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THE LSM1-7 COMPLEX DIFFERENTIALLY REGULATES ARABIDOPSIS 1

TOLERANCE TO ABIOTIC STRESS CONDITIONS BY PROMOTING 2

SELECTIVE mRNA DECAPPING 3

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Carlos Perea-Resa1*, Cristian Carrasco-López1*, Rafael Catalá1, Veronika 6

Turečková2, Ondrej Novak2, Weiping Zhang3, Leslie Sieburth3, José Manuel 7

Jiménez-Gómez4,5 and Julio Salinas1,† 8

9 1Departamento de Biología Medioambiental, Centro Investigaciones Biológicas, CSIC, 10

28040 Madrid, Spain 11 2Laboratory of Growth Regulators, Institute of Experimental Botany Academy of 12

Sciences of the Czech Republic, v.v.i. & Palacký University, 78371 Olomouc, Czech 13

Republic 14 3Department of Biology, University of Utah, Salt Lake City, UT 84112, USA 15 4Department of Plant Breeding and Genetics, Max Planck Institute for Plant Breeding 16

Research, 50829 Cologne, Germany 17 5INRA, Institut Jean-Pierre Bourgin, UMR 1318, 78026 Versailles, France. 18

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21 * These authors contributed equally to this work 22

23 †Corresponding author: salinas@cib.csic.es 24

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Short title: LSM1-7 promotes selective mRNA decapping 26

Synopsis: A decapping activator complex ensures the adequacy and specificity of the 27

response of Arabidopsis to different abiotic stresses by promoting selective mRNA 28

degradation. 29

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The author responsible for distribution of materials integral to the findings presented in 31

this article in accordance with the policy described in the Instructions for Authors 32

(www.plant cell.org) is: Julio Salinas (salinas@cib.csic.es) 33

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ABSTRACT 35

In eukaryotes, the decapping machinery is highly conserved and plays an essential role 36

in controlling mRNA stability, a key step in the regulation of gene expression. Yet, the 37

role of mRNA decapping in shaping gene expression profiles in response to 38

environmental cues and the operating molecular mechanisms are poorly understood. 39

Here, we provide genetic and molecular evidence that a component of the decapping 40

machinery, the LSM1-7 complex, plays a critical role in plant tolerance to abiotic 41

stresses. Our results demonstrate that, depending on the stress, the complex from 42

Arabidopsis thaliana interacts with different selected stress-inducible transcripts 43

targeting them for decapping and subsequent degradation. This interaction ensures the 44

correct turnover of the target transcripts and, consequently, the appropriate patterns of 45

downstream stress-responsive gene expression that are required for plant adaptation. 46

Remarkably, among the selected target transcripts of the LSM1-7 complex are those 47

encoding NCED3 and NCED5, two key enzymes in ABA biosynthesis. We demonstrate 48

that the complex modulates ABA levels in Arabidopsis exposed to cold and high salt by 49

differentially controlling NCED3 and NCED5 mRNA turnover, which represents a new 50

layer of regulation in ABA biosynthesis in response to abiotic stress. Our findings 51

uncover an unanticipated functional plasticity of the mRNA decapping machinery to 52

modulate the relationship between plants and their environment. 53

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INTRODUCTION 55

Plants have evolved sophisticated mechanisms to perceive and rapidly respond to the 56

numerous abiotic stresses to which they are continuously challenged. A decisive 57

component of all stress responses is the ability to reprogram transcriptomes. 58

Transcriptome reprogramming in response to adverse environments has been generally 59

considered as a complex process mainly based on the modulation of transcriptional 60

activity of stress-related genes (Yamaguchi-Shinozaki and Shinozaki, 2006; Gujjar et 61

al., 2014; Nakashima et al., 2014). Recent data, however, indicate that 62

posttranscriptional mechanisms also influential to make fine and timely adjustments of 63

the plant transcriptomes to unfavorable environments (Guerra et al., 2015). The control 64

of mRNA stability, in particular, is critical for the regulation of gene expression in 65

response to abiotic stress. Stability determinants for intrinsically unstable eukaryotic 66

mRNAs include the 3’ poly (A) tail and the 5’ cap. Transcript abundance is then fine-67

tuned by a major pathway of cytoplasmic mRNA degradation involving deadenylation, 68

decapping and subsequent exoribonuclease 5’-3’ activity (Parker, 2012). mRNA 69

decapping is achieved in distinct cytoplasmic foci named processing bodies (P-bodies) 70

by the action of the decapping complex, a protein conglomerate highly conserved 71

among eukaryotes (Coller and Parker, 2004; Fillman and Lykke-Andersen, 2005; Xu et 72

al., 2006; Goeres et al., 2007). This complex includes the decapping enzyme DCP2 and 73

its activators DCP1, DCP5, VCS, DHH1, PAT1 and the LSM1-7 complex (Coller and 74

Parker, 2004; Fillman and Lykke-Andersen, 2005; Xu et al., 2006; Goeres et al., 2007; 75

Xu and Chua, 2009). mRNA decapping has been shown to play important roles in 76

development (Xu et al., 2006; Goeres et al., 2007; Xu and Chua, 2009). By contrast, the 77

role of the decapping complex in abiotic stress signaling is barely documented. To date, 78

only DCP1 and DCP5 have been described to associate under drought conditions to 79

promote drought tolerance in Arabidopsis (Xu and Chua, 2012). Whether mRNA 80

decapping is involved in regulating eukaryotic responses to other abiotic stresses and 81

the molecular mechanisms operating in mRNA decapping during such responses is 82

largely unknown. 83

The SM-like proteins (LSMs) are implicated in numerous aspects of RNA metabolism 84

in eukaryotes. The Arabidopsis genome contains eleven LSM genes encoding eight 85

central, highly evolutionarily conserved LSM proteins (LSM1-LSM8) (Perea-Resa et 86

al., 2012). LSM1, LSM3 and LSM6 are each duplicated and code for pairs of 87

functionally redundant proteins (LSM1A, B; LSM3A, B; LSM6A, B). In Arabidopsis, 88

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as in yeast and animals, the eight conserved LSM proteins are organized in two 89

heteroheptameric ring complexes, LSM1-7 and LSM2-8, specifically localized in the 90

cytoplasm and nucleus, and defined by the subunits LSM1 and LSM8, respectively. The 91

LSM2-8 complex functions in pre-mRNA splicing through U6 snRNA stabilization, and 92

ensures normal Arabidopsis development (Perea-Resa et al., 2012). Moreover, 93

alternative splicing analysis in lsm4 and sad1/lsm5 mutants uncovered that this complex 94

acts as a positive regulator of salt tolerance in Arabidopsis (Zhang et al., 2011; Cui et 95

al., 2014). The LSM1-7 complex is involved in accurate mRNA turnover by promoting 96

decapping and subsequent 5’-3’ degradation, and is required for the formation of P-97

bodies (Perea-Resa et al., 2012). In the yeast Saccharomyces cerevisiae, the LSM1-7 98

complex has been described to operate through the interaction of LSM1 with 99

oligoadenylated mRNAs that are then targeted for degradation (Chowdhury et al., 100

2007). The analysis of an Arabidopsis lsm1a lsm1b double mutant defective in LSM1A 101

and LSM1B expression unveiled that LSM1 proteins are essential for the assembly of 102

the LSM cytoplasmic complex and that this complex is needed for correct plant 103

development (Perea-Resa et al., 2012). The participation of the LSM1-7 decapping 104

activator complex in abiotic stress responses, however, has not yet been established in 105

any organism. Here, we demonstrate that this complex regulates Arabidopsis tolerance 106

to freezing, drought and high salt by modulating the transcriptome reprogramming that 107

takes place in response to these adverse conditions. More important, RNA 108

immunoprecipitation (RIP) assays revealed that, depending on the abiotic stress to 109

which the plant is subjected, the LSM cytoplasmic complex targets selected stress-110

inducible transcripts for decapping and degradation, thus controlling their levels and, 111

therefore, ensuring the adequate transcriptomic response. Interestingly, among the 112

selected mRNAs that are differentially targeted for decapping in response to low 113

temperature, water deficiency and high salt are those encoding NCED3 and NCED5, 114

two key enzymes in ABA biosynthesis. We show that, as a consequence, the LSM1-7 115

complex determines the appropriate levels of ABA in Arabidopsis plants exposed to 116

different abiotic stresses. Our finding thus reveal a previously unrecognized layer of 117

regulation in the biosynthesis of this phytohormone and uncover unexpected functional 118

plasticity of the mRNA decapping machinery in influencing the responses of plants to 119

their environment. 120

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RESULTS 122

Arabidopsis LSM1 proteins localize to P-bodies in response to abiotic stress 123

The molecular characterization of Arabidopsis LSM1 genes uncovered that they were 124

responsive to low temperature. Quantitative RT-PCR (qPCR) assays showed that 125

LSM1A and LSM1B transcripts accumulated in response to 4ºC, reaching a peak of 126

accumulation after 1 day of treatment (Figure 1 A). This accumulation was detected 127

broadly through the adult Arabidopsis plant (Supplemental Figure 1 A). Transcripts 128

corresponding to LSM2-7 proteins exhibited similar cold-induced accumulation as 129

LSM1 mRNAs (Supplemental Figure 1 B). LSM transcripts did not accumulate, 130

however, in plants exposed to other related abiotic stresses, such as drought (55% PEG) 131

or high salt (150 mM NaCl) (Supplemental Figure 1 C). We concluded that the 132

expression of genes encoding the Arabidopsis cytoplasmic LSM complex is positively 133

regulated by low temperature. 134

Since LSM1A and LSM1B transcripts accumulated in response to low temperature, we 135

assessed whether this accumulation was followed by an increase of the corresponding 136

proteins. Immunoblot experiments using Arabidopsis plants containing genomic LSM1-137

GFP fusions driven by the corresponding LSM1 promoters (LSM1PRO) (Perea-Resa et 138

al., 2012) showed that LSM1A-GFP and LSM1B-GFP proteins were notably more 139

abundant after some days of cold exposure (Figure 1 B). Consistent with the expression 140

results, water and salt stresses did not alter the levels of LSM1 proteins (Supplemental 141

Figure 2 A). Therefore, concomitantly with the accumulation of their transcripts, the 142

levels of LSM1 proteins also increase in response to low temperature. 143

Previous data revealed that heat treatment promotes the localization of LSM1A and 144

LSM1B to P-bodies (Perea-Resa et al., 2012). We decided to investigate if LSM1 145

proteins also localize to these foci under cold, drought or high salt conditions by 146

examining the distribution of green fluorescence in root cells from the LSM1APRO-147

LSM1A-GFP and LSM1BPRO-LSM1B-GFP plants. In according with earlier results 148

(Perea-Resa et al., 2012), confocal microscopy analysis indicated that, at 20ºC, GFP 149

activity was in both cases broadly distributed in the cytoplasm (Figure 1 C). However, 150

when transgenic plants were exposed to 4ºC, water deficiency, or salt stress, LSM1A-151

GFP and LSM1B-GFP fusion proteins were found aggregated in discrete cytoplasmic 152

spots that resembled P-bodies. After cycloheximide treatment, which causes loss of P-153

bodies (Sheth and Parker, 2003), no cytoplasmic foci were observed in any case, 154

suggesting that the detected LSM1-GFP spots indeed corresponded to P-bodies (Figures 155

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1C and Supplemental Figure 2 B). The identity of these foci was confirmed by 156

colocalization studies with DCP1, a protein that accumulates in P-bodies (Xu et al., 157

2006; Weber et al., 2008; Motomura et al., 2015). The examination of LSM1APRO-158

LSM1A-GFP and LSM1BPRO-LSM1B-GFP plants cotransformed with a 35S-RED 159

FLUORESCENT PROTEIN (RFP)-DCP1 fusion (Perea-Resa et al., 2012) demonstrated 160

that, in fact, LSM1A-GFP and LSM1B-GFP colocalized with RFP-DCP1 in root cells 161

exposed to 4ºC (Figure 1 D). Identical results were obtained when LSM1APRO-LSM1A-162

GFP and LSM1BPRO-LSM1B-GFP transgenic plants were subjected to drought or high 163

salt (Supplemental Figure 2 C), providing evidence that LSM1 proteins localize to P-164

bodies in response to abiotic stress. 165

166

LSM1 proteins have been reported to be crucial for P-body integrity under heat stress 167

conditions (Perea-Resa et al., 2012). We examined whether they were also required for 168

P-body formation in response to low temperature. With this aim, the subcellular 169

localization of DCP2 and VCS, two P-body markers (Sheth and Parker, 2003; Xu et al., 170

2006), was compared in Col-0 (WT) and lsm1a lsm1b double mutant seedlings 171

containing 35S-GFP-DCP2 or 35S-GFP-VCS fusions (Perea-Resa et al., 2012) exposed 172

to 4ºC. While in WT plants both proteins accumulated in P-bodies, a mostly dispersed 173

cytosolic signal was observed in lsm1a lsm1b mutants (Figure 1 E), showing that, in 174

addition to accumulating in P-bodies, LSM1 proteins are also essential for the formation 175

of these cytoplasmic foci in response to low temperature. Similar results were obtained 176

when plants were subjected to water and salt stresses (Supplemental Figure 2 D), 177

demonstrating that LSM1 proteins also localize to P-bodies under these abiotic stresses, 178

and are necessary for the assembly of these foci in response to water deficiency and 179

high salt. 180

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The LSM1-7 complex regulates Arabidopsis tolerance to abiotic stress 182

To further characterize the role of LSM1 proteins and, therefore, of the LSM 183

cytoplasmic complex in plant adaptation to abiotic stress, we evaluated their possible 184

implication in Arabidopsis tolerance to freezing, drought and salinity. Freezing 185

tolerance was analyzed in non-acclimated and cold-acclimated (7 d, 4ºC) lsm1a lsm1b 186

mutants exposed for 6 h to different freezing temperatures (Catalá et al., 2014). Non-187

acclimated mutants presented a similar capacity to tolerate freezing as the WT, the LT50 188

(temperature that causes 50% lethality) values being in both cases around -4.5ºC 189

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(Supplemental Figure 3 A). By contrast, the freezing tolerance of cold-acclimated lsm1a 190

lsm1b mutants was significantly higher than that of WT plants. In this case, the 191

determined LT50 values were -11ºC and -9ºC, respectively (Figure 2A). 192

Drought tolerance was examined in lsm1a lsm1b seedlings transferred to plates 193

containing 25% PEG (Verslues et al., 2006). After one week, they exhibited 194

significantly enhanced tolerance compared with WT seedlings as revealed by the 195

quantification of their fresh weights and lateral roots (Figure 2 B). Tolerance to high salt 196

was assayed in lsm1a lsm1b seedlings grown one further week on plates containing 150 197

mM NaCl (Verslues et al., 2006). Mutants displayed lower fresh weights and shorter 198

main roots than WT seedlings (Figure 2 C), manifesting more sensitivity to salt stress. It 199

is worth noting that lsm1a lsm1b plants grown on soil showed identical drought-tolerant 200

and salt-sensitive tolerance phenotypes (Supplemental Figures 3 B and 3 C). 201

In all cases, lsm1a lsm1b mutants complemented with either LSM1APRO-LSM1A-GFP 202

(c-lsm1a) or LSM1BPRO-LSM1B-GFP (c-lsm1b) fusions (Perea-Resa et al., 2012) had 203

WT capacity to cold acclimate and to tolerate water deficiency and high salt (Figure 2 204

and Supplemental Figure 3), establishing that the stress-tolerance phenotypes of lsm1a 205

lsm1b were a direct consequence of the absence of LSM1A and LSM1B expression. 206

Together, these data provided genetic evidence that the LSM1-7 complex is 207

differentially involved in plant tolerance to abiotic stresses. It negatively regulates the 208

ability of Arabidopsis to cold acclimate and tolerate drought, but functions as a positive 209

regulator of salt tolerance. 210

211

The Arabidopsis LSM cytoplasmic complex differentially regulates gene 212

expression in response to abiotic stress 213

Since the Arabidopsis LSM1-7 complex influences gene expression by promoting RNA 214

decapping and decay (Perea-Resa et al., 2012), we examined whether it might affect 215

gene expression in response to abiotic stresses as a first step to understanding how it 216

differentially regulates plant tolerance to environmental challenges. High-throughput 217

RNA sequencing (RNAseq) was used to estimate the effect of lsm1a and lsm1b 218

mutations on the transcriptomes of Arabidopsis plants subjected to cold, drought or 219

high-salt conditions. For this, we sequenced cDNA libraries prepared from stress-220

treated lsm1a lsm1b and WT plants. The resulting reads (±12 Mbp/sample) were 221

mapped to the Arabidopsis genome (TAIR10 version) and gene expression changes in 222

the double mutant evaluated. In each treatment, the top 1000 upregulated and top 1000 223

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downregulated genes in lsm1a lsm1b, based on fold-change ratios with respect to their 224

corresponding controls, were considered for analysis. The expression levels of the top 225

1000 upregulated genes in mutant plants exposed for 24 h to 4ºC were increased at least 226

3.4-fold compared with the WT (Supplemental Data Set 1). Remarkably, 53.3% of these 227

genes (533) have been reported to be induced (≥2 fold) in response to cold (Kilian et al., 228

2007) (Supplemental Data Set 2). Many of them, moreover, have been associated with 229

the development of cold acclimation (Vogel et al., 2005; Cuevas et al., 2008; Miura and 230

Furumoto, 2013; Shi et al., 2015) and, therefore, could account for the tolerance 231

phenotype of the double mutant. The upregulation of some of these genes, including 232

LEA5, LEA7, ZAT12, NCED3, WRKY33, RD28, ERF53, PR5, WRKY46 and NCED5, in 233

lsm1a lsm1b was verified by qPCR, validating the RNAseq results (Figure 3 A). 234

Compared with the WT, the top 1000 downregulated genes in cold-treated mutants 235

displayed a decreased expression of at least 2.1-fold (Supplemental Data Set 3). Of 236

them, 311 (31.1%) have been described to be cold-induced (Kilian et al., 2007) 237

(Supplemental Data Set 4), but none as functioning as a negative regulator of cold 238

acclimation. Representative cold-inducible genes that were downregulated in lsm1a 239

lsm1b were also validated by qPCR (Supplemental Figure 4 A). 240

Under conditions of water deficiency (10 h, 55% PEG), the transcript levels of the top 241

1000 upregulated genes in lsm1a lsm1b plants were found to be higher, by at least 2-242

fold, than in the WT (Supplemental Data Set 5). Interestingly, 372 of these genes 243

(37.2%) have been shown to be induced (≥2 fold) in response to drought (Kilian et al., 244

2007) (Supplemental Data Set 6) and some of them, such as ABR1, ANAC019 or 245

ERF53, to have a positive role in Arabidopsis tolerance to drought (Tran et al., 2004; 246

Pandey et al., 2005; Cheng et al., 2012). qPCR experiments confirmed the upregulation 247

in lsm1a lsm1b plants of these and other genes that promote drought tolerance (Figure 3 248

B), which would account for the drought tolerance phenotype of the mutants and 249

validate the RNAseq data. Otherwise, the top 1000 downregulated genes in mutant 250

plants subjected to drought exhibited at least 1.9-fold lower expression than in WT 251

plants (Supplemental Data Set 7). Among them, 32.6% (326) have been previously 252

shown to be induced by water deficiency (Kilian et al., 2007) (Supplemental Data Set 253

8), although none acting as negative regulator of drought tolerance. These results were 254

also validated by analyzing representative drought-inducible genes that were 255

downregulated in lsm1a lsm1b using qPCR (Supplemental Figure 4 B). 256

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When comparing the transcriptome profiles of mutant and WT plants exposed to salt 257

stress (10 h, 150 mM NaCl), the expression levels of the top 1000 upregulated genes in 258

lsm1a lsm1b were at least 2.4-fold higher than in the WT (Supplemental Data Set 9). In 259

this case, 47% of the genes (470) have been reported to be salt-induced (≥2-fold) 260

(Kilian et al., 2007) (Supplemental Data Set 10), and several of them, such as 261

ANAC092, ATGSTU17 or AHK5, to act as negative regulators of salt tolerance in 262

Arabidopsis (Balazadeh et al., 2010; Chen et al., 2012; Pham et al., 2012). The 263

upregulation of these and other salt-responsive genes was verified by qPCR, validating 264

the RNAseq data (Figure 3 C). The top 1000 downregulated genes in mutant plants 265

showed at least 2.1-fold lower expression levels than in the WT (Supplemental Data Set 266

11). Strikingly, 457 (45.7%) of these genes were salt-induced (Kilian et al., 2007) 267

(Supplemental Data Set 12) and some of them, including DFR, PHI1 or CNI1 have been 268

described to be positive regulators of Arabidopsis tolerance to high salt (Cui et al., 269

2014; Peng et al., 2014). qPCR experiments confirmed that the expression of these and 270

other salt-regulated genes was lower in lsm1a lsm1b than in WT plants after salt 271

treatment, validating once again the RNAseq data (Figure 3 D). The altered expression 272

of the negative and positive regulators of salt tolerance described above was fully 273

consistent with the salt-sensitive phenotype exhibited by the double mutant. 274

In all cases, when RNAseq experiments were validated by qPCR we found a strong, 275

statistically significant correlation (Pearson r ≥ 0.909; P-value ≤ 0.0001) between the 276

fold-change results obtained from both kinds of analysis (Supplemental Figure 5). 277

Consistent with the close relationship existing between plant responses to low 278

temperature, water deficiency and high salt (Kilian et al., 2007), several genes were 279

regulated by the LSM1-7 complex in response to more than one stress condition. 280

Nonetheless, most LSM1-7-regulated genes were stress specific (Supplemental Figure 6 281

and Supplemental Data Sets 1-12). c-lsm1a plants presented WT expression patterns for 282

all validated genes (Figure 3). Overall, these results indicated that the Arabidopsis LSM 283

cytoplasmic complex regulates Arabidopsis tolerance to abiotic stresses by differentially 284

modulating stress-responsive gene expression. 285

286

The Arabidopsis LSM1-7 complex regulates the turnover of selected stress-287

dependent transcripts in response to abiotic stresses 288

Considering the capacity of the LSM cytoplasmic complex to control transcript turnover 289

through the interaction of LSM1 with target mRNAs, promoting their decapping and 290

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subsequent degradation (Chowdhury et al., 2007), we reasoned that it could 291

differentially regulate gene expression in response to abiotic stresses by inducing the 292

decay of selected transcripts in a stress-dependent manner. To investigate this idea, we 293

first identified direct targets of the complex under low temperature, drought and high 294

salt conditions by means of RIP assays. These assays were performed with c-lsm1a 295

plants grown under control conditions or exposed to 4ºC, 55% PEG or 150 mM NaCl. 296

Transcripts interacting with LSM1A-GFP were co-immunoprecipitated (co-IP) with 297

anti-GFP antibody and then identified by qPCR. Arabidopsis transgenic plants 298

expressing a GFP-tagged GRP7 protein (Streitner et al., 2012) were also used in RIP 299

experiments as a control for the specificity of the detected interactions. Given the 300

function of the LSM1-7 complex, we expected some of its direct targets to be among 301

the mRNAs upregulated in the stress-treated lsm1a lsm1b mutants. Targets, therefore, 302

were searched for among the cold-, drought- and salt-inducible transcripts whose levels 303

we had confirmed were upregulated in lsm1a lsm1b mutants in response to low 304

temperature, water stress and high salt, respectively (Figure 3A-C). As anticipated, 305

several cold-induced transcripts, including LEA7, ZAT12, NCED3, WRKY33, RD28, 306

ERF53, WRKY46, and NCED5 were significantly enriched (≥2 fold) in co-IP RNA 307

samples from cold-treated c-lsm1a plants but not in the RNA samples from GRP7-GFP 308

plants (Figure 4 A), providing evidence that they were direct targets of the Arabidopsis 309

LSM cytoplasmic complex in response to low temperature. Some mRNAs, such as 310

ZAT12, NCED3, RD28, ERF53, and NCED5, were already significantly detected, 311

although at lower levels, in co-IP RNA samples recovered from unstressed c-lsm1a 312

plants (Figure 4A), suggesting that they were also targets of the complex under control 313

conditions. 314

Compared to those obtained from GRP7-GFP plants, co-IP RNA samples obtained 315

from c-lsm1a plants exposed to water deficiency revealed a specific and significant 316

enrichment in ABR1, ANAC019, RD28, ERF53, and WRKY46 transcripts (Figure 4 B). 317

Consistent with the results obtained when looking for targets of the complex under cold 318

conditions (Figure 4A), RD28 and ERF53 mRNAs were also found to be significantly 319

detected in co-IP RNA samples from control c-lsm1a plants (Figure 4B). In the case of 320

RNA samples collected from c-lsm1a and GRP7-GFP plants subjected to high salt, RIP 321

assays uncovered a specific and significant enrichment of ANAC092, AHK5, ERF53, 322

and NCED5 mRNAs in c-lsm1a samples (Figure 4 C). ERF53 and NCED5 transcripts 323

were also significantly detected in co-IP RNA samples from non-stressed c-lsm1a plants 324

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(Figure 4C). 325

326

As anticipated from the function of the LSM cytoplasmic complex in mRNA 327

degradation by promoting the decapping of its targets (Perea-Resa et al., 2012), rapid 328

amplification of cDNA ends (RACE)-qPCR experiments revealed that LEA7, ZAT12, 329

NCED3, WRKY33, RD28, ERF53, WRKY46, and NCED5 messengers accumulated in 330

their capped forms in lsm1a lsm1b mutants compared with WT plants exposed to low 331

temperature (Figure 5 A). Additionally, the levels of capped ZAT12, NCED3, RD28, 332

ERF53, and NCED5 transcripts were higher in mutant than in WT plants grown under 333

control conditions (Figure 5A). RACE-qPCR experiments with RNAs from water-334

stressed lsm1a lsm1b and WT plants showed that capped ABR1, ANAC019, RD28, 335

ERF53, and WRKY46 messengers were significantly increased in the mutants. 336

Moreover, as expected, RD28 and ERF53 transcripts were also augmented in their 337

capped forms in unstressed mutants (Figure 5 B). The levels of capped ANAC092, 338

AHK5, ERF53 and NCED5 mRNAs were clearly higher in lsm1a lsm1b than in WT 339

plants exposed to NaCl (Figure 5 C). Furthermore, capped ERF53 and NCED5 340

transcripts also accumulated in mutants grown under standard conditions (Figure 5C). 341

The capped forms of all mRNAs analyzed were unaltered in c-lsm1a plants (Figure 5). 342

All of these results strongly suggested that the LSM1-7 complex regulates the turnover 343

of different selected stress-responsive target transcripts, including both specific and 344

non-specific ones, in response to different abiotic stresses. The existence of non-specific 345

target mRNAs, such as RD28, ERF53, WRKY46 and NCED5, has been already 346

evidenced (Figures 4 and 5). The existence of specific targets was established by 347

assessing the affinity of the complex for LEA7, ZAT12, NCED3, WRKY33, ABR1, 348

ANAC019, ANAC092 and AHK5 transcripts under different unfavorable conditions. 349

Thus, RIP experiments showed that LEA7, ZAT12, NCED3 and WRKY33, direct targets 350

of the LSM cytoplasmic complex in response to low temperature (Figures 4A and 5A), 351

were not enriched in co-IP RNA samples from c-lsm1a plants exposed to water 352

deficiency and high salt (Figure 6). In turn, ABR1 and ANAC019, direct targets of the 353

complex in response to drought (Figures 4B and 5B), were not recovered with LSM1-354

GFP in response to cold and salt stresses (Figure 6). Similarly, ANAC092 and AHK5, 355

direct targets of the complex in response to high salt (Figures 4C and 5C), were not 356

enriched in co-IP RNA samples from c-lsm1a plants subjected to 4ºC and drought 357

(Figure 6). In addition, as expected from the RIP assays, the levels of capped LEA7, 358

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ZAT12, NCED3 and WRKY33 messengers did not increase in lsm1a lsm1b mutants 359

under drought and high salt conditions (Figure 7 A), and ABR1 and ANAC019 mRNAs 360

did not accumulate in their capped forms in mutant plants in response to low 361

temperature and salt stress (Figure 7 B). Moreover, capped ANAC092 and AHK5 362

transcripts did not increase in lsm1a lsm1b in response to cold and water deficiency 363

(Figure 7 C). In all cases, c-lsm1a plants presented equivalent levels of capped mRNAs 364

as WT plants (Figure 7). 365

Together, our data indicated that the Arabidopsis LSM1-7 complex differentially 366

regulates gene expression in response to abiotic stresses, and consequently plant 367

tolerance to these challenging situations, by modulating in each case the decay of 368

selected, both specific and non-specific, stress-responsive transcripts. The fact that some 369

of these mRNAs are, furthermore, direct targets of the complex at 20ºC, suggests that it 370

also regulates stress-responsive gene expression under control conditions. 371

372

The Arabidopsis LSM cytoplasmic complex regulates ABA biosynthesis in 373

response to abiotic stress 374

Interestingly, among the mRNAs whose decay was differentially regulated by the 375

Arabidopsis LSM1-7 complex in response to abiotic stresses were those encoding 376

NCED3 and NCED5, two key enzymes in the biosynthesis of the phytohormone 377

abscisic acid (ABA) that plays a critical role in plant responses to abiotic stresses (Jia et 378

al., 2002; Cuevas et al., 2008; Wu et al., 2009; Frey et al., 2012). In fact, while NCED3 379

transcripts were direct targets of the complex specifically after cold treatment (Figures 6 380

and 7A), those of NCED5 were direct targets under both cold and high-salt conditions 381

(Figures 4A, 4C, 5A and 5C) but not following drought (Supplemental Figure 7). These 382

data strongly suggested that the LSM1-7 complex might differentially regulate ABA 383

biosynthesis in Arabidopsis in response to abiotic stresses. To test this prediction, we 384

measured the ABA content of WT and lsm1a lsm1b mutant plants grown under control 385

conditions or exposed to low temperature (4ºC), water deficiency (55% PEG), or high 386

salt (150 mM NaCl). When exposed to low temperature, ABA levels increased in both 387

WT and lsm1a lsm1b mutants but the increase was significantly higher in mutants than 388

in WT plants (Figure 8 A). Unstressed lsm1a lsm1b mutants also showed higher levels 389

of ABA than WT plants (Figure 8A). These levels, however, were much lower than 390

those detected after cold treatment, paralleling the lower binding affinity of the LSM 391

cytoplasmic complex for NCED3 and NCED5 mRNAs at 20ºC compared with 4ºC 392

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(Figures 4A and 5A). c-lsm1a plants exhibited similar ABA content as WT plants 393

(Figure 8A), providing evidence that the Arabidopsis LSM1-7 complex attenuates ABA 394

biosynthesis in response to low temperature and, to a lesser extent, under standard 395

conditions by promoting the decay of NCED3 and NCED5 messengers. 396

ABA levels also increased very prominently in Arabidopsis WT plants exposed to water 397

stress or high salt (Figure 8 B). In the case of lsm1a lsm1b mutants, water deficiency 398

caused an accumulation of ABA similar to that of WT plants. By contrast, salt stress 399

provoked an accumulation of ABA that was significantly higher than in WT plants. c-400

lsm1a plants exhibited similar ABA content as the WT in response to both drought and 401

salt stresses (Figure 8B). Therefore, as predicted from the expression and RIP analyses, 402

the LSM cytoplasmic complex also mediates the biosynthesis of ABA in Arabidopsis 403

plants exposed to salt stress but it is not involved in ABA biosynthesis in response to 404

water stress. Collectively, these findings demonstrated that the Arabidopsis LSM1-7 405

complex guarantees adequate levels of ABA in Arabidopsis plants exposed to different 406

abiotic stresses by differentially regulating the decay of NCED3 and NCED5 transcripts. 407

408

DISCUSSION 409

The control of mRNA stability is a key step in the regulation of gene expression. 410

However, the effect of mRNA decapping on plant transcriptome reprogramming in 411

response to abiotic stress, and its importance to stress tolerance are largely unknown. 412

Here, we show that the LSM1-7 decapping activator complex serves as an integration 413

node of regulatory pathways mediating plant tolerance to abiotic stresses. It regulates 414

Arabidopsis tolerance to freezing, drought and high salt by interacting with selected, 415

specific and nonspecific, stress-inducible transcripts under each stress condition to 416

promote their decapping and subsequent degradation, which ultimately ensures the 417

appropriate patterns of downstream stress-responsive gene expression. Interestingly, 418

one of the regulatory pathways mediated by the LSM1-7 complex is the one leading to 419

ABA biosynthesis. We demonstrate that this complex modulates the levels of ABA in 420

response to adverse environmental situations through the differential regulation of 421

NCED3 and NCED5 mRNA turnover. 422

The expression analysis described in this work revealed that LSM1-7 genes are 423

differentially regulated in response to abiotic stresses. LSM1-7 mRNAs accumulate in 424

response to cold but not in response to drought or high salt. The mechanisms underlying 425

this regulation are still under investigation. Consistent with the expression results, an 426

14

increase in LSM1A and LSM1B protein levels was observed only in plants exposed to 427

low temperature. Remarkably, however, Arabidopsis LSM1 proteins were found to 428

localize to P-bodies in response to the different stresses assayed, indicating that the 429

molecular mechanisms mediating LSM1 localization to these foci do not depend on 430

protein levels. In this way, DCP1 has also been reported to localize to P-bodies under 431

stress conditions even though its levels do not increase (Motomura et al., 2015). These 432

findings suggest that the localization of LSM1 to P-bodies, like that of DCP1 and other 433

Arabidopsis RNA-decay related proteins (Merret et al., 2013; Motomura et al., 2015), is 434

induced in plants exposed to abiotic stresses. Moreover, our results show that LSM1 435

proteins are required for P-body formation under challenging situations, indicating that 436

the assembly of these foci is also promoted by adverse environmental conditions. How 437

abiotic stresses regulate the cytoplasmic dynamics of proteins involved in RNA 438

degradation and, ultimately, P-body formation is poorly understood. Phosphorylation of 439

different components of human and Arabidopsis mRNA decapping machineries by 440

mitogen-activated protein kinases (MPKs) during stress responses seems to be 441

necessary for their cytoplasmic localization and for P-body assembly (Rzeczkowski et 442

al., 2011; Roux et al., 2015). The sequence of LSM1 proteins (Perea-Resa et al., 2012) 443

contains a consensus motif S/T-P for phosphorylation by MPK. It is, therefore, tempting 444

to speculate that the subcellular localization of LSM1 proteins could be regulated by 445

MPKs in response to abiotic stresses. 446

Our results demonstrate that the LSM1-7 complex differentially regulates Arabidopsis 447

tolerance to challenging environmental situations. In fact, the complex restrains the 448

plant’s capacity to cold acclimate and tolerate drought while promoting its tolerance to 449

high salt. The implication of a single component of the mRNA decay apparatus in a 450

range of abiotic stress responses has not been shown in any system, and uncovers the 451

enormous potential of this apparatus to precisely modulate the adaptation of a given 452

organism to its surroundings. The LSM cytoplasmic complex, therefore, seems to serve 453

as a regulatory node where pathways mediating abiotic stress responses converge and 454

integrate to guarantee the precise development of Arabidopsis tolerance to freezing, 455

drought and salinity. Our results, furthermore, provide evidence that the LSM1-7 456

complex operates in Arabidopsis tolerance to abiotic stresses by modulating stress-457

responsive gene expression. Hundreds of genes are specifically regulated by the 458

complex under low temperature, water deficiency or high salt. Moreover, the complex 459

also regulates the expression of many genes in response to more than one abiotic stress, 460

15

which indicates that it mediates gene expression under different unfavorable situations 461

via specific and shared signaling pathways. Interestingly, numerous specific and 462

nonspecific genes that are regulated by the LSM cytoplasmic complex under each stress 463

condition have been described to have a role in plant tolerance to the relevant condition, 464

substantiating a major and differential function for the complex in plant tolerance to 465

abiotic stresses by ensuring appropriate patterns of stress-responsive gene expression. 466

DCP5 has also been shown to act in abiotic stress tolerance through the regulation of 467

stress-related gene expression. It enhances Arabidopsis tolerance to dehydration by 468

modulating numerous dehydration-responsive genes (Xu and Chua, 2012). Whether 469

DCP5 and/or other components of the Arabidopsis mRNA decay apparatus have the 470

ability, similar to the LSM1-7 complex, to differentially regulate gene expression in a 471

stress-dependent manner remains unknown. 472

The role of the LSM cytoplasmic complex in the transcriptome reprogramming that 473

takes place in Arabidopsis when subjected to abiotic stresses is a consequence of its 474

capacity to modulate the turnover of stress-selected target transcripts. In fact, by 475

analyzing the interaction between mRNAs whose levels are attenuated by the complex 476

in response to abiotic stresses and the LSM1 protein, we have been able to identify 477

transcripts that are direct targets of the complex when Arabidopsis plants are exposed to 478

low temperature, drought or high salt, and may act as positive or negative regulators of 479

gene expression. Stress-selected targets are then decapped and subsequently degraded. It 480

is noteworthy that most of them are specific for each stress while others, however, are 481

selected by the complex in response to various adverse conditions. Modulating the 482

differential accumulation of the selected specific and nonspecific target transcripts, 483

therefore, ensures adequate stress-responsive gene expression under each abiotic stress, 484

and ultimately contributes to the capacity of the LSM1-7 complex to regulate 485

Arabidopsis tolerance to freezing, drought and salinity. The Arabidopsis decapping 486

activator DCP5 has also been reported to select target transcripts in response to an 487

individual abiotic stress, namely water deficiency (Xu and Chua, 2012). Our results, 488

nonetheless, unveil a more intricate scenario when considering the regulation of gene 489

expression and plant adaptation by the mRNA decay apparatus under unfavorable 490

circumstances. They provide evidence that one component can select different targets 491

depending on the stress situations, highlighting a previously unknown regulatory 492

mechanism of mRNA turnover during stress responses. 493

16

Remarkably, among the selected stress-dependent transcripts that are triggered by the 494

Arabidopsis LSM cytoplasmic complex for decapping in response to abiotic stresses, 495

we found those encoding NCED3 and NCED5, two key enzymes in ABA biosynthesis 496

(Tan et al., 2003; Frey et al., 2012). NCED3 and NCED5 messengers are direct targets 497

of the complex under low temperature but not under drought conditions. Under salt 498

stress only NCED5 is targeted by the LSM1-7 complex. ABA is biosynthesized de novo 499

in plants subjected to cold, drought or salinity, and plays a pivotal role in plant 500

responses to abiotic stresses (Jia et al., 2002; Cuevas et al., 2008; Wu et al., 2009). In 501

Arabidopsis, the accumulation of ABA after abiotic stress exposure seems to be the 502

result of NCED3 and NCED5 expression (Tan et al., 2003; Frey et al., 2012). So far, 503

available data had indicated that this expression is regulated at the transcriptional level 504

(Barrero et al., 2006; Cuevas et al., 2008; Jiang et al., 2012). The results reported here 505

show that the mRNA decay machinery, and in particular the decapping apparatus, has a 506

crucial function in establishing the levels of NCED3 and NCED5 mRNAs when plants 507

are exposed to stressful circumstances, indicating that these levels are also regulated by 508

post-transcriptional mechanisms. Our findings, therefore, reveal a layer of regulation of 509

ABA biosynthesis in response to abiotic stresses. Consistent with its role in 510

differentially modulating the turnover of NCED3 and NCED5 transcripts under adverse 511

environmental circumstances, our data also demonstrate that the LSM cytoplasmic 512

complex contributes to establish the appropriate levels of ABA under cold and high salt 513

conditions. It is not involved, however, in modulating the accumulation of ABA caused 514

by water deficiency. 515

Based on the results described in this work, a hypothetical model for the function of the 516

Arabidopsis LSM1-7 complex in plant adaptation to abiotic stresses is presented in 517

Figure 9. In response to low temperature, drought and salinity, the complex would 518

preferentially localize to P-bodies. There, depending on the stress, the complex would 519

interact with selected transcripts promoting their decapping and subsequent 5’-3’ 520

degradation. The vast majority of selected transcripts are stress specific; others, 521

however, interact with the complex under more than one stress condition. In most cases, 522

target transcripts would correspond to stress-inducible genes, and some of them, 523

moreover, encode proteins involved in regulating, positively or negatively, Arabidopsis 524

tolerance to abiotic stresses. The degradation of the LSM1-7 target transcripts selected 525

in response to a given abiotic stress would have, in turn, a substantial effect on the 526

downstream stress-regulated gene expression that would contribute to shape the 527

17

transcriptome reprogramming required for plant adaptation to that stress. In conclusion, 528

the results presented here reveal that the LSM1-7 decapping activator complex plays a 529

critical role in plant adaptation to abiotic stresses by modulating the turnover of selected 530

stress-specific and nonspecific target transcripts, and, consequently, stress-regulated 531

gene expression. Identifying the molecular mechanisms whereby the LSM cytoplasmic 532

complex selects different targets depending on the stress conditions is a clear goal of 533

future studies. 534

535

18

METHODS 536

Plant Materials, Growth Conditions, Treatments and Tolerance Assays 537

Arabidopsis Columbia (Col-0) ecotype was used in all experiments. The lsm1a lsm1b 538

double mutants, transgenic lines LSM1APRO-LSM1A-GFP (c-lsm1a) and LSM1BPRO-539

LSM1B-GFP (c-lsm1b), lines LSM1APRO-LSM1A-GFP and LSM1BPRO-LSM1B-GFP 540

expressing 35S-DCP1-RFP construct, and lsm1a lsm1b lines expressing the 35S-GFP-541

DCP2 or 35S-GFP-VCS constructs were previously described (Perea-Resa et al., 2012). 542

The Arabidopsis transgenic line expressing the GRP7PRO-GRP7-GFP fusion (Streitner 543

et al., 2012) was kindly provided by Dorothee Staiger (University of Bielefeld, 544

Germany). Plants were grown at 20ºC under long-day photoperiod (16 h of cool-white 545

fluorescent light, photon flux of 90 µmol m-2s-1) in pots containing a mixture of organic 546

substrate and vermiculite (3:1 v/v) or in Petri dishes containing Murashige and Skoog 547

medium supplemented with 1% sucrose (GM) and solidified with 0.8% (w/v) agar. 548

Low-temperature treatment for gene expression and immunoblot analyses was 549

performed by transferring plants growing in Petri dishes or on soil under control 550

conditions to a growth chamber set to 4°C for different periods of time under long-day 551

photoperiods with a photon flux of 40 mmol m-2s-1. For the rest of experiments, plants 552

were always exposed to 4ºC for 24 h. In all cases, water and salt stress treatments were 553

accomplished by transferring plants growing in Petri dishes under standard conditions to 554

plates containing GM medium supplemented with 55% PEG or 150 mM NaCl, 555

respectively, for 10 h. For immunoblot analysis, water and salt stress treatments were 556

also carried out for 24 h. Tolerance to freezing temperatures was determined on 2-week-557

old plants grown on soil as described (Catalá et al., 2014). In vitro tolerance to water 558

(25% PEG) and salt (150 mM NaCl) stresses was assayed on 7-day-old seedlings grown 559

on GM medium as reported (Verslues et al., 2006). The tolerance of 2-week-old plants 560

growing on soil to these stresses was estimated as the number of surviving individuals 561

after 10 days of water deprivation and 5 days of re-watering, or after watering with 250 562

mM NaCl for 10 days. In all cases, data reported are expressed as means of three 563

independent experiments with 50 plants each. 564

565

Microscopic Analysis 566

Subcellular localization of LSM1A-GFP, LSM1B-GFP, DCP1-RFP, GFP-DCP2 and 567

GFP-VCS fusion proteins was performed by confocal microscopy in roots from 6-day-568

old transgenic seedlings grown under control or subjected to stress conditions as 569

19

described above. Treatments with cycloheximide were performed by incubating stressed 570

seedlings in GM liquid medium supplemented with 200 µg/ml cycloheximide for 2 h at 571

20ºC or 4ºC. Microscopy images were collected using a Confocal Laser Spectral 572

microscope TCS SP5 (Leica Mycrosystem, Wetzlar, Germany). The excitation lines for 573

imaging GFP and RFP fusions were 488 and 561 nm, respectively. 574

575

ABA Measurements 576

ABA levels were determined in 20 mg of 2-week-old WT, lsm1a lsm1b and c-lsm1a 577

plants grown under control or stress conditions as described (Turečková et al., 2009). 578

All experiments were repeated as four biological replicates employing 10 pmol stable 579

isotope-labelled standard to validate the LC-MS method. 580

581

Immunoblot Analysis 582

Total protein was extracted from 2-week-old Arabidopsis LSM1APRO-LSM1A-GFP, 583

LSM1BPRO-LSM1B-GFP and WT plants grown under control or stress conditions as 584

reported (Catalá et al., 2014). Monoclonal anti-GFP (ab290; GR158277-1, Abcam) was 585

used as primary antibody, and horseradish peroxidase-conjugated anti-rabbit as 586

secondary antibody. Coomassie staining of the large subunit of Rubisco was used as a 587

loading control. All assays were carried out in triplicate employing three independent 588

protein samples. 589

590

Gene Expression Analysis and RNAseq Experiments 591

For gene expression, qPCR experiments were performed as described (Catalá et al., 592

2014). In all cases, the relative expression values were determined using AT4G24610 as 593

a control (Czechowski et al., 2005). Primers used are listed in Supplemental Data Set 594

13. All reactions were performed in triplicate employing three independent RNA 595

samples. 596

For RNAseq experiments, total RNA was extracted from 2-week-old WT and lsm1a 597

lsm1b plants subjected to stress conditions (see above) using TRIzol® Reagent (Life 598

Technologies) and cleaned with RNeasy Plant Mini Kit (Quiagen). cDNA libraries were 599

generated from 3 independent RNA preparations each. RNA quality, library preparation 600

and subsequent sequencing were performed by the staff of the Beijing Genome Institute 601

(BGI). RNA-seq reads were aligned to the TAIR10 Col-0 reference genome using 602

20

TopHat2 (Kim et al., 2013) with default parameters. Uniquely mapped reads 603

(Supplemental Data Set 14) were counted per representative gene model (excluding 604

introns) according to the TAIR10 annotation using custom R scripts. Only genes with 605

rpkm greater than 1 in at least one sample were used for differential expression analysis 606

between WT and lsm1a lsm1b plants in each condition using DEseq2 (Love et al., 607

2014). This package internally estimates size factors for each sample, calculates 608

dispersion for each gene and then fits a negative binomial GLM to detect differentially 609

expressed genes taking into account the size factors and dispersion values. 610

611

Capped mRNA Analysis and RIP Experiments 612

Capped transcript levels were determined essentially as reported (Perea-Resa et al., 613

2012). Total RNA from 2-week-old plants grown on GM plates under control or stress 614

conditions (see above) was employed. Capped mRNAs were quantified by qRT-PCR 615

using reverse specific-gene primers (Supplemental Data Set 13) and the 5´RACE Inner 616

Primer included in the First Choice RLM-RACE kit (Ambion). The levels of capped 617

transcripts were corrected with respect to the levels of their corresponding total 618

transcripts, determined by qPCR using gene-specific primers (Supplemental Data Set 619

13), and represented relative to the levels in WT plants under control conditions for 620

each gene. In all cases, reactions were performed in triplicate employing three 621

independent RNA samples. 622

In vivo RIP experiments were carried out essentially as described (Streitner et al., 2012). 623

Extracts from 2-week-old c-lsm1a and GRP7-GFP plants grown on GM plates under 624

control or stress conditions (see above) were incubated overnight at 4ºC with GFP-Trap 625

beads (Chromotek). Beads were subsequently washed three times prior to RNA 626

extraction using TRIzol® Reagent (Life Technologies). RNAs resulting from inputs, 627

prepared in parallel, and from coprecipitated samples, were subsequently quantified by 628

qPCR (see above) using gene-specific primers (Supplemental Data Set 13). Transcript 629

levels in coprecipitated samples from c-lsm1a plants were corrected with respect to their 630

corresponding input values and represented relative to their levels in coprecipitated 631

samples from GRP7-GFP plants for each gene. All assays were performed in triplicate 632

employing three independent RNA samples. 633

634

Statistical Analysis 635

21

Data sets were analyzed using Prism 6 software (GraphPad Software Inc., USA). 636

Comparisons between two groups were made using Student's t‐test. Comparisons 637

between multiple groups were made using one‐way or two-way analysis of variance 638

(ANOVA) test depending whether one or two different variables were considered, 639

respectively. Correlation plots were computed using Prism 6 software (GraphPad 640

Software Inc., USA) from log2-transformed values and shown the relationship between 641

qRT-PCR results (X-axis) and the corresponding data from RNA-seq (y-axis). 642

643

Accession Numbers 644

Sequence data from this article can be found in the GeneBank/EMBL data libraries 645

under the accession numbers listed in Supplemental Data Set 15. The full name of the 646

genes mentioned in this article are also included in Supplemental Data Set 15. The 647

complete genome-wide data from this publication have been submitted to the GEO 648

database (http://www.ncbi.nlm.nih.gov/geo/) and assigned the identifier accession 649

GSE70491. 650

651

Supplemental Data 652

Supplemental Figure 1. Expression analysis of Arabidopsis LSM1-7 genes in response 653

to abiotic stresses. 654

Supplemental Figure 2. Arabidopsis LSM1 proteins do not accumulate in response to 655

drought and salinity but localize to P-bodies under these abiotic stresses. 656

Supplemental Figure 3. Constitutive freezing tolerance and tolerance of drought and 657

salinity of lsm1a lsm1b plants grown on soil. 658

Supplemental Figure 4. Expression levels of genes downregulated in lsm1a lsm1b 659

plants in response to cold and drought. 660

Supplemental Figure 5. Correlation between RNAseq and qPCR results. 661

Supplemental Figure 6. Venn diagrams of de-regulated transcripts in lsm1a lsm1b 662

plants under cold, drought and salt stresses. 663

Supplemental Figure 7. The Arabidopsis LSM1-7 complex differentially interacts with 664

and promotes the decapping of NCED5 transcripts in response to drought and high salt. 665

Supplemental Data Set 1. Top 1000 upregulated genes in lsm1a lsm1b mutants in 666

response to low temperature. 667

Supplemental Data Set 2. Cold-inducible genes upregulated in lsm1a lsm1b mutants in 668

response to low temperature. 669

22

Supplemental Data Set 3. Top 1000 downregulated genes in lsm1a lsm1b mutants in 670

response to low temperature. 671

Supplemental Data Set 4. Cold-inducible genes downregulated in lsm1a lsm1b 672

mutants in response to low temperature. 673

Supplemental Data Set 5. Top 1000 upregulated genes in lsm1a lsm1b mutants in 674

response to drought. 675

Supplemental Data Set 6. Drought-inducible genes upregulated in lsm1a lsm1b 676

mutants in response to drought. 677

Supplemental Data Set 7. Top 1000 downregulated genes in lsm1a lsm1b mutants in 678

response to drought. 679

Supplemental Data Set 8. Drought-inducible genes downregulated in lsm1a lsm1b 680

mutants in response to drought. 681

Supplemental Data Set 9. Top 1000 upregulated genes in lsm1a lsm1b mutants in 682

response to salt stress. 683

Supplemental Data Set 10. Salt-inducible genes upregulated in lsm1a lsm1b mutants in 684

response to salt stress. 685

Supplemental Data Set 11. Top 1000 downregulated genes in lsm1a lsm1b mutants in 686

response to salt stress. 687

Supplemental Data Set 12. Salt-inducible genes downregulated in lsm1a lsm1b 688

mutants in response to salt stress. 689

Supplemental Data Set 13. Oligonucleotides used in this work. 690

Supplemental Data Set 14. Total mapped and unmapped reads obtained for each 691

sample by RNAseq experiments. 692

Supplemental Data Set 15. List of accession numbers appearing in this study. 693

694

ACKNOWLEDGEMENTS 695

We thank D. Staiger for the LSMGRP7PRO-GRP7-GFP Arabidopsis transgenic line, and 696

J.J. Sanchez-Serrano, J.A. Jarillo and R. Solano for discussions and comments. This 697

work was supported by grants BIO2010-17545 and BIO2013-47788-R from MINECO 698

to J.S., GA14-34792S from CSF to O. N. and MCB-1022435 from NSF to L.S. R.C. is 699

supported by a JAE-DOC contract from the CSIC and C.C. is recipient of a FPI 700

fellowship from MINECO. 701

702

AUTHOR CONTRIBUTIONS 703

23

C.P., C.C., O.N., L.S., and J.S. conceived and designed the experiments. C.P., C.C., 704

R.C., V.T., and W.Z. performed the experiments. C.P., C.C., R.C., O.N., L.S., J.M.M., 705

and J.S. analyzed the data. C.P. and J.S. wrote the paper. 706

707

24

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Peng, J., Li, Z., Wen, X., Li, W., Shi, H., Yang, L., Zhu, H., and Guo, H. (2014). 815

Salt-Induced Stabilization of EIN3/EIL1 Confers Salinity Tolerance by Deterring ROS 816

Accumulation in Arabidopsis. PLOS Genetics 10: e1004664. 817

818

Perea-Resa, C., Hernández-Verdeja, T., López-Cobollo, R., Castellano, M.M., and 819

Salinas, J. (2012). LSM Proteins Provide Accurate Splicing and Decay of Selected 820

Transcripts to Ensure Normal Arabidopsis Development. Plant Cell 24: 4930-4947. 821

822

Pham, J., Liu, J., Bennett, M.H., Mansfield, J.W., and Desikan, R. (2012), 823

Arabidopsis histidine kinase 5 regulates salt sensitivity and resistance against bacterial 824

and fungal infection. New Phytol. 194: 168-180. 825

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Roux, M.E., Rasmussen, M.W., Palma, K., Lolle, S., Mateu-Regué, A., Bethke, G., 827

Glazebrook, J., Zhang, W., Sieburth, L., Larsen, M.R., Mundy, J., and Petersen, 828

M. (2015). The mRNA decay factor PAT1 functions in a pathway including MAP 829

kinase 4 and immune receptor SUMM2. EMBO J. 34: 593-608. 830

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Rzeczkowski, K., Beuerlein, K., Müller, H., Dittrich-Breiholz, O., Schneider, H., 832

Ketter-Buhrow, D., Holtmann, H., and Kracht, M. (2011). C-Jun N-terminal kinase 833

phosphorylates DCP1a to control formation of P bodies. J. Cell Biol. 194: 581-596. 834

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Sheth, U., and Parker, R. (2003). Decapping and Decay of Messenger RNA Occur in 836

Cytoplasmic Processing Bodies. Science 300: 805-808. 837

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Shi, Y., Ding, Y., and Yang, S. (2015). Cold Signal Transduction and its Interplay with 839

Phytohormones During Cold Acclimation. Plant Cell Physiol. 56: 7-15. 840

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Streitner, C., Köster, T., Simpson, C.G., Shaw, P., Danisman, S., Brown, J.W.S., 842

and Staiger, D. (2012). An hnRNP-like RNA-binding protein affects alternative 843

splicing by in vivo interaction with transcripts in Arabidopsis thaliana. Nucleic Acis. 844

Res. 40: 11240-11255. 845

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Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and McCarty, 847

D.R. (2003). Molecular Characterization of the Arabidopsis 9-cis epoxycarotenoid 848

dioxygenase gene family. Plant J. 35: 44-56. 849

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Tran, L.S.P., Nakashima, K., Sakuma, Y., Simpson, S.D., Fujita, Y., Maruyama, 851

K., Fujita, M., Seki, M., Shinozaki, K., and Yamaguchi-Shinozaki, K. (2004). 852

Isolation and Functional Analysis of Arabidopsis Stress-Inducible NAC Transcription 853

Factors That Bind to a Drought-Responsive cis-Element in the early responsive to 854

dehydration stress 1 Promoter. Plant Cell 16: 2481-2498. 855

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Turečková, V., Novák, O., and Strnad, M. (2009). Profiling ABA metabolites in 857

Nicotiana tabacum L leaves by ultra-performance liquid chromatography–electrospray 858

tandem mass spectrometry. Talanta 80: 390-399. 859

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Verslues, P.E., Agarwal, M., Katiyar-Agarwal, S., Zhu, J., and Zhu, J.K. (2006). 861

Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic 862

stresses that affect plant water status. Plant J. 45: 523-539. 863

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Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G., and Thomashow, M.F. 865

(2005). Roles of the CBF2 and ZAT12 transcription factors in conFigureuring the low 866

temperature transcriptome of Arabidopsis. Plant J. 41: 195-211. 867

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Weber, C., Nover, L., and Fauth, M. (2008). Plant stress granules and mRNA 869

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processing bodies are distinct from heat stress granules. Plant J. 56, 517-530. 870

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Wu, Y., Deng, Z., Lai, J., Zhang, Y., Yang, C., Yin, B., Zhao, Q., Zhang, L., Li, Y., 872

Yang, C., and Xie, Q. (2009). Dual function of Arabidopsis ATAF1 in abiotic and 873

biotic stress responses. Cell Res. 19: 1279-1290. 874

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Xu, J., Yang, J.Y., Niu, Q.W., and Chua, N.H. (2006). Arabidopsis DCP2, DCP1, and 876

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Xu, J., and Chua, N.H. (2009). Arabidopsis decapping 5 is required for mRNA 880

decapping, P-body formation, and translational repression during postembryonic 881

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Xu, J., and Chua, N.H. (2012). Dehydration stress activates Arabidopsis MPK6 to 884

signal DCP1 phosphorylation. EMBO J. 31: 1975-1984. 885

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Yamaguchi-Shinozaki, K., and Shinozaki, K. (2006). Transcriptional regulatory 887

networks in cellular responses and tolerance to dehydration and cold stresses. Annu. 888

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890

Zhang, Z., Zhang, S., Zhang, Y., Wang, X., Li, D., Li, Q., Yue, M., Li, Q., Zhang, 891

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Confers High Salt Tolerance by Regulating Transcription and Pre-mRNA Splicing 893

through Altering Histone H4R3 and Small Nuclear Ribonucleoprotein LSM4 894

Methylation. Plant Cell 23: 396-411. 895

896

30

FIGURE LEGENDS 897

Figure 1. Arabidopsis LSM1 proteins accumulate in response to low temperature 898

and localize to P-bodies. (A) Expression of LSM1A and LSM1B in 2-week-old Col-0 899

plants exposed for the indicated hours (h) or days (d) to 4ºC. Levels, determined by 900

qPCR, are represented as relative to their corresponding values at 0 h. Error bars 901

indicate the standard deviation of the mean (n ≥ 3). In all cases, differences between 902

cold-treated and control (0 h) plants were significant (P ≤ 0.0001), as determined by 903

ANOVA test. (B) Immunoblots showing levels of LSM1A-GFP and LSM1B-GFP in 2-904

week-old transgenic Arabidopsis plants exposed for the indicated times to 4ºC. A lane 905

with Col-0 plants was added in the immunoblot as a negative control. Coomassie 906

staining of the large subunit of Rubisco was used as a loading control. (C) Subcellular 907

localization of LSM1A-GFP and LSM1B-GFP in root tip cells from 6-day-old 908

transgenic Arabidopsis seedlings grown under control conditions (20ºC), or exposed for 909

24 h to 4ºC in the presence or absence of cycloheximide (CHX). Bars = 20 µm. (D) 910

Colocalization of LSM1A-GFP and LSM1B-GFP with RFP-DCP1 in root tip cells from 911

6-day-old transgenic Arabidopsis seedlings exposed for 24 h to 4ºC. Bars = 20 µm. (E) 912

Subcellular localization of GFP-DCP2 and GFP-VCS in root tip cells from 6-day-old 913

Col-0 and lsm1a lsm1b Arabidopsis seedlings grown under control conditions and 914

subsequently exposed for 24 h to 4ºC. Bars = 20 µm. 915

916

Figure 2. The LSM1-7 complex differentially regulates abiotic stress tolerance in 917

Arabidopsis. (A) Freezing tolerance of cold-acclimated 2-week-old Col-0, lsm1a 918

lsm1b, c-lsm1a and c-lsm1b plants (left). Representative cold-acclimated plants 7 d after 919

being exposed to -11ºC for 6 h (right). (B) Drought tolerance of 7-d-old Col-0, lsm1a 920

lsm1b, c-lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or 921

exposed 7 d to 25% PEG (right). (C) Salt tolerance of 7-d-old Col-0, lsm1a lsm1b, c-922

lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or exposed 7 923

d to 150 mM NaCl (right). In all graphs, error bars indicate the standard deviation of the 924

mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, 925

***P ≤ 0.0001) between lsm1a lsm1b and the other plants, as determined by ANOVA-926

test. No significant differences between Col-0 and c-lsm1a or c-lsm1b plants were 927

observed in any case. 928

929

31

Figure 3. The Arabidopsis LSM1-7 complex differentially regulates gene 930

expression in response to abiotic stresses. (A) Expression of different cold-inducible 931

genes upregulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, 932

lsm1a lsm1b and c-lsm1a plants exposed for 24 h to 4ºC are represented relative to their 933

corresponding values in control plants (C). (B) Expression of different drought-934

inducible genes upregulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-935

old Col-0, lsm1a lsm1b and c-lsm1a plants exposed for 10 h to 55% PEG are 936

represented relative to their corresponding values in control plants (C). (C, D) 937

Expression of different salt-inducible genes up- (C) or down-regulated (D) in lsm1a 938

lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a 939

plants exposed for 10 h to 150 mM NaCl are represented relative to their corresponding 940

values in control plants (C). In all graphs, error bars indicate the standard deviation of 941

the mean (n ≥ 3). Differences between lsm1a lsm1b and Col-0 or c-lsm1a plants were 942

always significant (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001), as determined by ANOVA-943

test. No significant differences between Col-0 and c-lsm1a plants were observed in any 944

case. 945

946

Figure 4. The Arabidopsis LSM1-7 complex interacts with selected target 947

transcripts in response to abiotic stresses. (A-C) RIP assays of 2-week-old c-lsm1a 948

plants grown under control conditions (Control exposed for 24 h to 4ºC (A), 10 h to 949

55% PEG (B), or 10 h to 150 mM NaCl (C), using an anti-GFP antibody. RIP assays of 950

Arabidopsis containing a GRP7PRO-GRP7-GFP fusion grown under control and stress 951

conditions were also carried out as interaction specificity controls. Co-952

immunoprecipitated RNA samples corresponding to different cold- (A), drought- (B) 953

and salt-inducible (C) genes were quantified by qPCR. Transcript levels in c-lsm1a 954

plants were corrected with respect to their corresponding input values and are 955

represented relative to the levels obtained from RIP control assays. In all graphs, error 956

bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate 957

significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels 958

between RIP assays from stressed and control plants, as determined by t-test. 959

960

Figure 5. The Arabidopsis LSM1-7 complex promotes the decapping of selected 961

target transcripts in response to abiotic stresses. (A-C) Capped transcripts in 2-962

week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), 963

32

exposed for 24 h to 4ºC (A), 10 h to 55% PEG (B), or 10 h to 150mM NaCl (C). The 964

levels of capped transcripts corresponding to different cold- (A), drought- (B) and salt-965

inducible (C) genes were corrected with respect to the levels of their corresponding total 966

transcripts and are represented relative to control Col-0 plants. In all graphs, error bars 967

indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant 968

differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or 969

c-lsm1a plants, as determined by ANOVA tests. No significant differences between 970

Col-0 and c-lsm1a plants were observed in any case. 971

972

Figure 6. The interaction between the Arabidopsis LSM1-7 complex and some of 973

its target transcripts depends on the abiotic stress conditions. RIP assays of 2-week-974

old c-lsm1a plants grown under control conditions (Control, exposed for 24 h to 4ºC, 10 975

h to 55% PEG, or 10 h to 150 mM NaCl, using an anti-GFP antibody. RIP assays of 2-976

week-old Arabidopsis plants containing a GRP7PRO-GRP7-GFP fusion grown under 977

control and stressed conditions were also carried out as interaction specificity controls. 978

Co-immunoprecipitated RNA samples corresponding to LEA7, ZAT12, NCED3, 979

WRKY33, ABR1, ANAC019, ANAC092 and AHK5 genes were quantified by qPCR. 980

Transcript levels in c-lsm1a plants were corrected with respect to their corresponding 981

input values and are represented relative to the levels obtained from RIP control assays. 982

In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) 983

indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript 984

levels between RIP assays from stressed and control plants, as determined by ANOVA 985

tests. 986

987

Figure 7. Decapping of some target transcripts by the Arabidopsis LSM1-7 988

complex depends on the abiotic stress conditions. (A-C) Capped transcripts in 2-989

week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), 990

exposed for 24 h to 4ºC, 10 h to 55% PEG, or 10 h to 150 mM NaCl. The levels of 991

capped transcripts corresponding to LEA7, ZAT12, NCED3 and WRKY33 (A), ABR1 992

and ANAC019 (B), and ANAC092 and AHK5 (C) were corrected with respect to the 993

levels of their corresponding total transcripts and are represented relative to control Col-994

0 plants. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). 995

Asterisks (*) indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between 996

lsm1a lsm1b and Col-0 or c-lsm1a plants, as determined by ANOVA tests. No 997

33

significant differences between lsm1a lsm1b and the other plants were found in any 998

case. 999

1000

Figure 8. ABA biosynthesis is differentially regulated by the Arabidopsis LSM1-7 1001

complex in response to abiotic stresses. (A, B) ABA levels in 2-week-old Col-0, 1002

lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed for 24 h to 1003

4ºC (A), 10 h to 55% PEG (B) or 10 h to 150mM NaCl (B). In all graphs, error bars 1004

indicate the standard deviation of the mean (n = 4). Asterisks (*) indicate significant 1005

differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a 1006

plants, as determined by ANOVA tests. No significant differences between Col-0 and c-1007

lsm1a plants were observed in any case. 1008

1009

Figure 9. Proposed model for the function of LSM1-7 complex in plant responses 1010

to abiotic stresses. In response to abiotic stress conditions, the LSM1-7 complex would 1011

localize to P-bodies where it would interact with selected, specific and non-specific, 1012

mRNA targets to promote their decapping and subsequent decay. This function would 1013

contribute to shape the transcriptome reprogramming required for plant adaptation to 1014

abiotic stresses. Arrowheads and end lines indicate positive and negative regulation, 1015

respectively. 1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

Figure 1. Arabidopsis LSM1 proteins accumulate in response to low temperature and localize to P-bodies. (A) Expression ofLSM1A and LSM1B in 2-week-old Col-0 plants exposed for the indicated hours (h) or days (d) to 4ºC. Levels, determined by qPCR,are represented as relative to their corresponding values at 0h. Error bars indicate the standard deviation of the mean (n ≥ 3). In allcases, differences between cold-treated and control (0h) plants were significant (P ≤ 0.0001), as determined by ANOVA-test. (B)Levels of LSM1A-GFP and LSM1B-GFP in 2-week-old transgenic Arabidopsis plants exposed for the indicated times to 4ºC. A lanewith Col-0 plants was added in the western blot as a negative control. Comassie staining of the large subunit of Rubisco was used asa loading control. (C) Subcellular localization of LSM1A-GFP and LSM1B-GFP in root tip cells from 6-day-old transgenic Arabidopsisseedlings grown under control conditions (20ºC), exposed 24h to 4ºC, or exposed 24h to 4ºC with cycloheximide (CHX). Bars = 20µm. (D) Colocalization of LSM1A-GFP and LSM1B-GFP with RFP-DCP1 in root tip cells from 6-day-old transgenic Arabidopsisseedlings exposed 24h to 4ºC. Bars = 20 µm. (E) Subcellular localization of GFP-DCP2 and GFP-VCS in root tip cells from 6-day-oldCol-0 and lsm1a lsm1b Arabidopsis seedlings grown under control conditions and subsequently exposed 24h to 4ºC. Bars = 20 µm.

20ºC

4ºC

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lsm1a lsm1b

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*** ***

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Figure 2. The LSM1-7 complex differentially regulates abiotic stress tolerance in Arabidopsis. (A) Freezing tolerance of coldacclimated 2-week-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b plants (left). Representative cold acclimated plants 7d after beingexposed to -11ºC for 6h (right). (B) Drought tolerance of 7-day-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b seedlings (left).Representative seedlings grown on GM or exposed 7d to 25% PEG (right). (C) Salt tolerance of 7-day-old Col-0, lsm1a lsm1b, c-lsm1a and c-lsm1b seedlings (left). Representative seedlings grown on GM or exposed 7d to 150mM NaCl (right). In all graphs, errorbars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤0.0001) between lsm1a lsm1b and the other plants, as determined by ANOVA-test. No significant differences between Col-0 and c-lsm1a or c-lsm1b plants were observed in any case.

Figure 3. The Arabidopsis LSM1-7 complex differentially regulates gene expression in response to abiotic stresses. (A)Expression of different cold-inducible genes up-regulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1alsm1b and c-lsm1a plants exposed 24h to 4ºC are represented as relative to their corresponding values in control plants (C). (B)Expression of different drought-inducible genes up-regulated in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1alsm1b and c-lsm1a plants exposed 10h to 55% PEG are represented as relative to their corresponding values in control plants (C). (C,D) Expression of different salt-inducible genes up- (C) or down-regulated (D) in lsm1a lsm1b. Levels, determined by qPCR, in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants exposed 10h to 150mM NaCl are represented as relative to their corresponding values incontrol plants (C). In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Differences between lsm1a lsm1b andCol-0 or c-lsm1a plants were always significant (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001), as determined by ANOVA-test. No significantdifferences between Col-0 and c-lsm1a plants were observed in any case.

A

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/C)

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lsm1a lsm1bc-lsm1a

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* *****

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*** **

**

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**Col-0lsm1a lsm1bc-lsm1a

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**

***

**

*** ***

***

Figure 4. The Arabidopsis LSM1-7 complex interacts with selected target transcripts in response to abiotic stresses. (A-C)RIP assays on 2-week-old c-lsm1a plants grown under control conditions (Control), exposed 24h to 4ºC (A), 10h to 55% PEG (B), or10h to 150mM NaCl (C), using an anti-GFP antibody. RIP assays on Arabidopsis containing a GRP7PRO-GRP7-GFP fusion grownunder control and stressed conditions were also carried out as interaction specificity controls. Co-immunoprecipitated RNA samplescorresponding to different cold- (A), drought- (B) and salt-inducible (C) genes were quantified by qPCR. Transcript levels in c-lsm1aplants were corrected with respect to their corresponding input values and represented relative to the levels obtained from RIP controlassays. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels between RIP assays from stressed and control plants, as determined by t-test.

C

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-lsm

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*

**

* ***

***

*

**

****

*

****

**

*

Figure 5. The Arabidopsis LSM1-7 complex promotes the decapping of selected target transcripts in response to abioticstresses. (A-C) Capped transcripts in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed24h to 4ºC (A), 10h to 55% PEG (B), or 10h to 150mM NaCl (C). The levels of capped transcripts corresponding to different cold- (A),drought- (B) and salt-inducible (C) genes were corrected with respect to the levels of their corresponding total transcripts andrepresented relative to control Col-0 plants. In all graphs, error bars indicate the standard deviation of the mean (n ≥ 3). Asterisks (*)indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a plants, asdetermined by ANOVA-test. No significant differences between Col-0 and c-lsm1a plants were observed in any case.

B

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*******

*

Figure 6. The interaction between the Arabidopsis LSM1-7 complex and some of its target transcripts depends on the abioticstress conditions. RIP assays on 2-week-old c-lsm1a plants grown under control conditions (Control), exposed 24h to 4ºC, 10h to55% PEG, or 10h to 150mM NaCl, using an anti-GFP antibody. RIP assays on 2-week-old Arabidopsis plants containing a GRP7PRO-GRP7-GFP fusion grown under control and stressed conditions were also carried out as interaction specificity controls. Co-immunoprecipitated RNA samples corresponding to LEA7, ZAT12, NCED3, WRKY33, ABR1, ANAC019, ANAC092 and AHK5 geneswere quantified by qPCR. Transcript levels in c-lsm1a plants were corrected with respect to their corresponding input values andrepresented relative to the levels obtained from RIP control assays. In all graphs, error bars indicate the standard deviation of themean (n ≥ 3). Asterisks (*) indicate significant differences (*P ≤ 0.01, **P ≤ 0.001, ***P ≤ 0.0001) in transcript levels between RIPassays from stressed and control plants, as determined by ANOVA-test.

Figure 7. Decapping of some target transcripts by the Arabidopsis LSM1-7 complex depends on the abiotic stressconditions. (A-C) Capped transcripts in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C),exposed 24h to 4ºC, 10h to 55% PEG, or 10h to 150mM NaCl. The levels of capped transcripts corresponding to LEA7, ZAT12,NCED3 and WRKY33 (A), ABR1 and ANAC019 (B), and ANAC092 and AHK5 (C) genes were corrected with respect to the levels oftheir corresponding total transcripts and represented relative to control Col-0 plants. In all graphs, error bars indicate the standarddeviation of the mean (n ≥ 3). Asterisks (*) indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0or c-lsm1a plants, as determined by ANOVA-test. No significant differences between lsm1a lsm1b and the other plants were found inany case.

CCol-0lsm1a lsm1bc-lsm1a

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Figure 8. ABA biosynthesis is differentially regulated by the Arabidopsis LSM1-7 complex in response to abiotic stresses. (A,B) ABA levels in 2-week-old Col-0, lsm1a lsm1b and c-lsm1a plants grown under control conditions (C), exposed 24h to 4ºC (A), 10hto 55% PEG (B) or 10h to 150mM NaCl (B). In all graphs, error bars indicate the standard deviation of the mean (n = 4). Asterisks (*)indicate significant differences (**P ≤ 0.001, ***P ≤ 0.0001) between lsm1a lsm1b and Col-0 or c-lsm1a plants, as determined byANOVA-test. No significant differences between Col-0 and c-lsm1a plants were observed in any case.

ColdDrought

Salinity

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Drought Tolerance

12

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Transcriptomereprogramming

DECAPPING OF SELECTED STRESS‐SPECIFIC AND NONSPECIFIC TARGET 

TRANSCRIPTS 

PLANT ADAPTATION

ACCURATE STRESS‐RESPONSIVE GENE 

EXPRESSION

P‐bodies

Figure 9. Proposed model for the function of LSM1-7 complex in plant response to abiotic stresses. In response to abioticstress conditions the complex would localize in P-bodies where would interact with selected, specific and non-specific, mRNA targetsto promote their decapping and subsequent decay. This function would contribute to shape the transcriptome reprogramming requiredfor plant adaptation to abiotic stress. Arrowheads and end lines indicate positive and negative regulation, respectively.

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