Phosphoproteomics after nitrate treatments reveal an important role for PIN2 phosphorylation in control of root system architecture

Nitrate is an important signaling molecule that commands genome-wide gene expression changes that impact metabolism, physiology, plant growth and development. Although gene expression responses to nitrate at the mRNA level have been characterized in great detail, the impact of nitrate signaling at the proteome level has been much less explored. Most signaling pathways involve post-translational modifications of key protein factors and chiefly among these modifications is protein phosphorylation. In an effort to identify new components involved in nitrate responses in plants, we performed analyses of the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. We identified 268 phosphoproteins that show significant changes at 5 min or 20 min after nitrate treatments. The large majority of these proteins (96%) are coded by genes that are not modulated at the expression level in response to nitrate treatments in publicly available transcriptome data. Proteins identified by 5 min include potential signaling-components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with protein binding, transporter activity or hormone metabolism functions. Interestingly, the phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants in response to nitrate at 5 min was significantly different (95%) as compared to wild-type plants. This result is consistent with the role of NRT1.1 as a key component of a nitrate signaling pathway that involves phosphoproteomic changes. Our integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We experimentally validated the role of PIN2 phosphorylation in both primary and lateral root growth responses to nitrate. Our data provide new insights into the phosphoproteome and identifies novel protein components that are regulated post-translationally, such as PIN2, in nitrate responses in Arabidopsis thaliana roots.

protein phosphorylation. In an effort to identify new components involved in nitrate responses in 23 plants, we performed analyses of the Arabidopsis thaliana root phosphoproteome in response to 24 nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. We identified 25 268 phosphoproteins that show significant changes at 5 min or 20 min after nitrate treatments. The 26 large majority of these proteins (96%) are coded by genes that are not modulated at the expression 27 level in response to nitrate treatments in publicly available transcriptome data. Proteins identified 28 by 5 min include potential signaling-components such as kinases or transcription factors. In 29 contrast, by 20 min, proteins identified were associated with protein binding, transporter activity 30 or hormone metabolism functions. Interestingly, the phosphorylation profile of NITRATE 31 TRANSPORTER 1.1 (NRT1.1) mutant plants in response to nitrate at 5 min was significantly 32 different (95%) as compared to wild-type plants. This result is consistent with the role of NRT1.1 33 as a key component of a nitrate signaling pathway that involves phosphoproteomic changes. Our 34 integrative bioinformatics analysis highlights auxin transport as an important mechanism 35 Introduction 41 Nitrogen (N) is the mineral nutrient required in the greatest amounts by plants. N is often 42 scarce in natural and agricultural systems, constituting a major factor limiting plant growth and 43 agricultural yield. During the last 50 years, global demand for synthetic N fertilizers has 44 dramatically increased in response to growing agricultural demand. Depending on soil conditions 45 and plant species, less than 50% of the applied N fertilizer is taken up by crops. Excess N may 46 contaminate aquatic systems 1 or be released into the atmosphere as N-oxide gases 2,3 , both leading 47 to detrimental effects on the environment and human health. 48 The relevance of N for plants is exemplified by its effects on leaf growth 4 , senescence 5 , 49 root system architecture 6,7 , and flowering time 8,9 . Due to its importance, plants have evolved 50 sophisticated mechanisms to adapt to fluctuating N availability. Furthermore, growth and 51 developmental processes can be regulated by varying the amount of N supplied to plants. For 52 instance, exogenous nitrate applications stimulate lateral root elongation, enabling root growth and 53 colonization in nitrate-rich soil patches 10,11 . However, high nitrate concentrations reduce primary 54 and lateral root elongation under homogeneous growth-conditions 12 . Nitrate is the main form of 55 inorganic N for plants in natural and agricultural soils 13,14 . Besides its nutritional role, nitrate acts 56 as a signaling molecule that regulates several genes involved in a wide range of biological 57 processes 15,16 . With advances in genomic technologies and system approaches, thousands of 58 nitrate-responsive genes have been identified in Arabidopsis thaliana roots and shoots [17][18][19][20][21][22][23][24] . These 59 N-response genes include nitrate transporters, nitrate reductase (NR) and nitrite reductase (NiR), 60 putative transcription factors, and stress response genes, as well as genes whose products play roles 61 in glycolysis, N metabolism, and hormone pathways. Moreover, nitrate elicits local and systemic 62 signals to synchronize its availability with plant growth and development [25][26][27][28][29] . Although 63 transcriptional responses activated by nitrate have been described in great detail, it is clear that 64 regulation at the post-translational level is key for N-responses 30,31 . 65 The role of protein phosphorylation in response to nitrate was initially identified in post-66 translational modifications in N metabolism. The activity of NR, the enzyme that catalyzes the 67 first step of nitrate reduction, is modulated by protein phosphorylation and then inhibited by 14-3-68 3 protein interaction 32,33 . Studies in spinach leaves using 32 P labeling and kinase assays 69 demonstrated that the regulation of NR by light/dark and photosynthetic activity involves protein phosphorylation 34,35 . A subsequent study showed that a 14-3-3 family protein interacts with and 71 inactivates phosphorylated NR in the presence of covalent ions 32,36 . Earlier experiments also 72 indicated that changes in gene expression in response to nitrate treatments require kinase and 73 phosphatase activities. In maize leaves for example, treatments with inhibitors of calmodulin-74 dependent protein kinases repress nitrate induction of genes encoding nitrate assimilatory enzymes 75 such as NR, NiR, glutamine synthetase 2 (GS2) and ferredoxin glutamate synthase (Fd-76 GOGAT) 37 . Conversely, inhibition of protein phosphatases blocked the nitrate-response of NR, 77 NIR and GS2 37 . In another study, pharmacological inhibitors of serine-threonine protein 78 phosphatase and tyrosine protein kinases repressed the nitrate-induced accumulation of transcripts 79 for NR and NiR in barley leaves 38 . These early experiments suggested that changes in the status 80 of protein phosphorylation were important for the regulation of gene expression in response to 81 nitrate treatments. 82 The discovery that a kinase protein complex can directly phosphorylate the nitrate 83 transceptor NRT1.1/NPF6.3 demonstrated that phosphorylation plays an important role in nitrate 84 signaling. Under low-nitrate conditions, NRT1.1/NPF6.3 is phosphorylated in a threonine residue 85 (T101) by CIPK23-CBL9 complex (CIPK, CLB-interacting protein kinase; CBL, Calcineurin B-86 like protein), shifting into a high-affinity nitrate transporter 31,39 . In contrast, at high-nitrate levels, 87 NRT1.1/NPF6.3 is dephosphorylated at T101 and turns into a low-affinity transporter. 88 Experiments with a mutant mimicking the phosphorylated form of the transceptor showed the 89 importance of this phosphorylation for the regulation of gene expression at low nitrate 90 concentrations 39 . Phosphorylation of NRT1.1/NPF6.3 also appears to play a role in the modulation 91 of auxin transport and repression of lateral root emergence under low-nitrate conditions 40,41 . 92 Conversely, the dephosphorylated form of NRT1. 1 Another kinase involved in signaling is CIPK8 42 . In cipk8 mutants, the rapid induction of genes or primary nitrate response was strongly reduced (40-65% of WT levels), particularly in the low-102 affinity phase 42 . Both CIPK8 and CIPK23 are rapidly induced by nitrate treatments and 103 downregulated in the chl1-5 and chl1-9 mutants, respectively 39,42 . More recently, the calcium 104 sensor CBL1 and the protein phosphatase 2C (ABA-insensitive) ABI2 were also identified as 105 components of this signaling pathway, which regulates NRT1.1/NPF6.3 transport and sensing 43 . 106 The calcium sensor CBL1 also interacts with CIPK23 and this complex was dephosphorylated by 107 ABI2 43 . 108 Global-scale proteomic analysis performed in Arabidopsis seedlings, mostly shoot organs, 123 showed that nitrogen starvation and resupply (nitrate or ammonium) modulates protein 124 phosphorylation over a time course of 30 min 49 . In general, proteins such as receptor kinases and 125 transcription factors change their phosphorylation levels after nitrogen resupply at 5-10 min (fast 126 response). Proteins involved in protein synthesis and degradation, central and hormone metabolism 127 showed changes in their phosphorylation level after 10 min (late response). Another study showed 128 that nitrate deprivation affects both protein abundance and phosphorylation status 50 . Nitrate 129 deprivation assays revealed that some proteins, mostly involved in transport, contain sites that are 130 dephosphorylated early in the response 50 .
In this study, we performed quantitative time-course analyses of the Arabidopsis root 132 phosphoproteome in response to nitrate via liquid chromatography coupled to tandem mass 133 spectrometry detection (HPLC-MS/MS). We chose to focus on root-phosphoproteomics profiling 134 in response to nitrate for several reasons: (i) phosphoproteomics and proteomics studies describe 135 phosphorylation levels as more dynamic and mainly independent of protein abundance [51][52][53] , 136 suggesting that many proteins are regulated by phosphorylation independent of their changes in 137 protein abundance. (ii) Previous global studies of N treatment focused on the proteome and 138 phosphoproteome in Arabidopsis seedlings, which interrogates mostly shoot tissues 49,50 . In order 139 to search for new N-regulatory factors, our experimental approach focused on Arabidopsis roots 140 because early sensing and responses to N-supply occur in the roots. Several studies have shown 141 that HPLC-MS/MS provides accurate estimates of dynamic phosphorylation levels in vivo [54][55][56][57] . 142 We used HPLC-MS/MS to identify phosphorylated proteins with differential profiles in 143 response to nitrate treatments at 0, 5 or 20 min. We found that the nature of these phosphorylated 144 proteins differed significantly from those encoded by genes implicated in nitrate via transcriptomic 145 studies. We found different types of phosphoproteins changing at 5 or 20 min after nitrate 146 treatments. Interestingly, the large majority of these changes depend on NRT1.1/NPF6.3. Kinases 147 and transcription factors were over-represented at 5 min, while proteins involved in protein binding 148 and transporter activity were common by 20 min of nitrate treatments. We found several 149 phosphoproteins involved in auxin transport, including the auxin efflux-carriers PIN2 and PIN4. 150 We validated the role of PIN2 and found dephosphorylation of PIN2 to be important for 151 modulation of root system architecture in response to nitrate. Our analysis reveals that the nitrate 152 signaling pathway mediated by NRT1. . Briefly, phosphopeptides 171 were enriched using cerium oxide affinity capture and analyzed with a HPLC-MS/MS instrument 172 ( Figure S1). The spectra were assigned to specific peptide sequences by the MASCOT search 173 engine (FDR < 0.1%). We quantified the relative abundance of each phosphoprotein using average 174 normalized spectral counts (SPCs) of the total number of spectral-peptide matches to protein 175 sequences in three independent biological replicates for each treatment condition. In total, we 176 identified and measured 6,560 unique phosphopeptides which unambiguously mapped to 2,048 177 phosphoprotein groups (Supplemental Table S1). The majority of identified phosphopeptides 178 (82%) were phosphorylated in a single residue ( Figure S2A). The relative distribution of each 179 phosphorylated residue -80% serine, 18% threonine, and 2% tyrosine ( Figure S2B) -was 180 consistent with prior plant phosphoproteomic studies 50,54,61 . The identified phosphopeptides were 181 mapped and grouped in phosphoprotein groups, where proteins that shared peptides were clustered 182 together. A group leader was assigned to each group, based on having the highest number of 183 peptide identifications; throughout the remainder of the article, "phosphoproteins" is synonymous 184 with "group leaders". The majority of the identified phosphoproteins present one (50%), two phosphorylated residue (69% Ser, 28% Thr and 3% Tyr; Figure S2D). We recognized 187 phosphoproteins across several biological process, subcellular compartments and cellular 188 functions based on the Gene Ontology (GO) classification ( Figure S3). No overrepresented GO 189 categories were observed when comparing against the Arabidopsis genome, showing that our 190 experimental strategy was unbiased with regards to annotated protein functions, subcellular 191 locations or biological processes and represents an unbiased Arabidopsis proteome sampling. 192 To identify nitrate-regulated phosphoproteins in Arabidopsis roots we performed 193 statistical analysis using analysis of variance (significance: p < 0.05). We identified 268 194 phosphoproteins that were significantly altered under our experimental conditions. We found 62 195 phosphoproteins regulated at 5 min after nitrate treatments, 40 of which were induced ( Figure 1A).  To evaluate the biological significance of the phosphoproteome patterns observed in 220 response to nitrate, hierarchical clustering analysis was performed on the phosphoprotein dataset 221 at 5 and 20 min following nitrate treatments in Col-0 roots (Figure 2). In order to identify the most 222 prominent functional categories affected, we searched for overrepresented biological terms in each 223 cluster using the BioMaps program 63 and the PANTHER classification system 64,65 (significance: 224 p < 0.05, corrected by FDR). This analysis highlighted several signaling, regulatory or metabolic 225 functions differentially associated with early (5 min) and late (20 min) components in response to 226 nitrate treatments. "DNA binding" and "Nucleic acid binding" categories were overrepresented in 227 cluster 6, containing phosphoproteins that increase their levels at 5 min and do not change at 20 228 min. This cluster also includes previously undescribed transcription factors (TFs) in the N-229 response cascade in diverse transcriptomic studies [21][22][23][24]66 . In contrast, cluster groups containing 230 mostly phosphoproteins that changed their levels at 20 min in response to nitrate were enriched in 231 the functional categories: "transport" and "nitrogen compounds metabolism" (clusters 2 and 8). ) and was also identified in nitrate-238 deprivation experiments with an opposite phosphorylation response 50 . Also, phosphopeptides for 239 AMT1.3 with phosphorylation at Thr-464 and Ser-487 were identified as "up-regulated" by nitrate 240 treatments at 20 min. The phosphorylation of both sites inhibits transport function 69 and were also 241 identified as phosphopeptides in nitrate deprivation 50 and resupply 49 experiments. We also found 242 that nitrate strongly increased levels of phosphorylated nitrate reductase NIA2 at the highly 243 conserved and regulatory site Ser-534 70 . These results are consistent with previous studies and 244 suggest overall regulation of N metabolism by phosphorylation of key players by 20 min after 245 nitrate treatments. 246 The final group of clusters showed enrichment in signaling and regulatory pathways, with 247 different profiles at 5 min and 20 min. Clusters 1, 5, and 7, contained phosphoproteins with 248 opposite regulation at 5 and 20 min, and showed proteins related to microRNA processing, 249 phosphoinositide and phosphatidylinositol binding functions. Phosphoinositides can act in 250 signaling pathways and serve as precursors for phospholipase C (PLC)-mediated signaling. A 251 previous study in our laboratory implicated a PLC-activity in the nitrate signaling pathway 45 . Our 252 results are consistent with these results and show that PLC2, represented by the phosphopeptide 253 Ser-280, was identified as up-regulated in response to nitrate at 20 min (in Cluster 1). Cluster 10 254 is interesting for nitrate responses because it includes many components involved in classical 255 processes regulated by nitrate in Arabidopsis roots. For instance, auxin and lateral root 256 development are biological functions enriched in this group, which is consistent with auxin 257 pathways being modulated by nitrate 16,71 . Several reports indicate that auxin acts as regulator of 258 root system architecture in response to nitrate availability 7,71,72 . 259 Overall, our dataset captures the dynamic effects of N-signaling on phosphoproteome 260 profiles, which implicate a cascade of nonoverlapping processes in early and late responses. The 261 earliest steps in the N phospho-dynamics were involved in signal transduction and transcription 262 factor activity. In contrast, the later phosphoprotein data-set was enriched in metabolic, transport 263 and root developmental processes. These temporal mechanisms show a transition of 264 phosphorylation dynamics from phosphoproteins essential to signaling networks to proteins 265 associated with biological processes involved in nitrate response. 266

NRT1.1/AtNPF6.3 is essential for transient phosphoprotein changes in response to nitrate 267 treatments 268
The only nitrate sensor described to date is the nitrate transporter, NRT1.1/NPF6.3 39,73 . In 269 order to understand the importance of NRT1.1/NPF6.3 for nitrate elicited changes in the 270 phosphoproteome observed, we analyzed the phosphoproteomic profile of roots treated with 5 mM 271 KNO3 or KCl (control) in a nrt1.1-null background (mutant chl1-5), using the same experimental 272 conditions described above. 74% of phosphoproteins were detected in both datasets, yet only 5% 273 of the nitrate-phosphoproteome response observed in wild-type plants was maintained in the chl1-274 5 mutant ( Figure 3). Moreover, 95% of phosphoprotein levels were altered in the chl1-5 mutant 275 response to nitrate, in addition its established role as nitrate transceptor. 277 Our analysis indicates that NRT1.1/NPF6.3 is critical for maintaining the Arabidopsis 278 phosphoproteome in response to nitrate availability. It also denotes that NRT1.1/NPF6.3 function 279 is required for rapid changes in phosphorylation of key proteins in response to nitrate in 280 Arabidopsis roots. For example, phosphorylated peptides that map to proteins associated with 281 nitrogen metabolism (NRT2.1, AMT1-3 and NIA2) were identified in chl1-5 mutant roots, but 282 their levels were not affected in response to nitrate. 283

Network analysis reveals regulatory sub-networks connected to transcription factors and 284
potential kinases in response to nitrate. 285 To uncover key biological processes modulated by changes in phosphorylation, we 286 performed a multinetwork analysis with our phosphoproteomics data. We generated this network 287 by integrating different levels of information, including protein-protein interactions from 288 BioGRID 74 , predicted protein-DNA interactions of Arabidopsis TFs (DapSeq) 75,76 , Arabidopsis 289 metabolic pathways (KEGG), and miRNA-RNA, as described previously 16 . We also integrated 290 kinase-substrate predictions and identified the most significant phosphorylation motifs and their 291 predicted kinase families from our phosphoproteomic datasets using the Motif-X algorithm 77 and 292 the PhosPhAt Kinase-Target interactions database 78 ( Figure S5). We used the Cytoscape 79 293 software to visualize the resulting network, wherein genes that encoded each phosphoprotein were 294 represented as nodes linked by edges that signify any of the functional relationships annotated in 295 the various databases indicated above. We generated a network of 206 nodes with 700 interactions 296 ( Figure 4A). Although the majority of these genes are not regulated by nitrate at the mRNA level, 297 they form a highly interconnected network which includes potential regulatory transcription 298 factors and kinase components. They are connected by multiple edges, including protein-protein, 299 protein-DNA, and metabolic interactions. This result suggests that the products of these genes 300 form connected biological modules that are coordinately regulated at the post-translational level. 301 This network included several TFs with a high number of regulatory links. The most connected 302 were the Trihelix transcription factor 1 (GTL1), the WRKY DNA-binding protein 65 (WRKY65), 303 and the RELATED TO VERNALIZATION 1 (RTV1) transcription factors, which had not 304 previously been characterized in the context of nitrate response. Intriguingly, GTL1 regulates root 305 hair growth in Arabidopsis 80 , which has recently been described as a biological process modulated by nitrate treatments under the same experimental conditions 81 . A previous study indicated that 307 WRKY65 interacts at the protein level with the mitogen-activated protein kinase 10 (MPK10), 308 which binds with the lateral organ boundaries domain 16 (LBD16), LBD18, and LBD29 309 transcription factors 82,83 . These LBDs are inducible by auxin and play a role in the formation of 310 lateral roots 83 . In addition, MPK10 interacted with other genes involved in the auxin response 82 , 311 while LBD29 regulated genes involved in auxin transport, including auxin efflux-carriers PIN1 312 and PIN2 83 . This evidence suggests that WRKY65 could be involved in nitrate-auxin signaling 313 crosstalk. These three TFs appear to coordinate different subnetworks largely involved in auxin 314 transport and nitrogen metabolism. Consistent with this observation, analysis of over-represented 315 gene ontology annotations highlights the importance of auxin transport ( Figure 4B). Other over-316 represented biological functions in our network were mRNA binding and splicing, regulation of 317 translation and kinase activity ( Figure 4B). 318 Overall, this network analysis highlights a potential role of multiples TFs in linking the N 319 signal and regulatory nitrate responses that show significant enrichment for key functions in 320 signaling pathways and validates the important role auxin plays in the nitrate-response of . Moreover, the nitrate 333 transceptor NRT1.1/NPF6.3 not only senses and transports nitrate but can also transports auxin, a 334 process that regulates auxin-localization patterns and lateral-root elongation 84 . Consistent with 335 these prior observations, auxin transport was conspicuous throughout our entire phosphoproteomic analysis (Figure 2 and 4). PIN phosphorylation has been shown to be essential for auxin transport 337 and distribution [87][88][89][90][91][92] . PINs phosphorylation in conserved serine and/or threonine of the central loop 338 controls intracellular trafficking, recycling and polar membrane localization of PIN proteins 339 (Review by 87 ). Previous studies indicate that PIN polar localization explains auxin fluxes and 340 distribution patterns, which could mediate differential growth in diverse plant tissue such as 341 roots 91,93 . To validate the relevance of phosphorylation in auxin transport and its connection with 342 nitrate response, we chose the auxin efflux-carrier PIN2 (identified in our experimental dataset) 343 due to its potential role in linking nitrate and changes in root system architecture (RSA). We found 344 an uncharacterized PIN2 phosphorylation site (Ser439) at the end of the hydrophilic cytoplasmic 345 loop (C-loop, Figure 5A). Its phosphopeptide levels decreased by close to 75% in response to 346 nitrate treatments by 5 min ( Figure 5B). PIN2 belongs to the PIN-FORMED protein family of 347 auxin transporters and is the principal component mediating basipetal auxin transport in roots 94,95 . 348 This polar auxin transport is essential for root gravitropism 95 and lateral root formation 96 . 349 Intriguingly, we detected only one phosphorylated peptide for PIN2 in response to nitrate. Protein 350 sequence alignment indicated that this phosphosite is highly conserved in different plant species 351 representing gymnosperm, mono-and dicotyledonous plant lineages of seed plants ( Figure 5A). 352 This phosphopeptide has also been described as down-regulated after auxin treatment but its 353 function remains an open question 57 . 354 As a first step to understand the function of PIN2 phosphorylation in the nitrate response, 355 we performed a Phos-tag Western Blot analysis to confirm the changes in PIN2 phosphorylation 356 after nitrate treatment. We detected two, fast-and slow-mobility, PIN2 specific bands indicating 357 the presence of two phospho-populations in response to nitrate: one more and another less 358 phosphorylated PIN2. In contrast, only one slow-mobility band corresponding to the more 359 phosphorylated PIN2 subpopulation could be observed at time 0 (ammonium-supplied roots) or 360 under control conditions (KCl-treated roots) ( Figure 5C). To assess whether these changes in PIN2 361 phosphorylation status are a result of a change in protein abundance, we analyzed PIN2-GPF 362 protein levels by Western Blot under our experimental conditions. We introgressed the construct 363 PIN2::PIN2-GFP (PIN2 wt -GFP) into the pin2 loss-of-function mutant plant eir1-1 97 . No 364 differences in protein levels were observed in roots treated with nitrate as compared to roots at 365 time 0 ( Figure 5D). To understand the function of this specific PIN2 phosphosite Ser439, we also (PIN2::PIN2 S439A -GFP) or phospho-mimic (PIN2::PIN2 S439D -GFP) versions of PIN2-GFP. 368 Similarly, PIN2 protein concentrations were similar when our experimental conditions mimicked 369 PIN2 phospho-modifications ( Figure 5D). Moreover, no regulation at the mRNA level was 370 observed in PIN2 during nitrate responses ( Figure 5E). These results demonstrated that nitrate 371 regulates PIN2 at the posttranslational level, causing de-phosphorylation of PIN2 at a specific 372 phosphosite. 373 Next, we evaluated the role of PIN2 in root system architecture (RSA) in response to nitrate 374 treatments. We grew wild-type (Col-0) and pin2 mutant (eir1.1) plants for 2 weeks on ammonium 375 as sole N source (time 0) and evaluated RSA after nitrate treatments. We measured primary root 376 length 3 days after 5 mM KNO3 or KCl treatments. As expected for this experimental set up, we 377 found that nitrate-treated wild-type plants developed shorter primary roots as compared to KCl-378 treated plants, consistent with earlier results indicating that nitrate treatments inhibit primary root 379 elongation under these experimental conditions 7 ( Figure 6A). However, primary roots of eir1-1 380 plants were not significantly inhibited by nitrate treatments as compared to wild-type plants. We 381 also analyzed the density of lateral roots in response to nitrate treatments. In wild-type plants, 382 nitrate treatments increased the number of lateral roots (emerged and initiating) as compared with 383 KCl treatments ( Figure 6B). In contrast, the lateral root response to nitrate treatment was altered 384 in the eir1-1 mutant and the density of lateral roots was significantly reduced as compared with 385 wild-type plants ( Figure 6B To explore the impact of nitrate-regulated phosphorylation of PIN2 on cellular localization, 419 we examined the subcellular localization pattern in PIN2-GFP genotypes with phosphosite 420 substitutions. PIN2 WT -GFP proteins were accumulated at the plasma membrane of epidermal and 421 cortical cells, as previously described ( Figure 8A) 86 . PIN2 WT -GFP fluorescence signal increased 422 in epidermal and cortical cells 2 hours after nitrate treatments as compared to roots in control 423 conditions (roots without nitrate treatments, Figure 8A). Interestingly, mutations at S439 altered 424 this pattern. PIN2 S439A -GFP plants showed higher fluorescence in the plasma membrane even 425 without nitrate treatment as compared to PIN2 wt -GFP or PIN2 S439D -GFP ( Figure 8A). The total 426 fluorescence in all experimental conditions analyzed here was similar ( Figure 8B). In response to nitrate, PIN2 WT -GFP and PIN2 S439A -GFP were accumulated at the plasma membrane of epidermal 428 and cortical cells at comparable levels ( Figure 8C and 8D). On the contrary, PIN2 S439D -GFP plants 429 showed lower levels at epidermal and cortical cells in response to nitrate treatments as compared 430 to PIN2 WT -GFP or PIN2 S439A -GFP ( Figure 8C and 8D). 431 These results indicate that PIN2 phosphorylation status at S439 is important for a correct 432 subcellular localization-pattern in response to nitrate treatments. Moreover, these results indicate 433 that post-translational control impinging upon PIN2 localization is required for RSA changes in 434 response to nitrate treatments. 435

Discussion 437
A key plant nutrient, N also acts as a signal that regulates a myriad of plant growth and 438 developmental processes. Nitrate, a main N-source in natural and agriculture soils, elicits genome-439 wide changes in gene expression for thousands of genes involved in various biological functions. Interestingly, the majority of the transcription factors that we detected as differentially 458 phosphorylated had not been identified as part of the nitrate response. The phosphoproteomic 459 response to nitrate at 20 min revealed a group of different phosphoproteins, mostly involved in 460 protein binding and transport. In this dataset, we found proteins known to be involved in nitrogen 461 response as differentially phosphorylated, including the high-affinity nitrate transporter 462 . Furthermore, post-translational regulation does not require a change in gene 485 expression or de novo protein synthesis. Post-translational control is faster, allowing rapid 486 adaptation to environmental changes. Interestingly, the genes coding for nitrate-modulated 487 phosphoproteins identified in this study are highly co-expressed across many different 488 experimental conditions but not regulated by nitrate treatments 104 . This finding suggests that this 489 group of genes is functionally related and regulated at the mRNA level in response to several 490 endogenous or exogenous cues. In the context of nitrate responses, the products of these genes are 491 regulated at the post-translational level, uncovering a new layer of control that enables signal 492 crosstalk and fine tuning. Our results highlight the need for integrated analysis and data sets at 493 different levels to decipher plant responses to environmental cues. 494 It is now clear that auxin plays a central role in the plant root response to changes in nitrate 495 availability. Nitrate regulates primary root growth, lateral root initiation and elongation. Auxin in 496 turn is key during root development 96 , particularly in initiation and growth of lateral roots 93 .
Several reports show that auxin signaling, biosynthesis, transport, and accumulation are affected 498 during nitrate responses 72,84 , and transcriptomic analyses demonstrate that genes involved in auxin 499 response are controlled by nitrate 7, 16,71 . The main nitrate transporter NRT1.1/NPF6.3 can also 500 transport auxin 84,86 . A recent study also showed that NRT1.1/NPF6.3 negatively regulates the 501 TAR2 auxin-biosynthetic gene and the LAX3 auxin-influx transport gene at low nitrate 502 concentrations, repressing lateral root development 105 . These results suggest that an interplay 503 between nitrate signaling and auxin transport occurs at different levels [106][107][108] . Consistent with these 504 findings, we found that the molecular function "auxin transport" was overrepresented in our cluster 505 and network analyses. We showed that dephosphorylation of PIN2 in a novel phosphosite is a part 506 of a regulatory mechanism for RSA responses triggered by nitrate. The 507 phosphorylation/dephosphorylation of PIN proteins at specific sites (serine or threonine) located 508 in their higher loops has been shown to play important roles in modulating PIN functions 88,91,92,109 , 509 including trafficking 110 . We showed that phosphorylation/dephosphorylation of PIN2 at S439 is . The phosphorylation of PIN affects 517 PIN-mediated auxin transport, which controls plant growth 113 and other developmental 518 processes 114 . These results suggest that PIN phosphorylation is part of a regulatory switch that 519 strictly controls the directional transport of auxin and subsequent growth or developmental 520 processes. 521 Our results suggest a model (Figure 9) in which nitrate promotes dephosphorylation of 522 PIN2, which then impacts localization and auxin transport. Modulation of PIN2 function could 523 affect growth of primary and lateral roots for optimal nutrient uptake. Beyond this new regulatory 524 mechanism involving PIN2 protein, our phosphoproteomics results identify novel proteins, which 525 may be interesting targets for future studies or biotechnological developments for improved 526 nitrogen use-efficiency or crop yield.

548
Seeds were sterilized using 50% chlorine solution for 7 minutes and washed with sterile distilled water 549 three times. Then, 1,500 Arabidopsis seedlings were placed into a hydroponic system (Phytatrays) with

557
Root architecture analysis.

558
For root phenotyping, plants were scanned in plates using an Epson Perfection V700 Photo scanner, and 559 root length were measured using Fiji (v1.52). Initiating and emerging lateral roots were analyzed using DIC 560 optics on a Nikon Eclipse 80i microscope, as described 7 . The data were statistically analyzed in the Graph 561 Pad Prism 5 Program.

563
Protein extraction, phosphopeptide enrichment and mass spectrometry analysis.

564
Proteins were isolated from 1 g of frozen tissue per sample in each experimental condition (2-3 biological 565 replicates). Sample preparation and protein extraction were performed using previously described 566 methods 52,53 . Phosphopeptide enrichment was performed using 1% (w/v) colloidal CeO2 into an acidified 567 peptide solution at 1:10 (w/w). Mass spectrometry (MS) and peptide identification were based on protocols 568 described previously 60 . Briefly, the generated spectra were analyzed on LTQ Velos linear ion trap tandem 569 mass spectrometer (Thermo Electron) and phosphorylation sites were identified into a specific amino acid 570 within a peptide by using the variable modification localization score in Agilent Spectrum Mill software 118 .

571
Proteins were grouped based on their shared, common phophopeptides using principles of parsimony to 572 address redundancy in proteins. Proteins classified within the same group share the same subset of 573 phosphopeptides. Phosphorotein levels were quantified using spectral counting, as described 574 previously 52,53 . MS data were normalized using the total number of spectral counts (SPC) for each MS run.

575
Expressed phosphoproteins were defined by at least one SPC, after the application of quality score cutoff 576 in MS analysis, in minimum two of the tree biological replicates. To identify nitrate-regulated 577 phosphoproteins in Arabidopsis roots, raw data were log transformed and quantile normalized using R 578 studio (https://rstudio.com) and MEV (http://mev.tm4.org/) software. The statistical multifactor analysis of 579 variance (significance: p < 0.05) and pos-hoc analysis (significance: p < 0.1) were performed using R-