaining protein homeostasis, the levels of the 26S proteasome 103

are highly regulated to meet demand, which is achieved by the coordinated expression of all 104

core subunits and most, if not all, accessory factors. In yeast (Saccharomyces cerevisiae), this 105

regulon is driven by the C2H2-type zinc-finger transcription factor Rpn4, which binds to the 106

PACE (Proteasome Associated Control Element) cis element found upstream of most 107

proteasome subunit genes (Mannhaupt et al., 1999; Xie and Varshavsky, 2001; Shirozu et al., 108

2015). Rpn4 responds to proteotoxic stress by itself being a target of proteasomal breakdown. 109

When proteolytic demand is low, Rpn4 is rapidly degraded (t1/2 ~2.5 minutes), thus attenuating 110

expression of 26S proteasome genes (Xie and Varshavsky, 2001; Dohmen et al., 2007). 111

However, as 26S proteasome capacity is challenged by proteotoxic stress, Rpn4 is stabilized 112

thus allowing its levels to rise and up-regulate particle synthesis. 113

Although orthologs of Rpn4 are not obvious outside of yeasts, a similar ‘proteasome 114

stress regulon’ (PSR) exists in many other eukaryotes, including mammalian cells, Drosophila 115

melanogaster and Arabidopsis thaliana (Meiners et al., 2003; Yang et al., 2004; Lundgren et al., 116

2005; Kurepa et al., 2008; Book et al., 2009). In mice and humans, the Nrf1 (Nuclear Factor 117

Erythroid-derived 2-Related Factor 1) transcription factor, unrelated to Rpn4, was recently 118

shown to be a central effector (Radhakrishnan et al., 2010; 2014; Sha and Goldberg, 2014). 119

Nrf1 is bound to the endoplasmic reticulum (ER) and is constitutively degraded by the ER-120

associated protein degradation (ERAD) pathway, and like Δrpn4 yeast cells, nrf1-null cells are 121

more sensitive to proteasome inhibitors. Upon proteotoxic stress, Nrf1 is proteolytically 122

released from the ER and is now free to enter the nucleus and activate the PSR. 123

How the PSR is controlled by proteotoxic stress remains unclear in plants. One 124

candidate regulator in Arabidopsis is NAC78, a member of the NO APICAL 125

MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR-1/CUP-SHAPED 126

COTYLEDONS-2 (NAC) family of transcriptional regulators. NAC78 was first implicated by 127

overexpression studies showing that it positively regulates the expression of core 26S 128

proteasome subunit genes, and that its putative DNA-binding site is present within many, but 129

not all, associated promoters (Yabuta et al., 2011; Nguyen et al., 2013). Whereas NAC78-OX 130

plants are smaller than wild type, nac78 mutant plants are larger, which is consistent with a 131

5

reported role for 26S proteasome levels in controlling cell size (Kurepa et al., 2009; Sonoda et 132

al., 2009). 133

To more broadly define the plant PSR, we combined RNA-seq analysis with both 134

chemical inhibitors and proteasome mutants that induce proteotoxic stress. Gene co-135

expression combined with protein interactome network analyses revealed a complex network of 136

PSR genes/proteins enriched in protein chaperones, autophagy components and detoxification 137

enzymes that presumably help plants cope with proteotoxic stress, in addition to those encoding 138

the 26S proteasome and its assembly/capping components. Network and promoter-interaction 139

studies then identified the NAC78 paralog NAC53 as a key effector that, together with NAC78, 140

likely functions as homo- and heterodimers. Importantly, seedlings simultaneously lacking 141

NAC53 and NAC78 poorly activate the PSR during proteotoxic stress, and their growth is 142

strongly hypersensitive to proteasome inhibitors. As such, we propose that these two NAC 143

proteins are central regulators of an extended PSR in Arabidopsis by providing sufficient 26S 144

proteasomes and other protein homeostatic factors to mitigate proteotoxic stress. 145

146

RESULTS 147

26S Proteasome Subunits Are Up-Regulated During Proteotoxic Stress 148

To better understand how 26S proteasome gene expression is up-regulated by proteotoxic 149

stress, we examined the transcript levels for representative Arabidopsis subunits either upon 150

short (3 hr) or long term (24 hr) exposure of seedlings to the proteasome inhibitor MG132 (Yang 151

et al., 2004), or in mutant backgrounds that compromise 26S proteasome assembly (rpn10-1 152

and rpn12a-1) and elicit seedling phenotypes consistent with impaired capacity (Smalle et al., 153

2002; 2003). The mutant alleles were fortuitously generated by exon-trap mutagenesis; they 154

are viable but have stunted growth and show pleiotropic defects in hormone signaling. The 155

rpn12a-1 allele dampens expression of the full-length RPN12a transcript (Figure 2A), but is 156

relatively mild phenotypically (Smalle et al., 2002). The rpn10-1 allele expresses a truncation of 157

RPN10, the main Ub receptor in the 26S complex, and has much stronger developmental 158

consequences (Smalle et al., 2003). The translated polypeptide includes the N-terminal von 159

Willebrand Factor-A domain that links RPN10 to the rest of the RP but is missing the C-terminal 160

region containing the three Ub-interacting motifs that bind Ub, the autophagy adaptor ATG8, 161

and cargo receptors bearing Ub-like domains, respectively (Farmer et al., 2010; Fatimababy et 162

al., 2010; Marshall et al., 2015). 163

Previous studies demonstrated that both long-term exposure to MG132 and the rpn10-1 164

mutation elevate the steady-state levels of Ub conjugates and increase the abundance of 165

several core subunits of the 26S proteasome (Smalle et al., 2003; Yang et al., 2004; Kurepa et 166

6

al., 2008). We extended these results here by immunoblotting crude extracts from MG132-167

treated wild-type and untreated homozygous rpn10-1 and rpn12a-1 seedlings with antibodies 168

against Ub, the CP subunit PBA1(β1), the RP subunits RPN1, RPT2 and RPN12a, and the CP 169

regulator PA200 (known as Blm10 in yeast) (Figures 1A and 1B). In particular, accumulation of 170

the unprocessed form of PBA1 increased strongly upon MG132 treatment, consistent with the 171

need for active proteasomes to generate the mature, truncated β1 polypeptide (Finley, 2009; 172

Book et al., 2010). The rpn12a-1 allele had only a marginal effect on the abundance of Ub 173

conjugates and the PBA1, RPN1 and RPT2 subunits but its action was obvious based on its 174

strong effect on PA200 levels (Figure 1A). Why the effects on CP/RP subunit levels were mild 175

for the rpn12a-1 allele was unclear but it could reflect the relatively modest phenotype of the 176

mutant (Smalle et al., 2002), and/or the possibility that genetically compromised 26S 177

proteasomes generated by the rpn12a-1 allele more effectively induce autophagic turnover of 178

the complex as opposed to the rpn10-1 mutant, which blocks such turnover (Marshall et al., 179

2015). 180

When mRNA abundance was then examined by reverse-transcription quantitative PCR 181

(RT-qPCR) analysis of seedlings, levels of the CP subunit PBA1 and the RP subunits RPN5a, 182

RPN10 and RPN12a were found to rise rapidly upon short treatments with MG132 and were 183

constitutively up-regulated in the rpn10-1 and rpn12a-1 backgrounds (Figure 2A). (Throughout 184

this study, we used transcripts from the ACTIN (ACT)-2 and/or TYPE-2A SERINE/THREONINE 185

PROTEIN PHOSPHATASE (PP)-2A genes as controls given their relative immunity to 186

proteotoxic stress (see Supplemental Figure 1)). As with previous studies (Gallois et al., 2009; 187

Lee et al., 2011), this increased transcript abundance could also be demonstrated with 188

transgenic plants expressing the β-glucuronidase (GUS) reporter under the control of various 189

26S proteasome gene promoters. Up-regulation upon MG132 treatment was visualized 190

colorimetrically by staining the seedlings with X-Gluc, and quantitatively by 4-methylumbelliferyl-191

β-D-glucuronide (MUG)-based fluorescence activity assays of crude seedling extracts (Figures 192

2B and 2C). Interestingly, comparisons of several gene pairs that encode individual 193

proteasome subunits revealed that often only one responds to MG132, implying that the many 194

pairs have subfunctionalized with one locus mainly responsible for increasing subunit mRNA 195

levels during proteotoxic stress (e.g., RPN3a, RPT1a, RPT2a, and RPT4b (Figures 2B and 2C)). 196

197

RNA-seq Analysis of the Proteasome Stress-Induced Regulon (PSR) 198

To more fully identify the suite of genes that are up-regulated when proteasome capacity is 199

compromised, we performed RNA-seq analysis with our cohort of wild-type seedlings treated 200

with MG132 (3 and 24 hr) and untreated rpn10-1 and rpn12a-1 seedlings. These transcriptome 201

7

studies identified a large collection of mRNAs whose abundance was significantly affected as 202

compared to untreated wild-type seedlings based on negative binomial normalization using 203

edgeR differential expression analysis (p-value <0.01, FDR <0.05; see Supplemental Datasets 204

1 and 2). By merging the datasets, we identified 119 genes (including RPN10 and RPN12a 205

given their up-regulation in 2 of the 3 conditions) that were coordinately up-regulated under all 206

three conditions (3-hr treatment with MG132 and in the rpn10-1 and rpn12a-1 backgrounds), 207

which we designated as members of the PSR (Figure 3A; see Supplemental Dataset 3). RNA-208

seq analysis after a 24-hr exposure to MG132 identified an additional set of 865 up-regulated 209

genes; these loci were not included in the final PSR as their long-term induction might be more 210

indirectly related to the prolonged effects of the inhibitor and the downstream responses to 211

severe proteotoxic stress (see Supplemental Dataset 4). 212

Gene ontology analyses via DAVID (Database for Annotation, Visualization and 213

Integrated Discovery; https://david.ncifcrf.gov) of the 119 PSR up-regulated genes detected a 214

significant functional enrichment for the 26S proteasome, Ub-related events, and general stress 215

response processes, and identified several transcription factors that might transcriptionally 216

activate the PSR. This enrichment for 26S proteasome genes was especially strong in the more 217

robustly affected group with a ≥ 2-fold increase (44% of total; Figure 3B). Of the 53 genes 218

encoding core 26S proteasome subunits (Book et al., 2010; Russell et al., 2013), 48 (91%) were 219

significantly up-regulated (Figures 3B and 3C). We note that the rank order of the up-regulated 220

and down-regulated genes in the MG132 and rpn10-1/rpn12a-1 datasets based on their 221

strength of change differed markedly (Figure 3B); this deviation could reflect two distinct 222

subtypes of proteotoxic stress, one elicited rapidly and strongly by the inhibitor and the other 223

being a more subtle chronic stress elicited by the mutations. We also detected 33 genes whose 224

expression was down-regulated by all three conditions (Figures 3A and 3B; see Supplemental 225

Dataset 3); there was no significant functional enrichment in this group, thus leaving their 226

collective action(s) unclear. 227

From analysis of genes known or predicted to be associated with the Arabidopsis 26S 228

proteasome (Finley, 2009; Book et al., 2010), a strikingly coordinated up-regulation was evident 229

for most loci. This was apparent for both core CP and RP subunits as well as acknowledged 230

accessory factors (PA200 and ECM29) and chaperones (HSM3, NAS6, PBAC1, PBAC2 and 231

UMP1a) that are presumably needed during proteotoxic stress to assist in particle 232

regulation/assembly (Figure 3C). The proteasome-binding protein, PROTEASOME 233

REGULATOR (PTRE)-1, recently identified as a positive regulator of auxin signaling through 234

activation of the 26S proteasome (Yang et al., 2016), was also up-regulated, as was the 235

possible plant-specific chaperone PIP1. The only exception for subunits encoded by a single 236

8

gene was the locus encoding the Ub receptor RPN13, whose expression was relatively immune 237

to MG132 and was unchanged by the mutations. In agreement with the RT-qPCR and 26S-238

promoter:GUS reporter studies above, we found that multiple gene pairs encoding individual 239

subunits had at least one locus that was PSR regulated with the second locus sometimes non-240

responsive. Examples include the PAA2, PBB2, RPT1a, RPT6b, RPN3a, and RPN9b loci, 241

which were highly sensitive to proteotoxic stress, whereas their paralogs PAA1, PBB1, RPT1b, 242

RPT6a, RPN3, and RPN9a were comparatively silent (Figure 3C). 243

When the analysis was extended to non-proteasome transcripts, additional loci 244

potentially relevant to the PSR were detected (see Supplemental Dataset 3). Included were 245

mRNAs encoding members of the NAC, WRKY, and heat shock protein transcription factor 246

(HSF) families that often have prominent roles in various plant stress responses (Rushton et al., 247

2010; Nakashima et al., 2012; Scharf et al., 2012), other Ub/proteasome pathway components 248

including Ub, several Ub E3 ligases (AIP2, At1g71020, At4g27050 and At5g27920), 249

deubiquitylating enzymes (UBP6, UBP7 and UCH3), and extra-proteasomal Ub-binding proteins 250

(DSK2a and DDI1), and components of the autophagy system (see Supplemental Figure 2). 251

Notable autophagy factors included several members of the ATG8 family required for 252

autophagic vesicle dynamics and cargo delivery, and the autophagic receptor NBR1, which is 253

devoted to the recruitment of ubiquitylated cargo (Li and Vierstra, 2012; Zhou et al., 2013). 254

Strong transcriptional up-regulation of NBR1, especially by MG132, implied a role for autophagy 255

in removing Ub conjugates that become stabilized when proteasome capacity is limited. 256

257

The MG132-Induced PSR Network 258

To better visualize the MG132-induced PSR, we entered the 336 MG132-induced genes that 259

were significantly up-regulated ≥ 2 fold after a 3-hr treatment (see Supplemental Dataset 1) into 260

the Search Tool for the Retrieval of Interacting Genes/Protein (STRING) co-expression and 261

interactome database (Jensen et al., 2009). Upon visualization in Cytoscape v3.0 (Shannon et 262

al., 2003), an extensive collage of networks involving 275 output loci emerged that clustered into 263

three main groups (Figure 4A). As expected, one tight cluster included loci encoding 26S 264

proteasome subunits and accessory factors/proteasome chaperones. A second cluster 265

encompassed a large collection of general heat shock protein (HSP) chaperones (several 266

HSP70 isoforms, HSA32, HSP81, and HSP101, and their dedicated transcriptional regulator 267

HSFA8) that presumably direct protein refolding and/or minimize aggregation of insoluble 268

proteins awaiting proteasomal/autophagic turnover (Lin et al., 2014). A third more diffuse 269

cluster was enriched in the stress-related NAC, WRKY, and HSF transcription factors that might 270

help activate the PSR, and transferase activities (e.g., glutathione S-transferases and O-271

9

methyltransferases) that could mitigate proteotoxic stress (Livnat-Levanon et al., 2014). The 272

third cluster also contained several UDP-glycosyl transferases and the main mitochrondrial 273

alternative oxidase AOX1a, which were shown previously to be up-regulated upon mitochondrial 274

perturbation (De Clercq et al., 2013). Many of these loci were also seen in clusters generated 275

with the 24-hr MG132 treatment dataset, especially for the 26S proteasome and related factors 276

(see Supplemental Figure 3). Additions to the 24-hr MG132 network included a pronounced 277

autophagy cluster, and a large cluster enriched in protein kinases that could extend the PSR 278

signaling network given the roles of some in stress defense and developmental signal 279

transduction cascades (e.g., MEK1, MAPKKK1, RLP24, HLECRK, CRCK1 and CRCK11). 280

Under the assumption that many of the 336 loci are transcriptionally regulated by a 281

common set of transcription factors, we searched their promoter regions for shared cis 282

regulatory DNA elements using the motif-based sequence analysis tool MEME (Multiple 283

Expectation Maximization for Motif Elicitation; Bailey et al., 2006). Highly enriched were the 284

Proteasome-Related cis Element (PRCE) with a TGGGC core sequence, which was previously 285

implicated by Nguyen et al. (2013) in the regulation of proteasome genes, and the mitochondrial 286

dysfunction motif (MDM) associated with oxidative stress responses (De Clercq et al., 2013) 287

(Figures 4B and 4C). Both motifs were prominent in the 26S proteasome gene cluster, with few 288

promoters (14 of 48) predicted to be devoid of either motif (Figure 4D). When placed within the 289

region upstream of the translation start site for genes encoding core 26S proteasome subunits, 290

a wide dispersion of the PRCE and MDM motifs was seen, with some having these elements 291

close to the transcription start site and others placed >1000 bp away (see Supplemental Figure 292

4). Whereas single MDM elements were common, we often detected multiple PRCE-related 293

sequences in tandem, sometimes on opposite DNA strands, suggesting that PRCE is acted 294

upon by DNA-binding proteins that work palindromically. We also detected other potentially 295

relevant cis elements associated with the PSR at lower frequencies (see Supplemental Figure 296

5). Included were the SORLIP2 and T-Box motifs that are involved in light-induced 297

developmental responses (Chan et al., 2001; Hudson and Quail, 2003). 298

299

The NAC53 and NAC78 Transcription Factors Are Associated with the PSR. 300

To help identify the transcriptional regulators that might coordinate the PSR, we screened a 301

yeast one-hybrid (Y1H) library of ~1,700 known or predicted DNA-binding proteins from 302

Arabidopsis by an automated luciferase (LUC)-based expression system (Gaudinier et al., 303

2011), using both the PA200 or RPN12a promoter regions as bait (1000 and 253-bp fragments 304

upstream of the translation start codon, respectively). The activity of both promoters is strongly 305

up-regulated by MG132 exposure, and accordingly they contain multiple segments related to 306

10

the consensus PRCE sequence. (It should be noted that the RPN12a promoter is predicted to 307

be short given that only 253 bp separates its translation start site from the coding region 308

immediately upstream.) Among the list of 15 interactors that activated both Y1H assays was 309

NAC53 (At3g10500, also known as NTL4; see Supplemental Table 1), a 555-residue NAC 310

protein that we also discovered as a locus significantly up-regulated by brief MG132 exposure 311

and positioned by Cytoscape as a central hub within the PSR detoxification cluster (Figure 4A, 312

see Supplemental Figure 2). Upon scanning the Arabidopsis genome for related proteins, we 313

identified NAC78 (At5g04410, also known as NTL11/RPX1), a 584-residue NAC protein that is 314

its closest relative (~72% amino acid sequence identity) among the ~109 NAC-type 315

transcriptional regulators (Olsen et al., 2005a; Nakashima et al., 2012; Wu et al., 2012). Binding 316

of NAC53 and NAC78 to both promoters was confirmed by directed Y1H assays using 500 bp of 317

the PA200 promoter and 253 bp of the RPN12a promoter as baits. 318

Intriguingly, several previous studies implicated NAC53 and NAC78 in the PSR and/or in 319

binding PRCE/MDM-type sequences. NAC78 was first linked by over-expression screens that 320

searched for factors that regulate either plant size or seedling responses to light stress, and was 321

subsequently shown by transcriptome studies to impact proteasome gene expression (Morishita 322

et al., 2009; Yabuta et al., 2011; Nguyen et al., 2013). Nguyen et al. (2013) further connected 323

NAC78 to the PSR by reporting that it recognizes the same PRCE element identified here as 324

common within the regulon (Figure 3C). A further connection to the PSR was provided by DNA-325

binding studies showing that both NAC53 and NAC78 might also recognize MDM elements 326

found here to be common within PSR loci (De Clercq et al., 2013). Finally, NAC53 was 327

previously connected to heat stress, reactive oxygen species (ROS) production and senescence, 328

all of which are related to proteotoxic stress, through cursory phenotypic analyses of NAC53 329

mutant and overexpression lines (Lee et al., 2012; Lee et al., 2014), 330

To provide an evolutionary connection between NAC53 and NAC78, we examined the 331

two loci phylogenetically in the context of the entire NAC family. Bayesian analyses clustered 332

NAC53 and NAC78 together when either the 109 full-length NAC proteins were compared or 333

when just the DNA-binding NAM domains were analyzed, indicating that this pair forms a unique 334

subclade within the NAC family (Figure 5A; see Supplemental Figures 6 and 7). In fact, strong 335

amino acid sequence identity was seen throughout the NAM domain (~93%), implying that they 336

bind the same DNA sequence (see Supplemental Figure 8). Close sequence relatives (~50% 337

sequence identity) were also detected in other plant species, including various monocots and 338

dicots and the seedless plants Selaginella moelendorffii and Physcomitrella patens, but not in 339

the alga Chlamydomonas reinhardtii, implying that the pair has a conserved function in land 340

plants. Relatives of both NAC53 and NAC78 could be found in Arabidopsis lyrata that clustered 341

11

apart from likely orthologs in other dicots, suggesting that NAC53 and NAC78 originated from a 342

duplication that occurred recently within the Brassicaceae lineage. 343

The C-terminal ends of NAC53 and NAC78 are predicted to contain a 24-amino-acid 344

membrane-spanning sequence that is also found within 11 other Arabidopsis NAC proteins, 345

which are collectively designated as NTLs (NACs with transmembrane-like region (Kim et al., 346

2007; see Supplemental Figure 6). Like the NAM domain, this putative membrane-spanning 347

region is highly conserved between NAC53 and NAC78 (75% sequence identity), even when 348

aligning their sequences next to that of their closest NTL relative NAC13 (30% full polypeptide 349

sequence identify, 65% NAM domain sequence identity; see Supplemental Figure 8). Even 350

within the NTL subfamily, NAC53/NAC78 stand apart phylogenetically, suggesting that they 351

have distinct function(s) (Figure 5A; see Supplemental Figures 6 and 7). The role(s) of the 352

membrane-spanning domain is not yet clear; for at least one Arabidopsis NTL, it has been 353

proposed to facilitate binding of the NAC protein to cytosolic membranes, with proteolytic 354

release then permitting nuclear import (Kim et al., 2006; Kim et al., 2007). 355

356

NAC53 and NAC78 Regulate PSR Gene Expression 357

Given the aforementioned connections of NAC53/NAC78 to proteasome gene regulation and 358

their likely ability to bind PRCE-type motifs (Yabuta et al., 2011; De Clercq et al., 2013; Nguyen 359

et al., 2013; this report), we hypothesized that NAC53 and NAC78 work in concert as homo- 360

and heterodimers to up-regulate the PSR. As a first validation, we tested whether NAC53 and 361

NAC78 interact, as is common for paralogs within the NAC family (Nakashima et al., 2012; De 362

Clercq et al., 2013). By yeast two-hybrid assays (Y2H), we detected binding of NAC53 and 363

NAC78 to themselves and to each other, using the Gal4/LacZ pDEST system, thus generating 364

colonies that grew in the absence of His and were resistant to 50 mM 3-amino-1,2,4-triazole (3-365

AT) (Figure 5B). This assembly of homo- and heterodimers was then confirmed in planta by 366

bimolecular fluorescence complementation (BiFC) with the split YFP system transiently 367

expressed in Nicotiana benthamiana leaf epidermal cells. Only when the NAC53 or NAC78 368

coding regions were simultaneously expressed as fusions to either the nYFP or cYFP fragments 369

was fluorescence reconstituted (Figure 5C, see Supplemental Figures 9A and 9C). 370

Fluorescence was easily detected in the cytoplasm and nucleus, suggesting that the two 371

proteins interact in each compartment. 372

It had been reported that when a GFP fusion of full-length NAC78 is transiently 373

expressed in onion (Allium cepa) epidermal cells, it mainly localizes to the cytosol but becomes 374

concentrated in the nucleus when expressed without the membrane-spanning region (Morishita 375

et al., 2009). To examine whether proteotoxic stress could recapitulate this redistribution, we 376

12

attempted to generate transgenic plants that stably express GFP-NAC53 and GFP-NAC78 with 377

or without this transmembrane domain. Unfortunately no fluorescent signals were detected, in 378

agreement with a previous failure with another NTL, NAC13 (De Clercq et al., 2013). As an 379

alternative, we examined the BiFC signal from the NAC53/NAC78 pair in N. benthamiana cells 380

simultaneously treated with MG132. Unfortunately, under our conditions, MG132 treatment 381

neither impacted the strength nor the distribution of the BiFC signals (see Supplemental Figure 382

9B). 383

As the next step in connecting at least one of this NAC pair to the PSR, we examined 384

transgenic lines expressing NAC78 from the β-estradiol-inducible XVE promoter, which was 385

provided by the TRANSPLANTA collection (Coego et al., 2014). In our hands, NAC78 mRNA 386

levels rose ~60 fold after an overnight β-estradiol exposure. As shown in Figure 6, 387

pEST:NAC78 seedlings, but not wild-type seedlings, treated with β-estradiol also strongly 388

increased the mRNA abundance for several representative 26S proteasome subunits, as first 389

observed by Nguyen et al. (2013). Importantly, this up-regulation extended to other members of 390

the PSR outside of the core 26S particle, including genes encoding the proteasome accessory 391

factor PA200, the NAS6 assembly chaperone, the UPS component UFD1, and the HSP 392

transcriptional regulator HSF8A. Two other PSR genes encoding the proteasome accessory 393

factor PA200 and the glutathione S-transferase GSTU25 also displayed increases close to 394

significance, consistent with NAC78 more broadly controlling the PSR. 395

Assuming that the NAC53/NAC78 proteins work together, we generated double mutants 396

impacting the pair to observe any adverse effects on PSR regulation. Potentially useful alleles 397

were identified in the SALK collection of T-DNA insertion mutants that either disrupted the 398

coding region downstream from the N-terminal NAM domain (nac53-1), within the NAM domain 399

(nac78-1), just upstream of that for the C-terminal transmembrane sequence (nac53-2), or 400

toward the end of the coding region (nac78-2; Figure 7A). Genomic sequencing mapped the 401

insertion sites to 735 and 372 nucleotides downstream of the translational start site for the 402

nac53-1 and nac78-1 mutations, and 87 and 21 nucleotides upstream of the translational stop 403

site for the nac53-2 and nac78-2 mutations, respectively. 404

Transcript analyses by RT-PCR failed to detect the corresponding full-length mRNAs in 405

homozygous nac53-1 and nac78-1 seedlings, (Figure 7B). For nac53-1 we also could not 406

detect transcripts downstream of the insertion site, whereas for nac78-1 we could detect a 407

transcript of slightly greater length than expected. Collectively the RT-PCR data imply that 408

nac53-1 and nac78-1 represent null alleles. For homozygous nac53-2 and nac78-2 seedlings, 409

we detected low levels of near-full length transcripts originating upstream of the insertion sites; 410

these mRNAs included the NAM domain coding sequence. For the nac78-2 mutant, only a 411

13

small portion of the proposed C-terminal membrane-spanning region domain was eliminated, 412

whereas, nearly the entire membrane-spanning region was missing for the nac53-2 mutant (see 413

Supplemental Figure 8), suggesting that they represent weaker alleles under the assumption 414

that the full transmembrane domain is not essential (at least for NAC53). 415

When grown at 24°C under a normal 16-hr light/8-hr dark photoperiod on agar medium 416

or soil, all the single and double mutant combinations germinated well, generated phenotypically 417

normal rosettes, flowered at the same time, and were equally fertile as wild type, indicating that 418

NAC53 and NAC78 alone or in combination are not necessary for Arabidopsis growth, 419

development and fecundity under non-stressed conditions. At least under our growth conditions, 420

the mutant seedlings were not larger than wild type, as had been reported previously for a T-421

DNA insertion line disrupting NAC78 by itself (Nguyen et al., 2013). 422

Importantly, RT-qPCR analyses of PSR-related genes revealed that NAC53 and NAC78 423

combine to activate the PSR. Whereas the nac53-1 mutation had little effect on expression of 424

representative PSR genes after an overnight MG132 treatment, a mild but statistically significant 425

dampening (p-value <0.01) was seen for the nac78-1 line (Figure 8A). Moreover, when the 426

double nac53-1 nac78-1 mutant was tested, a substantially attenuated PSR response was 427

observed. Compromised loci included genes encoding subunits of the 26S particle (PBA1, 428

RPN5a, RPN10 and RPN12a), the accessory factors PA200 and CDC48, the NAS6 chaperone, 429

the UPS factor UFD1, the glutathione S-transferase GSTU25, and the heat shock transcription 430

factor HSFA8 (p-value <0.01). For some loci, the nac53-1 nac78-1 combination almost 431

completely ablated the transcriptional up-regulation. A smaller but significant drop in PSR gene 432

expression was also seen for the nac53-2 nac78-2 double mutant, consistent with the milder 433

effect of these mutations on the NAC53/NAC78 mRNAs (Figure 8A). 434

The diminished PSR in turn reduced the steady-state levels of some of the 435

corresponding proteins. Whereas MG132 treatment increased the seedling abundance of the 436

26S accessory protein PA200, the RP subunit RPN12a and the unprocessed form of the CP 437

subunit PBA1 in wild-type seedlings, this increase was substantially weaker in the nac53-1 438

nac78-1 background (Figure 8B). However, this drop did not appear to translate into a 439

substantial stabilization of Ub conjugates, as the pool of immunodetectable species was only 440

mildly increased in the nac53-1 nac78-1 plants as compared to wild type (see Supplemental 441

Figure 10). Consistent with the reduced strength of the nac53-2 and nac78-2 alleles on PSR 442

gene expression, the double mutants had little impact on the RPN12a and PBA1 protein levels, 443

and possibly only a mild effect on PA200 (Figure 8B). As the polypeptides derived from the 444

nac53-2 nac78-2 alleles should be missing all or part of the presumed transmembrane domain, 445

respectively, membrane association might not be essential for NAC53/NAC78 function. 446

14

447

Plants Missing NAC53 and NAC78 are Hypersensitive to Proteotoxic Stress 448

Phenotypic analysis of homozygous nac53-1 nac78-1 plants in turn revealed that both 449

transcription factors together help Arabidopsis survive proteotoxic stress. Whereas wild-type 450

plants could tolerate long-term exposure to sublethal doses of MG132 beginning at germination, 451

growth of the double mutant plants was substantially arrested (Figures 9A and 9B). In fact, 452

development of nac53-1 nac78-1 seeds stalled soon after germination, and for many, the 453

radicals failed to exit the seed coat after rupture. Quantification of the response also detected a 454

significant growth inhibition (p-value <0.01) for the nac78-1 single mutant and for the weaker 455

nac53-2 nac78-2 double mutant as compared to wild type (Figure 9B). 456

Given that the smaller plants observed for the nac53-1 nac78-1 line upon MG132 457

exposure might have arisen from defective or delayed germination, we also germinated and 458

grew the seedlings for 6 d in the absence of MG132 and then placed them on inhibitor-459

containing medium and measured seedling fresh weight 6 d afterwards. Even in this situation, 460

growth of the nac53-1 nac78-1 plants was significantly impaired as compared to wild type 461

(Figure 9C). As MG132 might impact other proteases besides those within the CP, we also 462

tested a second 26S proteasome inhibitor with potentially greater specificity and potency – 463

bortezomib (Kisselev et al., 2012). Here, growth of the nac53-1 nac78-1 plants was severely 464

inhibited at submicromolar concentrations, with a mild but significant effect also seen for the 465

nac53-2 nac78-2 plants (Figures 9D and 9E). 466

467

NAC53 and NAC78 Work Together and with Other Members of the NAC Family. 468

While our studies indicated that NAC53 and NAC78 work together, possibly as heterodimers 469

based on our Y2H assays, to activate the PSR, they could also have temporally distinct 470

functions within the response (e.g., early versus late) given that NAC53 but not NAC78 471

transcripts were increased rapidly by MG132 treatment. To test these possibilities, we 472

compared the time course for PSR activation by MG132 in the single mutant nac53-1 and 473

nac78-1 backgrounds by RT-qPCR analysis of representative PSR loci. As shown in 474

Supplemental Figure 11, the induction time courses were identical (but slightly diminished) for 475

genes encoding the CP and RP subunits, CDC48, the RP chaperone NAS6, and GSTU25, with 476

the double nac53-1 nac78-1 mutant lacking a significant response for all loci tested but GSTU25. 477

Together, the data imply that NAC53 and NAC78 work within the same time frame. 478

The mild attenuation of the PSR for the nac53-1 nac78-1 plants in response to MG132 479

as seen with GSTU25 and other loci (e.g., WRKY25) (Figure 8A; see Supplemental Figure 11), 480

suggested that other transcription factors also contribute to the PSR. Particularly notable were 481

15

several other members of the NAC family that were transcriptionally up-regulated by the 482

inhibitor, including NAC1, 13, 32, 44, 50, 55, 81/ATAF2, 82, and 87 (see Supplemental Figure 2). 483

To test whether they also heterodimerize with NAC53/NAC78, we subjected each to Y2H 484

assays using NAC53 and NAC78 as prey. Interestingly, NAC13 and NAC81, but not the others, 485

generated positive Y2H signals, suggesting that they also contribute to PSR activation in 486

combination with NAC53/NAC78 (Figure 5D). Whereas NAC13 bound to both NAC53 and 487

NAC78, NAC81 only showed an interaction with NAC53 under these conditions. NAC13 is 488

especially intriguing given its ability to recognize MDM cis elements common within the PSR 489

and its membership in the transmembrane-containing NTL-NAC subfamily like NAC53/NAC78. 490

491

DISCUSSION 492

Given the importance of protein quality control to maintaining healthy cellular functions, and the 493

likelihood that plants routinely experience proteotoxic stress (e.g., ERAD; Howell 2013), we 494

considered it likely that plants have strong protective responses. Examples include various 495

environmental insults such as heat, cold, salt, drought, excess light, photooxidative stress, and 496

ROS production that perturb protein folding, carbon and nitrogen starvation that limits synthesis 497

of new polypeptides, pathogen invasion which often coincides with the massive translation of 498

pathogen-associated polypeptides that might express improperly in the host, and exposure to 499

toxins made by the pathogen, such as the proteasome inhibitors epoxomicin and syringolin A 500

that block host protein turnover (Yang et al., 2004; Groll et al., 2008). Our RNA-seq studies on 501

Arabidopsis seedlings induced to experience proteotoxic stress through impairment of 26S 502

proteasome capacity (MG132 inhibition and the rpn10-1 and rpn12a-1 mutants) revealed an 503

intricate network of PSR genes/proteins that presumably represent parts of these protective 504

mechanisms. Many of the associated activities are also important for protein homeostasis in 505

yeast and animals, indicating that they reflect conserved protein homeostatic processes among 506

eukaryotes (Morimoto, 2008; Hipp et al., 2014). 507

A prominent PSR cluster is the 26S proteasome itself and its associated 508

assembly/regulatory factors that are needed to build a functional holoenzyme, demonstrating 509

that increasing 26S proteasome capacity is one crucial protective mechanism. In fact, given the 510

plethora of core subunits and assembly chaperones required to construct 26S proteasomes, a 511

remarkable coordination of expression has evolved to ensure faithful production of complete 512

particles. As shown by mutational studies affecting individual subunits, sub-stoichiometric 513

accumulation of only one subunit is sufficient to stall construction of the entire complex and 514

induce off-product accumulation of assembly intermediates (Smalle et al., 2002; 2003; Book et 515

al., 2009; Lee et al., 2011). The importance of 26S proteasomes to protein homeostasis is also 516

16

demonstrated by the fact that nearly all core subunits are encoded by at least one gene that is 517

strongly sensitive to proteotoxic stress. The presence of a non-responsive paralog likely reflects 518

sub-functionalization of the pair, which might be important for maintaining proteasome levels in 519

specific spatio-temporal contexts. 520

A second PSR cluster includes a collection of protein chaperones that help protein 521

folding and minimize protein aggregation, along with several HSF transcription factors that 522

promote their expression during stress. Their inclusion was anticipated given the central roles 523

of chaperones in protein quality control and maintaining protein homeostasis (Morimoto, 2008; 524

Hipp et al., 2014). A third PSR cluster is enriched in a collection of enzymes important for 525

cellular detoxification, such as glutathione-S-transferases, O-methyltransferases, and AOX1a, 526

the last of which is responsible for alternative respiration in mitochondria. Presumably, these 527

proteins help repair damaged proteins and reduce oxidative stress and ROS. Several 528

autophagy components are also members of the PSR, including ATG8 and the Ub receptor 529

NBR1, thus connecting autophagy to plant protein homeostasis. A role for NBR1 is in 530

agreement with studies on nbr1 mutants; they accumulate substantial amounts of high 531

molecular mass Ub conjugates during heat stress, which possibly represent ubiquitylated 532

protein aggregates awaiting autophagic clearance (Zhou et al., 2013). Interestingly, additional 533

autophagy genes appeared in the network created upon long-term MG132 exposure (see 534

Supplemental Figure 3), suggesting that autophagy represents a last line of defense during 535

proteotoxic stress. It should also be emphasized that we discovered a number of genes within 536

the Arabidopsis PSR whose function(s) remains to be discovered. Once their purposes become 537

clear, it is likely that additional protein homeostatic mechanisms will be revealed. 538

Combined with previous studies (Yabuta et al., 2011; Nguyen et al., 2013), our network 539

and promoter interaction studies identified NAC53 and NAC78 as key regulators of the PSR. 540

Support here includes: (i) Y1H binding of NAC53 to the PA200 and RPN12a promoters that 541

strongly respond to proteotoxic stress, (ii) the ability of NAC53 and NAC78 to homo- and hetero-542

dimerize, (iii) β-estradiol-induced transcription of an assortment of PSR genes in plants 543

harboring the pEST:NAC78 transgene, and (iv) strong transcriptional attenuation of 544

representative PSR genes in plants missing both NAC53 and NAC78. The ability to dimerize is 545

consistent with other NACs that require dimerization to bind a pair of palindromically oriented 546

DNA elements (Olsen et al., 2005b). Connections of NAC53 and/or NAC78 to heat, intense 547

light, drought stress, ROS production, and senescence have also been reported based on the 548

analysis of mutants and overexpression lines (Morishita et al., 2009; Yabuta et al., 2011; Lee et 549

al., 2012; 2014). It is conceivable that these phenotypes are indirectly manifested by direct 550

17

participation of the pair in proteotoxic stress protection and regulation of 26S proteasome 551

synthesis. 552

As expected for plants with a dampened PSR, we found that nac53 nac78 double 553

mutants are highly sensitive to proteasome inhibitors. Seedling growth was completely blocked 554

with 50 μM MG132 and 0.5 μM bortezomib for the null nac53-1 nac78-1 plants, with partial 555

inhibition seen for plants harboring the weaker nac53-2 nac78-2 alleles. A similarly modest 556

growth inhibition was seen for the nac78-1 single mutant but not for the single nac53-1 mutant, 557

suggesting that the NAC78 protein is more critical to PSR activation. Although the proteotoxic 558

stress conditions studied here activated expression of NAC53 more robustly than NAC78, we 559

consider it likely that they work simultaneously within the PSR given the similar temporal 560

induction seen for the nac53-1 and nac78-1 single mutants. Regardless of their functions, we 561

note that nac53 nac78 double null mutants are phenotypically normal under non-stress 562

conditions and do not hyper-accumulate Ub conjugates upon MG132 treatment (see 563

Supplemental Figure 10). These observations imply that other transcription factors are 564

responsible for the basal synthesis of 26S proteasomes and other protein homeostasis 565

regulators, and that during proteotoxic stress, additional recycling pathways are engaged to 566

eliminate the excess Ub conjugates that accumulate if 26S proteasome capacity is insufficient 567

(e.g., autophagy). As an aside, we note that Arabidopsis growth is more sensitive to bortezomib 568

than MG132 and thus might represent a better agent to block 26S proteasome activity in planta. 569

We consider it likely that NAC53 and NAC78 recognize the same cis element given their 570

strong amino acid sequence similarity within the NAM DNA-binding motif. Unfortunately, the 571

nature of the element is unclear. Prior studies identified the PRCE element with the consensus 572

core TGGGC sequence as the preferred NAC78 binding site (Yabuta et al., 2010; Nguyen et al., 573

2013), whereas more recent interaction studies with NAC53 and NAC78 reported that both 574

prefers MDM-type motifs (Lee et al., 2012; De Clercq et al., 2013). Understanding this 575

discrepancy will certainly require more in-depth binding studies using NAC53 and NAC78 alone 576

and in combination. Both cis motifs were common in many of the PSR loci and are present 577

upstream of most 26S proteasome genes. Given the close proximity of PRCE and MDM 578

sequences within proteasome subunit promoters, it is conceivable that the NAC53/NAC78 579

dimers bind both motifs simultaneously and/or work with other transcription factors that bind. 580

How NAC53 and NAC78 activate the PSR during proteotoxic stress is unclear. In yeast, 581

their functional counterpart Rpn4 participates in a simple negative feedback circuit whereby 582

Rpn4 becomes stabilized during proteotoxic stress as proteasome capacity becomes 583

overloaded. In mice and humans, its potential counterpart is Nrf1, a transmembrane-containing 584

basic leucine zipper transcription factor that resides on the ER membrane under non-stressed 585

18

conditions. Upon proteotoxic stress, the DNA-binding region of Nrf1 is proteolytically released 586

from the membrane-spanning segment, which then permits its nuclear import to drive 26S 587

proteasome gene expression. Given that NAC53 and NAC78, as part of the Arabidopsis NTL 588

subfamily, are also predicted to have a transmembrane domain, and that others within this 589

subfamily have been reported to use proteolytic release from cytoplasmic stores to regulate 590

their transcriptional activity (Kim et al., 2006; Kim et al., 2007), it is plausible that a shuttle 591

mechanism similar to that of Nrf1 exists. However, this proteolytic step and membrane release, 592

if they occur, might not be essential as the truncated nac53-2 protein described here (Figure 8A), 593

and an engineered truncation of NAC78 by Nguyen et al. (2013) did not appear to constitutively 594

activate the PSR even in the absence of MG132. A similar lack of effect of the C-terminal 595

truncation was seen for another NAC-NTL, NAC13, in its ability to activate transcription (De 596

Clercq et al., 2013), thus questioning the role(s) of the transmembrane domain in NTL-NAC 597

action. 598

Interestingly, the PSR is populated with transcripts for a number of other NAC 599

transcription factors besides NAC53, including NAC1, 13, 32, 44, 50, 55, 81/ATAF2, 82, and 87, 600

that are robustly up-regulated upon MG132 exposure, and/or in the rpn10-1 and rpn12a-1 601

backgrounds. These factors could represent additional transcriptional regulators that assist in 602

the immediate activation of the PSR, or reflect part of a transcriptional cascade working 603

downstream that helps activate the full suite of PSR loci. In support of the former, Y2H 604

analyses of these PSR-associated NAC proteins identified NAC13 and NAC81 as 605

NAC53/NAC78 binding partners that could directly participate in PSR activation. NAC13 is 606

particularly intriguing as it, like NAC53 and NAC78, is a member of the NTL subclade, and has 607

been implicated in the transcriptional response to mitochondrial dysfunction and binding to MDM 608

sequences (De Clercq et al., 2013). Notably, a number of the gene targets of the mitochondrial 609

dysfunction response are shared with the PSR (e.g., encoding UDP-glycosyl transferases and 610

AOX1a), which combined with the prevalence of MDM sequences in PSR genes, suggests that 611

the two stress responses share a number of protective functions (e.g., oxidative stress defense). 612

A similar overlap between proteotoxic stress and mitochondrial dysfunction has also been 613

observed in mammals (Livnat-Levanon et al., 2014). Taking these results together, an attractive 614

hypothesis is that the PSR loci containing both PRCE and MDM elements are activated by 615

heterodimers bearing NAC13 in combination with either NAC53 or NAC78. 616

We also note that a subpopulation of PSR genes are likely immune or only weakly 617

responsive to NAC53 and NAC78 regulation (e.g., WRKY25), indicating that other transcription 618

factors are responsible for their activation under proteotoxic stress. One or more of these 619

factors might be found within the PSR (e.g., WRKY6, 25, 33 and 45; see Supplemental Figure 620

19

2), or present in the list of DNA-binding proteins identified as common in our Y1H screen with 621

the PA200 and RPN12a promoters (see Supplemental Table 1 and Supplemental Figure 5). 622

Consequently, it is possible that the PSR is controlled by an additional suite of transcriptional 623

regulators beyond NAC53/NAC78 that generate a multi-layered system to fully activate the 624

regulon during the various iterations of proteotoxic stress. 625

626

MATERIALS AND METHODS 627

Plant Materials and Growth Conditions 628

The T-DNA insertion mutants for NAC53 (nac53-1, SALK_009578C; nac53-2, SALK_018311C) 629

and NAC78 (nac78-1, SALK_025098; nac78-2, SALK_040812C) in the Arabidopsis thaliana 630

ecotype Col-0 (Alonso and Stepanova, 2003) were obtained from the Arabidopsis Biological 631

Resource Center (ABRC) at Ohio State University (https://abrc.osu.edu/). The rpn10-1 and 632

rpn12a-1 exon-trap lines in the C24 background were generated as previously described 633

(Smalle et al., 2002; 2003). The NAC78 overexpression line (Col-0 background) driven by the 634

β-estradiol-inducible XVE promoter (NAC78 #2138 and #2319) was provided by the 635

TRANSPLANTA resource (Coego et al., 2014). 636

The proteasome promoter:GUS transgenic lines were generated by PCR amplification of 637

the 5’ upstream region for representative proteasome subunit loci, starting at the end of the 638

upstream coding region (or 2 kb) and terminating at the transcriptional start site. The PCR 639

products were introduced upstream of the full GUS coding region present in the pCAMBIA3301 640

or pMDC163 vectors. The chimeric genes were transformed into Arabidopsis (Col-0 ecotype) 641

by the floral dip method using the Agrobacterium tumefaciens strain GV3101 (Lee et al., 2011). 642

Basta-resistant seedlings were screened for GUS activity by histochemical staining with the 643

substrate X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronic acid; Sigma Aldrich). For quantitative 644

studies, total extracts from 10-d-old seedlings were assayed for GUS activity using the 645

fluorescence-based MUG (Sigma Aldrich) assay (Lee et al., 2011). At least 30 independent 646

transformants were examined for each construction to avoid artifacts generated by the site of 647

the transgene insertion. For examination of GUS staining patterns, 6-d-old seedlings were 648

incubated overnight in X-Gluc following a 12-hr exposure to 100 μM MG132 dissolved in DMSO, 649

using an equivalent volume of DMSO as the control. 650

Unless otherwise noted, seeds were surface sterilized, stratified in the dark at 4°C for 2 651

d, and then germinated on 0.7% agar containing half-strength Murashige and Skoog (MS) 652

medium (Caisson Labs), 1% sucrose and 0.5% MES (pH 5.7), with or without various 653

concentrations of MG132 (N-(benzyloxycarbonyl)-leucinyl-leucinyl-leucinal; SelleckChem) or 654

bortezomib ((1R)-3-methyl-1-(((2S)-3-phenyl-2-(pyrazin-2-carbonylamino) 655

20

propanoyl)amino)butyl)boronic acid; SelleckChem). For the RNA-seq studies, the seedlings 656

were grown for 5 d in 12-well liquid cultures plates under continuous fluorescent white light at 657

22°C prior to RNA isolation. For studies on the phenotypic effects of MG132 or bortezomib, 658

stratified seeds were plated on the same medium with the addition of 0.7% agar. After 6 to 10 d 659

growth under a 16-hr light/8-hr dark photoperiod at 22°C, the plants were weighed individually or 660

in batches of 2-5 seedlings to determine fresh weight. One-way ANOVA was used to determine 661

statistical significance among the various genetic backgrounds and treatments. 662

663

RT-qPCR and RNA-seq Analyses 664

Following various treatments, liquid-grown seedlings were pressed dry, frozen in liquid nitrogen, 665

and pulverized. Total RNA was extracted with the QIAzol Lysis Reagent (Qiagen), treated with 666

DNaseI (Promega), and then converted to cDNA using Superscript III Reverse Transcriptase 667

(Life Technologies). RT-qPCR amplifications on the cDNA populations were performed with a 668

Roche LightCycler 480 using either Roche LightCycler 480 SYBR Green Master Mix or MidSci 669

Bullseye SYBR Green Master Mix. Appropriate priming sites for each locus were identified 670

using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi). See 671

Supplemental Table 2 for the list of primers. Primer efficiencies were experimentally determined 672

to be between 1.90-2.10, based on assay of a standard dilution series (Marshall et al., 2015). 673

The relative abundance of each transcript was determined by the comparative threshold cycle 674

method (Pfaffl, 2001) using the ACT2 and PP2A reference genes as internal controls. All data 675

was normalized to untreated wild type. 676

mRNA enrichment and library generation for RNA-seq studies were performed using the 677

TruSeq RNA library sample preparation kit v2 (Illumina) with help from the University of 678

Wisconsin-Madison Gene Expression Center (http://www.biotech.wisc.edu/services/gec). 679

Multiplexed sequencing was performed on the Illumina HiSeq 2500 platform using 100-bp 680

single-ended reads. At least 2 biological replicates were analyzed for each condition with each 681

amplification yielding between 10-25 million raw sequencing reads. FASTQ files were quality 682

checked with the Trimmomatic package (Bolger et al., 2014). Between 70-80% of reads were 683

retained in all biological replicates. Trimmed reads were aligned by Tophat-Bowtie 2 to the 684

TAIR 10 genome using the Ensemble 2013 Arabidopsis.gtf file annotations (Langmead and 685

Salzberg, 2012). Read counts were generated through HTSeq (Anders et al., 2015), quantified 686

via edgeR (Robinson et al., 2010), and then normalized to the wild-type Col-0 untreated control. 687

Only those transcripts that met a FDR ≤ 0.05 cutoff were included in the analyses. 688

All available gene co-expression and protein interaction data was collected from the 689

STRING database (Jensen et al., 2009) and mapped using the organic layout option from 690

21

Cytoscape 3.0.1 (Shannon et al., 2003). GO and functional term enrichments were determined 691

using DAVID (Huang da et al., 2009). Highly interconnected gene node clusters were identified 692

using the MCODE plugin for Cytoscape (Bader and Hogue, 2003). Heat maps of significantly 693

expressed genes were generated by Java TreeView v1.1643 (Saldanha, 2004). 694

695

Immunoblot Analysis 696

Immunoblot analyses were conducted with 6-d-old seedlings homogenized directly into SDS-697

PAGE sample buffer. Following SDS-PAGE, proteins were transferred onto Millipore 698

Immobilon-P or Immobilon-FL membranes and probed with antibodies against PA200 (Book et 699

al., 2010), Ub (van Nocker et al., 1996), RPN1, RPN5, RPN10, RPN12a and RPT2a (Smalle et 700

al., 2002; Yang et al., 2004), and histone H3 (AbCam; AB1791). 701

702

Phylogenetic Comparisons of NAC Protein Sequences 703

Amino acid sequences were aligned by MEGA v6.0 under the MUSCLE default settings 704

(Tamura et al., 2013). Alignments are provided in Supplemental Data Sets 5 and 6. The 705

locations of the possible NAM and membrane spanning regions were predicted by PFAM v28.0 706

(http://pfam.xfam.org). Phylogenetic analyses of the predicted full-length NAC proteins or only 707

their NAM DNA-binding regions from Arabidopsis thaliana and other plant species available in 708

Phytozome (www.phytozome.jgi.doe.gov/) or NCBI (www.ncbi.nlm.nih.gov/genbank) were 709

performed using MrBayes 3.2 (Ronquist et al., 2012) and the mixed amino acid model 710

(aamodelpr = mixed) until convergence (average standard deviation of split frequencies) 711

reached below 0.05 or plateaued. The trees were rooted with the Physcomitrella patens NAC 712

protein gi_168025227 as the outgroup, and visualized using FigTree v1.4.2. Nucleotide 713

sequences related to the consensus PRCE and MDM cis motifs (De Clercq et al., 2013; Nguyen 714

et al., 2013) were detected in the region upstream of the translation start site using the MEME 715

sequence enrichment identification algorithm with a 7th-order background model calculated from 716

all Arabidopsis thaliana intergenic sequences (Bailey et al., 2006). 717

718

Bimolecular Fluorescence (BiFC) and Yeast One (Y1H)- and Two-Hybrid (Y2H) Analyses 719

For BiFC, the full-length NAC53 or NAC78 coding sequences in the pDONR221 plasmid were 720

introduced into either the pSITE-N-EYFP-C1 or pSITE-C-EYFP-C1 vectors (ARBC stock 721

numbers CD3-1648 (nYFP) or CD3-1649 (cYFP), respectively), and transformed into the 722

Agrobacterium strain GV3101 (Li et al., 2014). Overnight cultures were diluted to OD600nm of 0.5 723

in re-suspension buffer (10 mM MgCl2, 10 mM MES (pH 5.7) and 100 μM acetosyringone) and 724

syringe-infiltrated into 4 to 6-week-old Nicotiana benthamiana leaves. Fluorescence within the 725

22

infiltrated regions was visualized after 36 hr using a Zeiss 510 Meta confocal laser-scanning 726

microscope. For MG132 treatments and DAPI staining, the re-suspension buffer without 727

acetosyringone and containing 100 μM MG132 or 1 μM DAPI (4',6-diamidino-2-phenylindole) 728

was infiltrated into the same leaf regions 24 or 1.5 hr prior to visualization, respectively. 729

Y2H assays were performed using the ProQuest Two-Hybrid System (Life Technologies). 730

The indicated NAC genes were amplified by PCR from Arabidopsis cDNA generated as 731

described above and recombined into pDONR221 via the Gateway BP clonase II reaction. 732

These fragments were then recombined in-frame to either the GAL4 activation domain or GAL4 733

binding domain in the pDEST22 or pDEST32 vectors (Life Technologies). Constructs were 734

verified by sequencing, and pairwise combinations of genes in pDEST22 and pDEST32 (or the 735

empty vectors as controls) were co-transformed into the Saccharomyces cerevisiae strain 736

MaV203 (Vidal et al., 1996). Y2H assays were performed by diluting overnight yeast cultures 737

with either YPD medium lacking Leu and Trp or lacking Leu, Trp and His and containing the 738

indicated concentrations of 3-AT (3-amino-1,2,4-triazole), and then growing the yeast for 2 d at 739

30°C on the same medium for non-selective and selective growth, respectively. 740

For a non-biased Y1H screen, 1000-bp and 253-bp fragments of the PA200 and 741

RPN12a upstream regions, respectively, were cloned into a pLacZ plasmid and screened 742

individually against 1,700 Arabidopsis transcription factors cloned into the MaV203 yeast strain 743

(Gaudinier et al., 2011). For confirmatory Y1H screens, lacZ activation was quantified using the 744

Miller β-galactosidase assay and the substrate 5-bromo-4-chloro-3-indolyl- β -D 745

galactopyranoside as previously described (Zhang and Bremer, 1996). At least three biological 746

replicates were averaged for each measurement. 747

748

Accession Numbers 749

Sequence data from this article can be found in the Arabidopsis Genome Initiative or 750

GenBank/EMBL databases under the following accession numbers: ACT2, (At3g18780), 751

CDC48a (At3g09840), GSTU25 (At1g17180), HSPF8a (At1g67970), NAC1 (At1g01010), 752

NAC13 (At1g32870), NAC32 (At1g77450), NAC44 (At3g01600), NAC50 (At3g10480), NAC53 753

(At3g10500), NAC55 (At3g15500), NAC78 (At5g04410), NAC81 (At5g08790), NAC82 754

(At5g09330), NAC87 (At5g18270), NAS6 (At2g03430), PA200, (At3g13330), PBA1 (At4g31300), 755

PP2A (At1g13320), RPN1a (At2g20580), RPN1b (At4g28470), RPN3a (At1g20200), RPN3b 756

(At1g75990), RPN5a (At5g09900), RPN5b (At5g64760), RPN10 (At4g38630), RPN12a 757

(At1g64520), RPT1a At1g53750), RPT1b (At1g53780), RPT2a (At4g29040), RPT2b 758

(At2g20140), RPT4a (At5g43010), RPT4b (At1g45000), UFD1 (At2g21270), and WRKY25 759

(At2g30250). 760

23

761

762

Supplemental Data 763

764

Supplemental Figure 1. RNA-seq Comparison of Proteotoxic Stress on the Expression of 765

Several Transcripts Commonly Used as Standards for RT-qPCR Studies. 766

Supplementary Figure 2. Expression Heat Maps of Representative PSR Genes Outside of 767

Those Encoding 26S Proteasome Subunits. 768

Supplemental Figure 3. Description of the 24-hr MG132 Up-regulated Gene Interaction and 769

Co-expression Network. 770

Supplemental Figure 4. PRCE and MDM Sequences are Located in the Upstream DNA 771

Region of 26S Proteasome Subunits. 772

Supplemental Figure 5. Occurrence of AGRIS-Annotated DNA-binding Motifs in the Upstream 773

Region of 26S Proteasome and Other PSR Genes. 774

Supplemental Figure 6. Phylogenetic Tree of the 109 Arabidopsis NAC Transcription Factors 775

using the Full Amino Acid Sequences for Comparison. 776

Supplemental Figure 7. Phylogenetic Tree of the 109 Arabidopsis NAC Transcription Factors 777

using just the NAM Domain Sequences for Comparison. 778

Supplemental Figure 8. Amino Acid Sequence Alignment of NTL Proteins NAC53, NAC78 779

and NAC13. 780

Supplemental Figure 9. Bimolecular Fluorescence Complementation of NAC53 and NAC78 781

With or Without MG132 Treatment. 782

Supplemental Figure 10. Ub Conjugate Levels in nac53-1 and nac78-1 Mutants With or 783

Without MG132 Treatment. 784

Supplemental Figure 11. The PSR in Single nac53 and nac78 Mutants Have the Same 785

Temporal Response to MG132 as Wild Type. 786

787

Supplemental Table 1. List of Y1H Prey in Common when using the Arabidopsis PA200 and 788

RPN12a Promoters as Bait. 789

Supplemental Table 2. List of Oligonucleotide Primers used for RT-qPCR Analysis. 790

791

Supplemental Dataset 1. List of Genes Whose Expression was Significantly Affected by 3-hr 792

Exposure to MG132 793

Supplemental Dataset 2. List of Genes Whose Expression was Significantly Up-regulated in 794

the rpn10-1 and rpn12a-1 Mutant Backgrounds. 795

24

Supplemental Dataset 3. List of Genes Within the PSR. 796

Supplemental Dataset 4. List of Genes Whose Expression was Significantly Affected by Long- 797

Term Exposure to MG132. 798

Supplemental Dataset 5. Text File of the Full NAC Protein Alignment Corresponding to the 799

Phylogenetic Analysis in Supplemental Figure 6. 800

Supplemental Dataset 6. Text File of the NAM Domain Alignment Corresponding to the 801

Phylogenetic Analysis in Supplemental Figure 7. 802

803

ACKNOWLEDGEMENTS 804

The authors wish to thank David C. Gemperline and Joseph M. Walker for helpful discussions 805

and Lucas M. Slivicke for technical assistance. This work and N.P.G. were supported by a 806

grant from the U.S. Department of Energy Office of Science; Office of Basic Energy Sciences; 807

Chemical Sciences, Geosciences, and Biosciences Division (DE-FG02-88ER13968). N.P.G 808

was also funded by a NIH Training fellowship provided to the University of Wisconsin-Madison 809

Department of Genetics. 810

811

812

813

AUTHORS CONTRIBUTIONS 814

N.P.G and R.D.V. designed the research. N.P.G. performed most research, analyzed data, and 815

performed all the computational analyses. R.S.M. assisted with the Y2H assays, RT-PCR, and 816

RT-qPCR analyses. K.H.L. generated the transgenic lines expressing the promoter:GUS 817

fusions and analyzed their responses. N.P.G. and R.D.V. wrote the paper. 818

819

REFERENCES 820

Alonso, J.M., and Stepanova, A.N. (2003). T-DNA mutagenesis in Arabidopsis. Methods Mol. 821

Biol. 236: 177-188. 822

Amm, I., Sommer, T., and Wolf, D.H. (2014). Protein quality control and elimination of protein 823

waste: the role of the ubiquitin-proteasome system. Biochim. Biophys. Acta 1843: 182-824

196. 825

Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq: a Python framework to work with high-826

throughput sequencing data. Bioinformatics 31: 166-169. 827

Bader, G.D., and Hogue, C.W. (2003). An automated method for finding molecular complexes 828

in large protein interaction networks. BMC Bioinformatics 4: 2. 829

25

Bailey, T.L., Williams, N., Misleh, C., and Li, W.W. (2006). MEME: discovering and analyzing 830

DNA and protein sequence motifs. Nucleic Acids Res. 43: W369-W373. 831

Bhattacharyya, S., Yu, H., Mim, C., and Matouschek, A. (2014). Regulated protein turnover: 832

snapshots of the proteasome in action. Nat. Rev. Mol. Cell. Biol. 15: 122-133. 833

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina 834

sequence data. Bioinformatics 30: 2114-2120. 835

Book, A.J., Gladman, N.P., Lee, S.S., Scalf, M., Smith, L.M., and Vierstra, R.D. (2010). 836

Affinity purification of the Arabidopsis 26S proteasome reveals a diverse array of plant 837

proteolytic complexes. J. Biol. Chem. 285: 25554-25569. 838

Book, A.J., Smalle, J., Lee, K.H., Yang, P., Walker, J.M., Casper, S., Holmes, J.H., Russo, 839

L.A., Buzzinotti, Z.W., Jenik, P.D., and Vierstra, R.D. (2009). The RPN5 subunit of the 840

26S proteasome is essential for gametogenesis, sporophyte development, and complex 841

assembly in Arabidopsis. Plant Cell 21: 460-478. 842

Chan, C.S., Guo, L., and Shih, M.C. (2001). Promoter analysis of the nuclear gene encoding 843

the chloroplast glyceraldehyde-3-phosphate dehydrogenase B subunit of Arabidopsis 844

thaliana. Plant Mol. Biol. 46: 131-141. 845

Coego, A., Brizuela, E., Castillejo, P., Ruiz, S., Koncz, C., del Pozo, J.C., Pineiro, M., 846

Jarillo, J.A., Paz-Ares, J., Leon, J., and the TRANSPLANTA Consortium (2014). The 847

TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of 848

transcription factors based on their conditional overexpression. Plant J. 77: 944-953. 849

Cuanalo-Contreras, K., Mukherjee, A., and Soto, C. (2013). Role of protein misfolding and 850

proteostasis deficiency in protein misfolding diseases and aging. Int. J. Cell Biol. 2013: 851

638083. 852

De Clercq, I., Vermeirssen, V., Van Aken, O., Vandepoele, K., Murcha, M.W., Law, S.R., 853

Inze, A., Ng, S., Ivanova, A., Rombaut, D., van de Cotte, B., Jaspers, P., Van de 854

Peer, Y., Kangasjarvi, J., Whelan, J., and Van Bruesegem, F. (2013). The membrane-855

bound NAC transcription factor ANAC013 functions in mitochondrial retrograde 856

regulation of the oxidative stress response in Arabidopsis. Plant Cell 25: 3472-3490. 857

Dohmen, R.J., Willers, I., and Marques, A.J. (2007). Biting the hand that feeds: Rpn4-858

dependent feedback regulation of proteasome function. Biochim. Biophys. Acta 1773: 859

1599-1604 860

Farmer, L.M., Book, A.J., Lee, K.H., Lin, Y.L., Fu, H., and Vierstra, R.D. (2010). The RAD23 861

family provides an essential connection between the 26S proteasome and ubiquitylated 862

proteins in Arabidopsis. Plant Cell 22: 124-142. 863

26

Fatimababy, A.S., Lin, Y.L., Usharani, R., Radjacommare, R., Wang, H.T., Tsai, H.L., Lee, 864

Y., and Fu, H. (2010). Cross-species divergence of the major recognition pathways of 865

ubiquitylated substrates for ubiquitin/26S proteasome-mediated proteolysis. FEBS J. 866

277: 796-816. 867

Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the 868

proteasome. Annu. Rev. Biochem. 78: 477-513. 869

Gallois, J.L., Guyon-Debast, A., Lecureuil, A., Vezon, D., Carpentier, V., Bonhomme, S., 870

and Guerche, P. (2009). The Arabidopsis proteasome RPT5 subunits are essential for 871

gametophyte development and show accession-dependent redundancy. Plant Cell 21: 872

442-459. 873

Gaudinier, A., Zhang, L., Reece-Hoyes, J.S., Taylor-Teeples, M., Pu, L., Liu, Z., Breton, G., 874

Pruneda-Paz, J.L., Kim, D., Kay, S.A., Walhout, A.J., Ware, D., and Brady, S.M. 875

(2011). Enhanced Y1H assays for Arabidopsis. Nat. Methods 8: 1053-1055. 876

Groll, M., Schellenberg, B., Bachmann, A.S., Archer, C.R., Huber, R., Powell, T.K., Lindow, 877

S., and Dudler, R. (2008). A plant pathogen virulence factor inhibits the eukaryotic 878

proteasome by a novel mechanism. Nature 452: 755–758. 879

Hanssum, A., Zhong, Z., Rousseau, A., Krzyzosiak, A., Sigurdardottir, A., and Bertolotti, A. 880

(2014). An inducible chaperone adapts proteasome assembly to stress. Mol. Cell 55: 881

566-577. 882

Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress 883

and beyond. Nat. Rev. Mol. Cell Biol. 13: 89-102. 884

Hipp, M.S., Park, S.H., and Hartl, F.U. (2014). Proteostasis impairment in protein-misfolding 885

and aggregation diseases. Trends Cell Biol. 24: 506-514. 886

Howell, S.H. (2013). Endoplasmic reticulum stress responses in plants. Annu. Rev. Plant Biol. 887

64: 477-499. 888

Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis 889

of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4: 44-57. 890

Hudson, M.E., and Quail, P.H. (2003). Identification of promoter motifs involved in the network 891

of phytochrome A-regulated gene expression by combined analysis of genomic 892

sequence and microarray data. Plant Physiol. 133: 1605-1616. 893

Jensen, L.J., Kuhn, M., Stark, M., Chaffron, S., Creevey, C., Muller, J., Doerks, T., Julien, 894

P., Roth, A., Simonovic, M., Bork, P., and von Mering, C. (2009). STRING 8: a global 895

view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 896

37: D412-416. 897

27

Khaminets, A., Behl, C., and Dikic, I. (2015). Ubiquitin-dependent and independent signals in 898

selective autophagy. Trends Cell Biol. 16: 6-16.. 899

Kim, S.Y., Kim, S.G., Kim, Y.S., Seo, P.J., Bae, M., Yoon, H.K., and Park, C.M. (2007). 900

Exploring membrane-associated NAC transcription factors in Arabidopsis: implications 901

for membrane biology in genome regulation. Nucleic Acids Res. 35: 203-213. 902

Kim, Y.E., Hipp, M.S., Bracher, A., Hayer-Hartl, M., and Hartl, F.U. (2013). Molecular 903

chaperone functions in protein folding and proteostasis. Annu. Rev. Biochem. 82: 323-904

355. 905

Kim, Y.S., Kim, S.G., Park, J.E., Park, H.Y., Lim, M.H., Chua, N.H., and Park, C.M. (2006). A 906

membrane-bound NAC transcription factor regulates cell division in Arabidopsis. Plant 907

Cell 18: 3132-3144. 908

Kisselev, A.F., van der Linden, W.A., and Overkleeft, H.S. (2012). Proteasome inhibitors: an 909

expanding army attacking a unique target. Chem. Biol. 19: 99-115. 910

Kraft, C., Peter, M., and Hofmann, K. (2010). Selective autophagy: ubiquitin-mediated 911

recognition and beyond. Nat. Cell Biol. 12: 836-841. 912

Kurepa, J., Toh-e, A., and Smalle, J. (2008). 26S proteasome regulatory particle mutants have 913

increased oxidative stress tolerance. Plant J. 53: 102-114. 914

Kurepa, J., Wang, S., Li, Y., Zaitlin, D., Pierce, A.J., and Smalle, J.A. (2009). Loss of 26S 915

proteasome function leads to increased cell size and decreased cell number in 916

Arabidopsis shoot organs. Plant Physiol. 150: 178-189. 917

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. 918

Methods 9: 357-359. 919

Lee, K.H., Minami, A., Marshall, R.S., Book, A.J., Farmer, L.M., Walker, J.M., and Vierstra, 920

R.D. (2011). The RPT2 subunit of the 26S proteasome directs complex assembly, 921

histone dynamics, and gametophyte and sporophyte development in Arabidopsis. Plant 922

Cell 23: 4298-4317. 923

Lee, S., Seo, P.J., Lee, H.-J., and Park, C.-M. (2012). A NAC transcription factor NTL4 924

promotes reactive oxygen species production during drought-induced leaf senescence in 925

Arabidopsis. Plant J. 70: 831-844. 926

Lee, S., Lee, H.-J., Huh, S.U., Paek, K.-H., Ha, J.-H., and Park, C.-M. (2014). The Arabidopsis 927

NAC transcription factor NTL4 participates in a positive feedback loop that induces 928

programmed cell death under heat stress conditions. Plant Sci. 227: 76-83. 929

Li, F., and Vierstra, R.D. (2012). Autophagy: a multifaceted intracellular system for bulk and 930

selective recycling. Trends Plant Sci 17: 526-537. 931

28

Li, F., Chung, T., and Vierstra, R.D. (2014). AUTOPHAGY-RELATED11 plays a critical role in 932

general autophagy and senescence-induced mitophagy in Arabidopsis. Plant Cell 26: 933

788-807. 934

Lin, M.Y., Chai, K.H., Ko, S.S., Kuang, L.Y., Lur, H.S., and Charng, Y.Y. (2014). A positive 935

feedback loop between HEAT SHOCK PROTEIN101 and HEAT STRESS-936

ASSOCIATED 32-KD PROTEIN modulates long-term acquired thermotolerance 937

illustrating diverse heat stress responses in rice varieties. Plant Physiol. 164: 2045-2053. 938

Livnat-Levanon, N., Kevei, E., Kleifeld, O., Krutauz, D., Segref, A., Rinaldi, T., 939

Erpapazoglou, Z., Cohen, M., Reis, N., Hoppe, T., and Glickman, M.H. (2014). 940

Reversible 26S proteasome disassembly upon mitochondrial stress. Cell. Rep. 7: 1371-941

1380. 942

Lundgren, J., Masson, P., Mirzaei, Z., and Young, P. (2005). Identification and 943

characterization of a Drosophila proteasome regulatory network. Mol. Cell. Biol. 25: 944

4662-4675. 945

Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I., and Feldmann, H. (1999). Rpn4p acts as a 946

transcription factor by binding to PACE, a nonamer box found upstream of 26S 947

proteasomal and other genes in yeast. FEBS Lett. 450: 27-34. 948

Marshall, R.S., Li, F., Gemperline, D.C., Book, A.J., and Vierstra, R.D. (2015). Autophagic 949

degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor 950

RPN10 in Arabidopsis. Mol. Cell 58: 1053-1066. 951

Meiners, S., Heyken, D., Weller, A., Ludwig, A., Stangl, K., Kloetzel, P.M., and Kruger, E. 952

(2003). Inhibition of proteasome activity induces concerted expression of proteasome 953

genes and de novo formation of mammalian proteasomes. J. Biol. Chem. 278: 21517-954

21525. 955

Morimoto, R.I. (2008). Proteotoxic stress and inducible chaperone networks in 956

neurodegenerative disease and aging. Genes Dev. 22: 1427-1438. 957

Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi, A., Yabuta, Y., and Shigeoka, S. 958

(2009). Arabidopsis NAC transcription factor, ANAC078, regulates flavonoid 959

biosynthesis under high light. Plant Cell Physiol. 50: 2210-2222. 960

Nakashima, K., Takasaki, H., Mizoi, J., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2012). 961

NAC transciption factors in plant abiotic stress responses. Biochim. Biophys. Acta 1819: 962

97-103. 963

Nguyen, H.M., Schippers, J.H., Goni-Ramos, O., Christoph, M.P., Dortay, H., van der 964

Hoorn, R.A., and Mueller-Roeber, B. (2013). An upstream regulator of the 26S 965

proteasome modulates organ size in Arabidopsis thaliana. Plant J. 74: 25-36. 966

29

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005a). NAC transcription factors: 967

structurally distinct, funcitonally diverse. Trends Plant Sci. 10: 79-87. 968

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005b). DNA-binding specificity and 969

molecular functions of NAC transcription factors. Plant Sci. 169: 785-797. 970

Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time PCR. 971

Nucleic Acids Res. 29, 45. 972

Radhakrishnan, S.K., den Besten, W., and Deshaies, R.J. (2014). p97-dependent 973

retrotranslocation and proteolytic processing govern formation of active Nrf1 upon 974

proteasome inhibition. eLife 3: e01856. 975

Radhakrishnan, S.K., Lee, C.S., Young, P., Beskow, A., Chan, J.Y., and Deshaies, R.J. 976

(2010). Transcription factor Nrf1 mediates the proteasome recovery pathway after 977

proteasome inhibition in mammalian cells. Mol. Cell 38: 17-28. 978

Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for 979

differential expression analysis of digital gene expression data. Bioinformatics 26: 139-980

140. 981

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B., 982

Liu, L., Suchard, M.A., and Huelsenbeck, J.P. (2012). MrBayes 3.2: efficient Bayesian 983

phylogenetic inference and model choice across a large model space. Syst. Biol. 61: 984

539-542. 985

Rushton, P.J., Somssich, I.E., Ringler, P., and Shen, Q.J. (2010). WRKY transcription factors. 986

Trends Plant Sci. 15: 247-258. 987

Russell, J.D., Scalf, M., Book, A.J., Ladror, D.T., Vierstra, R.D., Smith, L.M., and Coon, J.J. 988

(2013). Characterization and quantification of intact 26S proteasome proteins by real-989

time measurement of intrinsic fluorescence prior to top-down mass spectrometry. PLoS 990

One 8: e58157. 991

Saldanha, A.J. (2004). Java Treeview: extensible visualization of microarray data. 992

Bioinformatics 20: 3246-3248. 993

Scharf, K.D., Berberich, T., Ebersberger, I., and Nover, L. (2012). The plant heat stress 994

transcription factor (Hsf) family: structure, function and evolution. Biochim. Biophys. Acta 995

1819: 104-119. 996

Seifert, A., Schofield, P., Barton, G.J., and Hay, R.T. (2015). Proteotoxic stress reprograms 997

the chromatin landscape of SUMO modification. Science Sig. 8: rs7. 998

Sha, Z., and Goldberg, A.L. (2014). Proteasome-mediated processing of Nrf1 is essential for 999

coordinate induction of all proteasome subunits and p97. Curr. Biol. 24: 1573-1583. 1000

30

Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., 1001

Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for 1002

integrated models of biomolecular interaction networks. Genome Res. 13: 2498-2504. 1003

Shirozu, R., Yashiroda, H., and Murata, S. (2015). Identification of minimum Rpn4-responsive 1004

elements in genes related to proteasome functions. FEBS Lett. 589: 933-940. 1005

Smalle, J., Kurepa, J., Yang, P., Babiychuk, E., Kushnir, S., Durski, A., and Vierstra, R.D. 1006

(2002). Cytokinin growth responses in Arabidopsis involve the 26S proteasome subunit 1007

RPN12. Plant Cell 14: 17-32. 1008

Smalle, J., Kurepa, J., Yang, P., Emborg, T.J., Babiychuk, E., Kushnir, S., and Vierstra, 1009

R.D. (2003). The pleiotropic role of the 26S proteasome subunit RPN10 in Arabidopsis 1010

growth and development supports a substrate-specific function in abscisic acid signaling. 1011

Plant Cell 15: 965-980. 1012

Sonoda, Y., Sako, K., Maki, Y., Yamazaki, N., Yamamoto, H., Ikeda, A., and Yamaguchi, J. 1013

(2009). Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit. 1014

Plant J. 60: 68-78. 1015

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: molecular 1016

evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 30: 2725-2729. 1017

van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., 1018

and Vierstra, R.D. (1996). The multiubiquitin-chain-binding protein Mcb1 is a 1019

component of the 26S proteasome in Saccharomyces cerevisiae and plays a 1020

nonessential, substrate-specific role in protein turnover. Mol. Cell. Biol. 16: 6020-6028. 1021

Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E., and Boeke, J.D. (1996). A reverse two- 1022

hybrid and one-hybrid system to detect dissociation of protein-protein and DNA-protein 1023

interactions. Proc. Natl. Acad. Sci. USA 93, 10315-10320. 1024

Wu, A., Allu, A.D., Garapati, P., Siddiqui, H., Dortay, H., Zanor, M.I., Asensi-Fabado, M.A., 1025

Munne-Bosch, S., Antonio, C., Tohge, T., Fernie, A.R., Kaufmann, K., Xue, G.P., 1026

Mueller-Roeber, B., and Balazadeh, S. (2012). JUNGBRUNNEN1, a reactive oxygen 1027

species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant 1028

Cell 24: 482-506. 1029

Xie, Y., and Varshavsky, A. (2001). RPN4 is a ligand, substrate, and transcriptional regulator 1030

of the 26S proteasome: a negative feedback circuit. Proc. Natl. Acad .Sci. USA 98: 1031

3056-3061. 1032

Yabuta, Y., Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi, A., and Shigeoka, S. 1033

(2010). Identification of recognition sequence of ANAC078 protein by the cyclic 1034

amplification and selection of targets technique. Plant Sig. Behav. 5: 695-697. 1035

31

Yabuta, Y., Osada, R., Morishita, T., Nishizawa-Yokoi, A., Tamoi, M., Maruta, T., and 1036

Shigeoka, S. (2011). Involvement of Arabidopsis NAC transcription factor in the 1037

regulation of 20S and 26S proteasomes. Plant Sci. 181: 421-427. 1038

Yang, B.-J., Han, X.-X., Yin, L.-L., Xing, M.-Q., Xu, Z.-H., and Xue, H.-W. (2016). Arabidopsis 1039

PROTEASOME REGULATOR1 is required for auxin-mediated suppression of 1040

proteasome activity and regulates auxin signaling. Nat. Commun. 1041

doi:10.1038/ncomms11388. 1042

Yang, P., Fu, H., Walker, J., Papa, C.M., Smalle, J., Ju, Y.M., and Vierstra, R.D. (2004). 1043

Purification of the Arabidopsis 26S proteasome: biochemical and molecular analyses 1044

revealed the presence of multiple isoforms. J. Biol. Chem. 279: 6401-6413. 1045

Zhang, X., and Bremer, H. (1996). Effects of Fis on ribosome synthesis and activity and on 1046

rRNA promoter activities in Escherichia coli. J. Mol. Biol. 259: 27-40. 1047

Zhou, J., Wang, J., Cheng, Y., Chi, Y.J., Fan, B., Yu, J.Q., and Chen, Z. (2013). NBR1-1048

mediated selective autophagy targets insoluble ubiquitinated protein aggregates in plant 1049

stress responses. PLoS Genet. 9: e1003196. 1050

1051

Figure 1. Both the Proteasome Inhibitor MG132 and Proteasome Mutants Increase the

Accumulation of Proteasome Components and Ub Conjugates in Arabidopsis.

Wild-type seedlings treated with 100 µM MG132 or untreated rpn10-1 and rpn12a-1 seedlings

were grown for 5 d. Total extracts were probed by immunoblotting with the indicated antibodies,

using anti-histone H3 antibodies to verify near equal protein loading.

(A) Levels of individual subunits of the proteasome. The open and closed arrowheads identify

the unprocessed and processed forms of the β1 subunit PBA1.

(B) Levels of Ub conjugates. Closed arrowheads locate free Ub and poly-Ub chains assembled

with varying numbers of Ub monomers. Bracket locates high molecular mass Ub conjugates.

Figure 2. Expression of Arabidopsis Proteasome Genes is Up-Regulated in Response to

Proteasome Stress.

(A) Expression of 26S proteasome subunit genes following proteasome inhibition with MG132

or in rpn10-1 or rpn12a-1 mutant backgrounds that compromise assembly. Total RNA from 5-d-

old seedlings, either untreated or treated for 3 and 24 hr with 100 µM MG132, was subjected to

qRT-PCR. The expression values were calculated using the ACT2 transcript as a reference

and normalized to those from untreated wild-type (WT) seedlings. Each bar represents the

average of at least three biological replicates (± SD).

(B) Effects of MG132 on the expression of proteasome promoter:GUS transgenes. Transgenic

WT seedlings expressing the fusions were grown for 3 d, treated for 1 d with 100 µM MG132,

and then incubated overnight with the X-Gluc substrate.

(C) Quantitative measure of proteasome promoter:GUS expression following MG132 treatment.

Ten-d-old seedlings were incubated overnight with or without 100 µM MG132 and homogenized,

and the GUS activity in the resulting cell extracts was assayed using the MUG substrate. Each

bar represents the analysis of at least 30 independent T1 lines, each assayed in triplicate (± SD).

The data in panels B and C for the RPT2a and RPT2b promoter-GUS fusions were reported

previously and are included here for comparison (Lee et al., 2011).

Figure 3. Characterization of the Proteasome-Stress Regulon (PSR) in Arabidopsis.

(A) Venn diagrams of transcripts that were differentially expressed significantly (p-value < 0.01,

FDR < 0.05) under three proteasome-stress conditions: wild-type (WT) after a 3-hr treatment

with 100 µM MG132, and the rpn10-1 and rpn12a-1 genetic backgrounds. The shared 119 up-

regulated (including RPN10 and RPN12a) and 33 down-regulated genes determined by edgeR

analysis comprise the PSR.

(B) A heat map of the PSR for the three treatments ranked by the fold-change in expression

obtained with the MG132-treated seedlings. The brackets indicate genes with more or less than

a two-fold increase (log2(fold-change) = 1) in expression as compared to WT. Categories of

enriched gene classes are indicated (TFs = transcription factors). See Supplemental Dataset 3

online for the full list.

(C) Expression change heat maps of 26S proteasome subunit and assembly factor genes after

proteasome stress as compared to untreated WT. EST values obtained from The Arabidopsis

Information Resource (TAIR v10) are listed.

Figure 4. Description of the MG132 Up-regulated Gene Interaction and Co-expression

Network.

(A) An interaction and co-expression map of Arabidopsis proteins encoded by MG132 up-

regulated genes. The connections reflect known protein/protein interactions and co-expression

data collected from the STRING database for the available 275 of the total 336 significantly up-

regulated genes (≥ 2-fold up as compared to untreated wild type) after a 3-hr exposure to 100

µM MG132. Classifications of major functional groups in the network are highlighted. Members

of the NAC transcription factor family are in red. Portions of the map enclosed by the dashed

lines are statistically enriched for genes with the denoted functional categories (DAVID, p-value

<0.01). If available, specific gene names were used instead of the TAIR locus identifier. See

Supplemental Dataset 3 for the full list.

(B) Members of the network that contain the consensus PRCE or MDM cis motifs within their

promoter regions (motif-containing gene nodes are in red).

(C) Sequence descriptions of the PRCE and MDM sequences as determined by MEME.

(D) Interactome maps focusing on the statistically significant central hubs present in the

proteasome, chaperone, and ‘detoxification’ clusters as determined by MCODE analysis, and

colored based on the presence of PRCE and/or MDM sequences.

Figure 5. NAC53 and NAC78 Are Closely Related and Physically Interact.

(A) A Bayesian phylogenetic tree of all 109 NAC proteins in Arabidopsis rooted to a

Physcomitrella patens NAC protein (gi_168025227). Nodes highlighted in red indicate the NTL

subclass with a predicted transmembrane-spanning motif. The same tree with the included

names for each protein is in Supplementary Figure 6 and a text file of the alignment used is

presented as Supplemental Data Set 5.

(B) Y2H assays showing that NAC53 and NAC78 homo- and hetero-dimerize. The full-length

proteins were expressed as N-terminal fusions with either the GAL4 activating (AD) or binding

(BD) domains. Shown are cells grown on selective medium lacking Leu and Trp, or lacking Leu,

Trp and His, and containing 50 mM 3-AT.

(C) Bimolecular fluorescence complementation (BiFC) analysis showing that NAC53 and

NAC78 homo- and hetero-dimerize in planta and partially localize to the nucleus. N.

benthamiana leaf epidermal cells were co-infiltrated with plasmids expressing the N- and C-

terminal fragments of YFP (nYFP and cYFP, respectively) fused to the N-terminus of NAC53 or

NAC78. Shown are reconstituted BiFC signals, as detected by confocal fluorescence

microscopy of leaf epidermal cells 36 hr after infiltration, along with DAPI staining of nuclei

(white arrowheads) and a bright field (BF) image of the cells. Scale bar = 10 µm.

(D) Y2H assays testing interactions between NAC53 and NAC78 and other NAC proteins within

the PSR. The assays were conducted as in panel (B) with the selective medium lacking Leu,

Trp and His, and containing 25 mM 3-AT.

Figure 6. NAC78 Overexpression Induces the Expression of Some Arabidopsis PSR

Genes.

Up-regulation of proteasome subunit and other PSR genes in seedlings expressing NAC78 from

an estradiol-inducible promoter. Total RNA from 6-d-old wild type (WT) and pEST:NAC78

seedlings incubated for 24 hr with or without 10 µM β-estradiol were subjected to RT-qPCR.

The expression values were calculated using ACT2 (black) and PP2A (grey) transcripts as

references and normalized to those obtained from untreated wild-type (WT) seedlings. Bars

represent the average of at least three biological replicates (± SD), each measured in triplicate.

Asterisks indicate significant differences between pEST:NAC78 and WT based on Student’s t-

test (p <0.05). Dashed lines indicate the average values for untreated WT seedlings.

Figure 7. Generation of Mutants Impacting NAC53 and NAC78.

(A) Diagrams of the NAC53 and NAC78 transcribed regions. Boxes represent coding regions

(colored) and predicted UTRs (white). The blue and orange boxes identify the DNA-binding

NAM domains and the predicted membrane-spanning regions, respectively. Lines represent

introns. The positions of the T-DNA insertions are indicated by the red triangles; their exact

locations in the amino acid sequences are indicated in Supplemental Figure 8.

(B) RT-PCR analysis of the NAC53 and NAC78 transcripts in the single and double mutants.

Total RNA isolated from wild-type (WT) or homozygous mutant plants was subjected to RT-PCR

using the primer pairs indicated in (A). RT-PCR with primers specific for ACT2 was included to

confirm analysis of equal amounts of cDNA.

Figure 8. Loss of NAC53 and NAC78 Compromises Activation of the Proteasome Stress

Regulon.

(A) RT-qPCR analysis of representative PSR mRNAs during proteasome stress. Total RNA

was extracted from 6-d-old wild-type (WT) and nac mutant seedlings after a 24-hr incubation

with or without 100 µM MG132. Transcript abundance was determined via RT-qPCR using the

ACT2 (black) and PP2A (grey) mRNAs as references and normalized to those obtained from

untreated wild-type (WT) seedlings. Bars represent the average of at least three biological

replicates (± SD), each measured in triplicate. Asterisks indicate significant differences between

the nac51-1 nac78-1 seedlings and WT (+MG132) based on Student’s t-test (p-value <0.05).

Dashed lines indicate the average values for untreated WT seedlings.

(B) Increased levels of several 26S proteasome subunits during proteasome stress depends on

NAC53 and NAC78. Seedlings were treated +/- MG132 as in panel (A) and the resulting crude

extracts were immunoblotted with the indicated antibodies. Histone H3 was included to confirm

near equal loading.

Figure 9. Plants Lacking Both NAC53 and NAC78 Are Hypersensitive to Proteasome

Inhibitors.

(A) Double homozygous nac53-1 nac78-1 plants are hypersensitive to MG132. Ten-day old

seedlings of the indicated genotypes were germinated and grown on MS medium plus sucrose

and containing either DMSO (control) or 30 or 50 µM MG132.

(B) Quantification of seedling fresh weight in shown in panel A.

(C) Growth inhibition of 6-d-old nac53-1 nac78-1 seedlings first germinated on MG132-free

medium and then transferred to medium containing 50 µM MG132 2 d after germination. .

(D) Double homozygous nac53-1 nac78-1 plants are strongly hypersensitive to bortezomib.

Seedlings were germinated and grown for 7 d on various concentrations of bortezomib.

(E) Fresh weight of 7-d-old seedlings of WT, nac53-1 nac78-1 and nac53-2 nac78-2 seedlings

grown on 1 µM bortezomib (Btz).

Asterisks in panels B, C, and E indicate a p-value <0.01 based on one-way ANOVA.

Parsed CitationsAlonso, J.M., and Stepanova, A.N. (2003). T-DNA mutagenesis in Arabidopsis. Methods Mol. Biol. 236: 177-188.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Amm, I., Sommer, T., and Wolf, D.H. (2014). Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system. Biochim. Biophys. Acta 1843: 182-196.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq: a Python framework to work with high-throughput sequencing data.Bioinformatics 31: 166-169.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bader, G.D., and Hogue, C.W. (2003). An automated method for finding molecular complexes in large protein interaction networks.BMC Bioinformatics 4: 2.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bailey, T.L., Williams, N., Misleh, C., and Li, W.W. (2006). MEME: discovering and analyzing DNA and protein sequence motifs.Nucleic Acids Res. 43: W369-W373.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bhattacharyya, S., Yu, H., Mim, C., and Matouschek, A. (2014). Regulated protein turnover: snapshots of the proteasome in action.Nat. Rev. Mol. Cell. Biol. 15: 122-133.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Bolger, A.M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Book, A.J., Gladman, N.P., Lee, S.S., Scalf, M., Smith, L.M., and Vierstra, R.D. (2010). Affinity purification of the Arabidopsis 26Sproteasome reveals a diverse array of plant proteolytic complexes. J. Biol. Chem. 285: 25554-25569.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Book, A.J., Smalle, J., Lee, K.H., Yang, P., Walker, J.M., Casper, S., Holmes, J.H., Russo, L.A., Buzzinotti, Z.W., Jenik, P.D., andVierstra, R.D. (2009). The RPN5 subunit of the 26S proteasome is essential for gametogenesis, sporophyte development, andcomplex assembly in Arabidopsis. Plant Cell 21: 460-478.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Chan, C.S., Guo, L., and Shih, M.C. (2001). Promoter analysis of the nuclear gene encoding the chloroplast glyceraldehyde-3-phosphate dehydrogenase B subunit of Arabidopsis thaliana. Plant Mol. Biol. 46: 131-141.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Coego, A., Brizuela, E., Castillejo, P., Ruiz, S., Koncz, C., del Pozo, J.C., Pineiro, M., Jarillo, J.A., Paz-Ares, J., Leon, J., and theTRANSPLANTA Consortium (2014). The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis oftranscription factors based on their conditional overexpression. Plant J. 77: 944-953.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Cuanalo-Contreras, K., Mukherjee, A., and Soto, C. (2013). Role of protein misfolding and proteostasis deficiency in proteinmisfolding diseases and aging. Int. J. Cell Biol. 2013: 638083.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

De Clercq, I., Vermeirssen, V., Van Aken, O., Vandepoele, K., Murcha, M.W., Law, S.R., Inze, A., Ng, S., Ivanova, A., Rombaut, D., vande Cotte, B., Jaspers, P., Van de Peer, Y., Kangasjarvi, J., Whelan, J., and Van Bruesegem, F. (2013). The membrane-bound NACtranscription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis.Plant Cell 25: 3472-3490.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Dohmen, R.J., Willers, I., and Marques, A.J. (2007). Biting the hand that feeds: Rpn4-dependent feedback regulation of proteasomefunction. Biochim. Biophys. Acta 1773: 1599-1604

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Farmer, L.M., Book, A.J., Lee, K.H., Lin, Y.L., Fu, H., and Vierstra, R.D. (2010). The RAD23 family provides an essential connectionbetween the 26S proteasome and ubiquitylated proteins in Arabidopsis. Plant Cell 22: 124-142.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Fatimababy, A.S., Lin, Y.L., Usharani, R., Radjacommare, R., Wang, H.T., Tsai, H.L., Lee, Y., and Fu, H. (2010). Cross-speciesdivergence of the major recognition pathways of ubiquitylated substrates for ubiquitin/26S proteasome-mediated proteolysis.FEBS J. 277: 796-816.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78: 477-513.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gallois, J.L., Guyon-Debast, A., Lecureuil, A., Vezon, D., Carpentier, V., Bonhomme, S., and Guerche, P. (2009). The Arabidopsisproteasome RPT5 subunits are essential for gametophyte development and show accession-dependent redundancy. Plant Cell 21:442-459.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Gaudinier, A., Zhang, L., Reece-Hoyes, J.S., Taylor-Teeples, M., Pu, L., Liu, Z., Breton, G., Pruneda-Paz, J.L., Kim, D., Kay, S.A.,Walhout, A.J., Ware, D., and Brady, S.M. (2011). Enhanced Y1H assays for Arabidopsis. Nat. Methods 8: 1053-1055.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Groll, M., Schellenberg, B., Bachmann, A.S., Archer, C.R., Huber, R., Powell, T.K., Lindow, S., and Dudler, R. (2008). A plantpathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature 452: 755-758.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hanssum, A., Zhong, Z., Rousseau, A., Krzyzosiak, A., Sigurdardottir, A., and Bertolotti, A. (2014). An inducible chaperone adaptsproteasome assembly to stress. Mol. Cell 55: 566-577.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nat. Rev. Mol. CellBiol. 13: 89-102.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hipp, M.S., Park, S.H., and Hartl, F.U. (2014). Proteostasis impairment in protein-misfolding and aggregation diseases. Trends CellBiol. 24: 506-514.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Howell, S.H. (2013). Endoplasmic reticulum stress responses in plants. Annu. Rev. Plant Biol. 64: 477-499.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVIDbioinformatics resources. Nat. Protoc. 4: 44-57.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Hudson, M.E., and Quail, P.H. (2003). Identification of promoter motifs involved in the network of phytochrome A-regulated geneexpression by combined analysis of genomic sequence and microarray data. Plant Physiol. 133: 1605-1616.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Jensen, L.J., Kuhn, M., Stark, M., Chaffron, S., Creevey, C., Muller, J., Doerks, T., Julien, P., Roth, A., Simonovic, M., Bork, P., andvon Mering, C. (2009). STRING 8: a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res.37: D412-416.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Khaminets, A., Behl, C., and Dikic, I. (2015). Ubiquitin-dependent and independent signals in selective autophagy. Trends Cell Biol.16: 6-16..

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim, S.Y., Kim, S.G., Kim, Y.S., Seo, P.J., Bae, M., Yoon, H.K., and Park, C.M. (2007). Exploring membrane-associated NACtranscription factors in Arabidopsis: implications for membrane biology in genome regulation. Nucleic Acids Res. 35: 203-213.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim, Y.E., Hipp, M.S., Bracher, A., Hayer-Hartl, M., and Hartl, F.U. (2013). Molecular chaperone functions in protein folding andproteostasis. Annu. Rev. Biochem. 82: 323-355.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kim, Y.S., Kim, S.G., Park, J.E., Park, H.Y., Lim, M.H., Chua, N.H., and Park, C.M. (2006). A membrane-bound NAC transcription factorregulates cell division in Arabidopsis. Plant Cell 18: 3132-3144.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kisselev, A.F., van der Linden, W.A., and Overkleeft, H.S. (2012). Proteasome inhibitors: an expanding army attacking a uniquetarget. Chem. Biol. 19: 99-115.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kraft, C., Peter, M., and Hofmann, K. (2010). Selective autophagy: ubiquitin-mediated recognition and beyond. Nat. Cell Biol. 12:836-841.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kurepa, J., Toh-e, A., and Smalle, J. (2008). 26S proteasome regulatory particle mutants have increased oxidative stress tolerance.Plant J. 53: 102-114.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Kurepa, J., Wang, S., Li, Y., Zaitlin, D., Pierce, A.J., and Smalle, J.A. (2009). Loss of 26S proteasome function leads to increased cellsize and decreased cell number in Arabidopsis shoot organs. Plant Physiol. 150: 178-189.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Langmead, B., and Salzberg, S.L. (2012). Fast gapped-read alignment with Bowtie 2. Nat. Methods 9: 357-359.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lee, K.H., Minami, A., Marshall, R.S., Book, A.J., Farmer, L.M., Walker, J.M., and Vierstra, R.D. (2011). The RPT2 subunit of the 26Sproteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis. PlantCell 23: 4298-4317.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lee, S., Seo, P.J., Lee, H.-J., and Park, C.-M. (2012). A NAC transcription factor NTL4 promotes reactive oxygen species productionduring drought-induced leaf senescence in Arabidopsis. Plant J. 70: 831-844.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lee, S., Lee, H.-J., Huh, S.U., Paek, K.-H., Ha, J.-H., and Park, C.-M. (2014). The Arabidopsis NAC transcription factor NTL4participates in a positive feedback loop that induces programmed cell death under heat stress conditions. Plant Sci. 227: 76-83.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li, F., and Vierstra, R.D. (2012). Autophagy: a multifaceted intracellular system for bulk and selective recycling. Trends Plant Sci 17:

526-537.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Li, F., Chung, T., and Vierstra, R.D. (2014). AUTOPHAGY-RELATED11 plays a critical role in general autophagy and senescence-induced mitophagy in Arabidopsis. Plant Cell 26: 788-807.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lin, M.Y., Chai, K.H., Ko, S.S., Kuang, L.Y., Lur, H.S., and Charng, Y.Y. (2014). A positive feedback loop between HEAT SHOCKPROTEIN101 and HEAT STRESS-ASSOCIATED 32-KD PROTEIN modulates long-term acquired thermotolerance illustratingdiverse heat stress responses in rice varieties. Plant Physiol. 164: 2045-2053.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Livnat-Levanon, N., Kevei, E., Kleifeld, O., Krutauz, D., Segref, A., Rinaldi, T., Erpapazoglou, Z., Cohen, M., Reis, N., Hoppe, T., andGlickman, M.H. (2014). Reversible 26S proteasome disassembly upon mitochondrial stress. Cell. Rep. 7: 1371-1380.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Lundgren, J., Masson, P., Mirzaei, Z., and Young, P. (2005). Identification and characterization of a Drosophila proteasomeregulatory network. Mol. Cell. Biol. 25: 4662-4675.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Mannhaupt, G., Schnall, R., Karpov, V., Vetter, I., and Feldmann, H. (1999). Rpn4p acts as a transcription factor by binding to PACE,a nonamer box found upstream of 26S proteasomal and other genes in yeast. FEBS Lett. 450: 27-34.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Marshall, R.S., Li, F., Gemperline, D.C., Book, A.J., and Vierstra, R.D. (2015). Autophagic degradation of the 26S proteasome ismediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol. Cell 58: 1053-1066.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Meiners, S., Heyken, D., Weller, A., Ludwig, A., Stangl, K., Kloetzel, P.M., and Kruger, E. (2003). Inhibition of proteasome activityinduces concerted expression of proteasome genes and de novo formation of mammalian proteasomes. J. Biol. Chem. 278: 21517-21525.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Morimoto, R.I. (2008). Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging. Genes Dev.22: 1427-1438.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi, A., Yabuta, Y., and Shigeoka, S. (2009). Arabidopsis NAC transcription factor,ANAC078, regulates flavonoid biosynthesis under high light. Plant Cell Physiol. 50: 2210-2222.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nakashima, K., Takasaki, H., Mizoi, J., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2012). NAC transciption factors in plant abioticstress responses. Biochim. Biophys. Acta 1819: 97-103.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Nguyen, H.M., Schippers, J.H., Goni-Ramos, O., Christoph, M.P., Dortay, H., van der Hoorn, R.A., and Mueller-Roeber, B. (2013). Anupstream regulator of the 26S proteasome modulates organ size in Arabidopsis thaliana. Plant J. 74: 25-36.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005a). NAC transcription factors: structurally distinct, funcitonally diverse.Trends Plant Sci. 10: 79-87.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Olsen, A.N., Ernst, H.A., Leggio, L.L., and Skriver, K. (2005b). DNA-binding specificity and molecular functions of NAC transcription

factors. Plant Sci. 169: 785-797.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Pfaffl, M.W. (2001). A new mathematical model for relative quantification in real-time PCR.

Nucleic Acids Res. 29, 45.

Radhakrishnan, S.K., den Besten, W., and Deshaies, R.J. (2014). p97-dependent retrotranslocation and proteolytic processinggovern formation of active Nrf1 upon proteasome inhibition. eLife 3: e01856.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Radhakrishnan, S.K., Lee, C.S., Young, P., Beskow, A., Chan, J.Y., and Deshaies, R.J. (2010). Transcription factor Nrf1 mediates theproteasome recovery pathway after proteasome inhibition in mammalian cells. Mol. Cell 38: 17-28.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Robinson, M.D., McCarthy, D.J., and Smyth, G.K. (2010). edgeR: a Bioconductor package for differential expression analysis ofdigital gene expression data. Bioinformatics 26: 139-140.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B., Liu, L., Suchard, M.A., and Huelsenbeck,J.P. (2012). MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61:539-542.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Rushton, P.J., Somssich, I.E., Ringler, P., and Shen, Q.J. (2010). WRKY transcription factors. Trends Plant Sci. 15: 247-258.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Russell, J.D., Scalf, M., Book, A.J., Ladror, D.T., Vierstra, R.D., Smith, L.M., and Coon, J.J. (2013). Characterization andquantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down massspectrometry. PLoS One 8: e58157.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Saldanha, A.J. (2004). Java Treeview: extensible visualization of microarray data. Bioinformatics 20: 3246-3248.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Scharf, K.D., Berberich, T., Ebersberger, I., and Nover, L. (2012). The plant heat stress transcription factor (Hsf) family: structure,function and evolution. Biochim. Biophys. Acta 1819: 104-119.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Seifert, A., Schofield, P., Barton, G.J., and Hay, R.T. (2015). Proteotoxic stress reprograms the chromatin landscape of SUMOmodification. Science Sig. 8: rs7.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sha, Z., and Goldberg, A.L. (2014). Proteasome-mediated processing of Nrf1 is essential for coordinate induction of all proteasomesubunits and p97. Curr. Biol. 24: 1573-1583.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., Schwikowski, B., and Ideker, T. (2003). Cytoscape:a software environment for integrated models of biomolecular interaction networks. Genome Res. 13: 2498-2504.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Shirozu, R., Yashiroda, H., and Murata, S. (2015). Identification of minimum Rpn4-responsive elements in genes related toproteasome functions. FEBS Lett. 589: 933-940.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Smalle, J., Kurepa, J., Yang, P., Babiychuk, E., Kushnir, S., Durski, A., and Vierstra, R.D. (2002). Cytokinin growth responses inArabidopsis involve the 26S proteasome subunit RPN12. Plant Cell 14: 17-32.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Smalle, J., Kurepa, J., Yang, P., Emborg, T.J., Babiychuk, E., Kushnir, S., and Vierstra, R.D. (2003). The pleiotropic role of the 26Sproteasome subunit RPN10 in Arabidopsis growth and development supports a substrate-specific function in abscisic acidsignaling. Plant Cell 15: 965-980.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Sonoda, Y., Sako, K., Maki, Y., Yamazaki, N., Yamamoto, H., Ikeda, A., and Yamaguchi, J. (2009). Regulation of leaf organ size by theArabidopsis RPT2a 19S proteasome subunit. Plant J. 60: 68-78.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version6.0. Mol. Biol. Evol. 30: 2725-2729.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

van Nocker, S., Sadis, S., Rubin, D.M., Glickman, M., Fu, H., Coux, O., Wefes, I., Finley, D., and Vierstra, R.D. (1996). Themultiubiquitin-chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays anonessential, substrate-specific role in protein turnover. Mol. Cell. Biol. 16: 6020-6028.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Vidal, M., Brachmann, R.K., Fattaey, A., Harlow, E., and Boeke, J.D. (1996). A reverse two-

hybrid and one-hybrid system to detect dissociation of protein-protein and DNA-protein

interactions. Proc. Natl. Acad. Sci. USA 93, 10315-10320.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Wu, A., Allu, A.D., Garapati, P., Siddiqui, H., Dortay, H., Zanor, M.I., Asensi-Fabado, M.A., Munne-Bosch, S., Antonio, C., Tohge, T.,Fernie, A.R., Kaufmann, K., Xue, G.P., Mueller-Roeber, B., and Balazadeh, S. (2012). JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell 24: 482-506.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Xie, Y., and Varshavsky, A. (2001). RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: a negativefeedback circuit. Proc. Natl. Acad .Sci. USA 98: 3056-3061.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yabuta, Y., Morishita, T., Kojima, Y., Maruta, T., Nishizawa-Yokoi, A., and Shigeoka, S. (2010). Identification of recognition sequenceof ANAC078 protein by the cyclic amplification and selection of targets technique. Plant Sig. Behav. 5: 695-697.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yabuta, Y., Osada, R., Morishita, T., Nishizawa-Yokoi, A., Tamoi, M., Maruta, T., and Shigeoka, S. (2011). Involvement of ArabidopsisNAC transcription factor in the regulation of 20S and 26S proteasomes. Plant Sci. 181: 421-427.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yang, B.-J., Han, X.-X., Yin, L.-L., Xing, M.-Q., Xu, Z.-H., and Xue, H.-W. (2016). Arabidopsis PROTEASOME REGULATOR1 isrequired for auxin-mediated suppression of proteasome activity and regulates auxin signaling. Nat. Commun.doi:10.1038/ncomms11388.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Yang, P., Fu, H., Walker, J., Papa, C.M., Smalle, J., Ju, Y.M., and Vierstra, R.D. (2004). Purification of the Arabidopsis 26Sproteasome: biochemical and molecular analyses revealed the presence of multiple isoforms. J. Biol. Chem. 279: 6401-6413.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhang, X., and Bremer, H. (1996). Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia

coli. J. Mol. Biol. 259: 27-40.Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

Zhou, J., Wang, J., Cheng, Y., Chi, Y.J., Fan, B., Yu, J.Q., and Chen, Z. (2013). NBR1-mediated selective autophagy targets insolubleubiquitinated protein aggregates in plant stress responses. PLoS Genet. 9: e1003196.

Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title

DOI 10.1105/tpc.15.01022; originally published online May 18, 2016;Plant Cell

Nicholas P Gladman, Richard S Marshall, Kwang-Hee Lee and Richard D. VierstraArabidopsis

The Proteasome Stress Regulon Is Controlled by a Pair of NAC Transcription Factors in

 This information is current as of February 26, 2020

 

Supplemental Data /content/suppl/2016/05/18/tpc.15.01022.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists