key: cord-0309950-kvidu6fv
authors: Gao, Zhi-Fang; Shen, Zhuo; Chao, Qing; Yan, Zhen; Ge, Xuan-Liang; Lu, Tiancong; Zheng, Haiyan; Qian, Chun-Rong; Wang, Bai-Chen
title: Large-scale Proteomic and Phosphoproteomic Analysis of Maize Seedling Leaves During De-etiolation
date: 2020-03-14
journal: bioRxiv
DOI: 10.1101/2020.03.13.977843
sha: 46b62d2db6718caf1bc13969930ae44295e11e1a
doc_id: 309950
cord_uid: kvidu6fv

De-etiolation consists of a series of developmental and physiological changes that a plant undergoes in response to light. During this process light, an important environmental signal, triggers the inhibition of mesocotyl elongation and the production of photosynthetically active chloroplasts, and etiolated leaves transition from the “sink” stage to the “source” stage. De-etiolation has been extensively studied in maize (Zea mays L). However, little is known about how this transition is regulated. In this study, we describe a quantitative proteomic and phosphoproteomic atlas of the de-etiolation process in maize. We identified 16,420 proteins and quantified 14,168. In addition, 8,746 phosphorylation sites within 3,110 proteins were identified. From the proteomic and phosphoproteomic data combined, we identified a total of 17,436 proteins, 27.6% of which are annotated protein coding genes in the Zea_mays AGPv3.28 database. Only 6% of proteins significantly changed in abundance during de-etiolation. In contrast, the phosphorylation levels of more than 25% of phosphoproteins significantly changed; these included proteins involved in gene expression and homeostatic pathways and rate-limiting enzymes involved in photosynthesis light and carbon reactions. Based on phosphoproteomic analysis, 34% (1,057) of all phosphoproteins identified in this study contained more than three phosphorylation sites, and 37 proteins contained more than 16 phosphorylation sites, which shows that multi-phosphorylation is ubiquitous during the de-etiolation process. Our results suggest that plants might preferentially regulate the level of PTMs rather than protein abundance for adapting to changing environments. The study of PTMs could thus better reveal the regulation of de-etiolation.

Leaves during De-etiolation 28 29 a ORCID: 0000-0001-8384-4927. (Figure 4) . Four pathways were highly enriched in cluster 2 proteins, which 239 continuously increased in abundance after illumination: response to freezing, 240 photosynthesis, homeostatic process and generation of precursor metabolites and 241 energy. Numerous studies have shown that there is a complex cross-talk between 242 pathways in response to light and low temperature although the mechanism remains 243 poorly understood. For example, PIF3 and HY5 are key regulators in light response, 244 besides they both play vital roles in response to low temperature in Arabidopsis [36−38] . 245

In present study, when etiolated maize undergone photoporphogenesis, lots of proteins 246 involving in response to light signals were changed in abundance, which night also play 247 roles in resisting cold stress, so terms of response to freezing was enriched in cluster 2 248 proteins. In contrast, DNA replication initiation and regulation of macromolecule 249 metabolic process were the most highly enriched pathways for cluster 4 proteins, which 250 dramatically decreased in abundance after illumination. Though we did not find 251 significantly enriched pathways containing photoreceptors, we also followed with 252 interest the changes in the abundance of photoreceptors during the de-etiolation process. 253

The abundances of PHYA, PHYB, PHYC and CRY2 were sharply downregulated after 254 12 hours of light treatment (Supplementary Table 1 ). This is consistent with the 255 previous finding that photoreceptors are activated by light-induced phosphorylation, 256 which eventually initiates their ubiquitination and degradation [17, 39, 40] . 257 258

The number of phosphorylation sites per phosphorylated peptide and protein varied 260 greatly. We found 8,746 phosphorylation sites in 9,528 phosphopeptides that matched 261 Table 2 ). The most abundant phosphorylation site was S (7,639 or 268 77.78% of phosphosites), followed by T (1,067 or 12.20%) and Y (40 or 0.46%) ( Figure  269 5A). This suggests that S is the chief site modified by phosphorylation in maize leaves. 270

In the interest of revealing the pathways regulated by phosphorylation during the 271 de-etiolation of etiolated maize seedlings, GO enrichment analysis of all 3,110 272 phosphoproteins was performed ( Figure 5D ). Eleven biological processes were highly 273 enriched in phosphorylated proteins expressed during the de-etiolation process, such as 274 protein amino acid phosphorylation, signaling pathway, and regulation of signaling 275 process. The highest protein ratio (the number of phosphoproteins annotated to a certain 276 GO term to the total number of proteins) of phosphoproteins was observed for the 277 potassium ion transport term. This high ratio, 0.67, is because of the relatively low 278 number of proteins (24) assigned to the potassium ion transport term. The ratios of 279 phosphoproteins in the homeostatic process, response to cold, chromatin organization, 280 and signaling pathway categories (0.31, 0.27, 0.22 and 0.25, respectively) were also 281 relatively high. This indicates that phosphorylation modification may play a crucial role 282 in the regulation of these pathways during the de-etiolation process. 283

To investigate which proteins bring about changes in phosphorylation during maize 284 leaf de-etiolation, we screened our identified proteins for kinases and phosphatases 285 (Supplementary Table 5 (Figure 6 , Supplementary Table 6) . 300

Predicted kinase-motif interactions and protein quantification/phosphorylation 301 analysis can provide the basis for identifying possible substrates of different kinases. 302

To identify the pathways that are potentially regulated by protein phosphorylation, we 303 also identified phosphorylation motifs and the kinases that potentially phosphorylate 304 these sites. Kinases that phosphorylate phospho-motifs where S or T is the central 305 amino acid were classified into three major subgroups, namely proline-directed (pro-306 directed), basophilic (basic), and acidophilic (acidic), based on the types of substrate 307 sequences preferred [43] , and also into other families that we collectively refer to here 308 as "other" (Supplementary Table 7 ). The pS-and pT-containing sites (99.6%) were also 309 classified as pro-directed (33.82%), acidic (32.25%), basic (12.06%), and other 310 (21.47%) (Figure 7) . In this study, only 40 phosphorylated tyrosine peptides were 311 identified, accounting for 0.46% of all phosphopeptides (Figure 7) . 312

Using a method described previously [44], we identified phosphorylation motifs 313 centered on S, T or Y residues that were overrepresented in phosphopeptides using 314 motif-X (http://motif-x.med.harvard.edu) with the Zea mays AGPv3.28 protein 315 database as the background. Using stringent criteria for S and T and looser criteria for 316 Y, we identified 64 pS motifs, 13 pT motifs and 1 pY motif (Figure 7 , Supplementary 317 Table 7 ). The kinases that potentially phosphorylate these motifs were identified as 318 described in the Materials and Methods. Casein kinase II substrates (sxE, sDxE, and 319 sDxD) and basic motifs (RSxs, Rxxs, RxxsxD, and RxxsxG), which are phosphorylated 320 by PKA and PKC kinases, were identified in our phosphopeptide dataset. Pro-directed 321 substrates, which are mainly recognized by CDKs and MAPKs, were predominantly 322 found among the phosphopeptides containing S phosphorylation sites (Figure 7 , 323 Supplementary 

The phosphorylation modification of proteins may be highly complex. Some 334 phosphoproteins have multiple phosphosites, and phosphorylation may occur at 335 different phosphosites under different conditions. There may also be differences in the 336 change in NPL of peptides during de-etiolation. Here we describe the change in HY5 337 phosphorylation as an example. We identified three isoforms of HY5 338 (GRMZM2G039828_P01, GRMZM2G137046_P01 and GRMZM2G171912_P01) in 339 maize leaves, and three pS sites were conserved in all three isoforms (Supplementary 340 Figure 2 ). Six phosphopeptides of GRMZM2G039828_P01 were identified, which 341 contained a total of four phosphosites. We identified two phosphorylated forms of the 342 peptide "TSTTSSLPSSSER". One was phosphorylated at the fifth S and the other was 343 phosphorylated at the ninth S, and the changes in NPL were different for each peptide. 344

Moreover, two other peptides containing the same pS or pT plus pS sites, which were 345 possibly derived from different isoforms, showed very different changes in NPL. 346

In order to reveal what types of proteins are regulated by phosphorylation in 347 etiolated seedlings exposed to light, we screened for significantly changed 348 phosphopeptides (SCPPs) using stringent cutoff criteria (see Materials and Methods). 349

In brief, the phosphopeptides with a fold change in NPL ≥1.5 or ≤0.67 were considered 350 significantly changed. The proteins matching these peptides were considered proteins 351 with significantly changed phosphorylation (PSCPs). We identified 1,475 PSCPs 352 matching 826 proteins encoded by 823 genes. In fact, the number of PSCPs is likely 353 much higher because many phosphorylated peptides were filtered out because the 354 proteins they corresponded to were not quantified. 355

Firstly, we performed hierarchical clustering analyses of SCPPs using the average 356 fold change in intensity ratios that were normalized by protein abundance (Figure 8A) . 357

PSCPs were assigned to six clusters, and the first three clusters contained approximately 358 80% (1167) of the phosphopeptides, in which phosphorylation was immediately 359 downregulated after illumination. The phosphorylation levels of the remaining SCPPs 360 belonging to clusters 4, 5, and 6 increased after illumination. We also performed GO 361 We next analyzed GO categories enriched in the phosphoproteins in each cluster 368 ( Figure 8C ). In cluster 1, phosphorylation of SCPPs was reduced to the lowest level at 369 1 h and enrichment was observed for three BP categories (G-protein coupled receptor 370 signaling pathway, response to freezing, and nucleosome assembly) and six MF 371 categories. For SCPPs in clusters 3 and 2, phosphorylation was downregulated to the 372 lowest level at 6 h and 12 h, respectively. The GO categories enriched in cluster 3 and 373 2 proteins were response to freezing and nucleosome assembly of BP, respectively. The 374 phosphoproteins assigned to cluster 6, the phosphorylation of which was upregulated 375 by light at 1 h, were enriched in protein tyrosine kinase activity. The categories enriched 376 in cluster 4 SCPPs, the phosphorylation of which gradually increased, reaching the 377 highest level at 12 h, were protein tyrosine kinase activity, protein serine/threonine kinase activity, ATP binding, and structural molecule activity. These results suggest 379 that the phosphorylation modification of proteins involved in the response to signaling 380 pathway and kinase activities is affected by light and that changes in phosphorylation 381 in response to light may regulate the de-etiolation process. Table 8 ). The abundance of 37 (7.4%) out of 498 quantified TFs 389 significantly changed during de-etiolation (Supplemental Table 8 Table 8 ). In Arabidopsis, phosphorylation on three serine residues (Ser- showed that NPH3 is phosphorylated in dark-grown seedlings; its dephosphorylation is 452 stimulated by blue light and appears to be correlated with phototrophism [58, 59]. In 453

Arabidopsis, three phosphosites on the NPH3 (S212, S222 and S236) were identified 454 by immunoblotting analysis, which were phosphorylated under dark conditions [60] . 455

Here we identified 11 members of the NPH3 family in maize, and the abundance of one 456 of them was significantly downregulated in response to light. Moreover, eight of these 457 NPH3 proteins were found to be phosphorylated, and the NPLs of five phosphopeptides 458 Table 9 ). In 480 particular, the NPL of Thr377 in CAS increased 2.8-fold at 1 h, 13.7-fold at 6 h, and 481 20.0-fold at 12 h compared with the 0 h sample (Supplemental Table 9 ). Sequence 482 alignment indicated that Thr377 of maize CAS corresponds to Thr380 of Arabidopsis 483 CAS, which is one of the target sites of the thylakoid protein kinase STN8 484 (Supplementary Figure 7) . In Arabidopsis, CAS is essential for regulating the 485 transcription of photosynthetic electron transport-related genes, the formation of the 486 photosynthetic electron transport (PET) system, and water use efficiency [61] . 487 Therefore, the drastically increased NPL of Thr377 in CAS might be tightly related to 488 the formation or the regulation of the PET system. Table  512 11). After 6 h of illumination, the NPLs of these three PEPC proteins decreased to a 513 level similar to that of the 0 h sample, which suggests that phosphorylation rapidly 514 adjusts the enzyme activities of PEPC proteins in response to light. identified phosphorylated peptides contained more than two phosphosites; these 599 peptides corresponded to 1,057 (34%) phosphoproteins containing three or more 600 phosphosites, and 128 of them contained more than 10 phosphosites. These phosphosites may regulate different aspects of protein function by activating or 602 inhibiting protein activity, which may in turn regulate the functions of these proteins in 603 different pathways. Moreover, these effects may be enhanced by phosphorylation at 604 multiple sites in the same protein. Our data suggest that the regulation of PTM levels on proteins might be more 606 efficient than the regulation of protein abundance for adapting to changing 607 environments. Reversible PTMs allow plants to rapidly respond to internal and external 608

cues. In addition, PTM is more economical in terms of energy use than transcriptional 609 regulation, which involves several steps from the initiation of gene transcription to the 610 formation of a mature protein; only a little energy (ATP or GTP) is needed to add or to 611 remove a functional group (PTM) on a protein in order to change its physical and 612 chemical properties. Therefore, the study of protein PTM is important to fully explore 613 the mechanisms of plant adaptation to environmental changes. 614 615

The maize inbred line B73 was used in this study. The seedlings were planted and 618 samples were collected as described previously [32] . Under the same conditions, two 619 biological replicates were performed and the first seedling leaves from each replicate 620 were rapidly sampled. All samples were frozen in liquid nitrogen and stored at -80 °C 621 until further use. 622 623

Total proteins were extracted from maize seedling leaves using a 10% (w:v) 625 trichloroacetic acid (TCA)/acetone solution as described previously [32] . The protein 626 concentration of each sample was determined using the 2-D Quant kit (GE Healthcare). 627

Protein samples were stored at -80 °C for further experiments. 628

Protein extraction of two sets of maize samples (0 h, 1 h, 6 h, and 12 h, ~5 mg each) 631 and trypsin digestion were performed as previously described [32] .According to the 632 manufacturer's instructions, samples were labeled with iTRAQ 4plex reagent (ABSciex, 633 MA, US) and then combined. Phosphopeptide sequences were extended to 13 aa with a central S, T or Y using the 711 Zea mays database (V3.28) [78] . Pre-aligned peptides were submitted to the Motif-X algorithm (http://motif-x.med.harvard.edu/). Sites that were located at the N-or C-713 terminus and could not be extended to 13 aa were excluded. The significance was set 714 to P<10 -6 , and the minimum number of motif occurrences was set to 20 for S and T and 715

to 15 for Y. Motifs were classified as proline-directed, acidic, basic and other as 716 

Reconstruction of 772 protein networks from an atlas of maize seed proteotypes

Distinguishing protein-774 coding and noncoding genes in the human genome

Plant phosphoproteomics: a long 776 road ahead

Status of large-scale analysis of post-translational modifications 778 by mass spectrometry

Parallel proteomic and 780 phosphoproteomic analyses of successive stages of maize leaf development

Cross-783 talk between phosphorylation and lysine acetylation in a genome-reduced bacterium

Serinephosphoric acid obtained on hydrolysis of vitellinic 786 acid

Electrochemical methods 788 for detection of post-translational modifications of proteins

Recent advances and challenges in plant 791 phosphoproteomics

Multisite protein phosphorylation-from molecular mechanisms 793 to kinetic models

Yellow to red, significant enrichment; white, not significant. C1-C6: cluster 1-cluster 1361 6; GPCR: G-protein coupled receptor. 1362 (PEP)/phosphate translocator; RBCL: large subunit of Rubisco; RBCS: small subunit 1396 of Rubisco; PGK: Phosphoglycerate kinase; GAPDH: Glyceraldehyde-3-phosphate 1397 dehydrogenase

FBP: Fructose-1,6-bisphosphatase; TKL: Transketolase; SBPase: Seduheptulose 1399 bisphosphatase; RPI: Ribose-5-phosphate isomerase; RPE: Ribulose-phosphate 3-1400 epimerase; PRK: Phosphoribulokinase; PEPCK: Phosphoenolpyruvate carboxykinase

Supplementary Figure 7 Alignment of the AtCAS and ZmCAS proteins

The amino acid sequences of AtCAS (AT5G23060.1) and ZmCAS 1537 (GRMZM2G122715_T02) were aligned. The "T" outlined by a red box with asterisk 1538 is the phosphorylation site identified in both the Arabidopsis and Zea mays proteins

Single phosphorylation motifs were identified using the Motif-X algorithm [78] and 1067 overrepresented motifs were extracted. The background was the Zea mays.AGP v3.28 1068 protein database. The width was set to 13, the significance was set to 1×10 -6 , and the 1069 occurrence was set to 20 for serine and threonine motifs and to 15 for tyrosine motifs.