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. 2014 Apr 3;65(8):2093–2106. doi: 10.1093/jxb/eru082

The phosphoproteome in regenerating protoplasts from Physcomitrella patens protonemata shows changes paralleling postembryonic development in higher plants

Xiaoqin Wang 1,2, Meiyan Qi 2, Jingyun Li 2, Zhongzhong Ji 2, Yong Hu 2, Fang Bao 2, Ramamurthy Mahalingam 3, Yikun He 2,*
PMCID: PMC3991745  PMID: 24700621

Summary

During protoplast regeneration, proteins related to cell morphogenesis, organogenesis and development adjustment were phosphorylated in Physcomitrella patens. These proteins play important roles in regulating postembryonic development in higher plants.

Key words: LC-MS/MS, Physcomitrella patens, phosphoproteome, postembryonic development, protoplast regeneration, TiO2 enrichment.

Abstract

The moss Physcomitrella patens is an ideal model plant to study plant developmental processes. To better understand the mechanism of protoplast regeneration, a phosphoproteome analysis was performed. Protoplasts were prepared from protonemata. By 4 d of protoplast regeneration, the first cell divisions had ensued. Through a highly selective titanium dioxide (TiO2)-based phosphopeptide enrichment method and mass spectrometric technology, more than 300 phosphoproteins were identified as protoplast regeneration responsive. Of these, 108 phosphoproteins were present on day 4 but not in fresh protoplasts or those cultured for 2 d. These proteins are catalogued here. They were involved in cell-wall metabolism, transcription, signal transduction, cell growth/division, and cell structure. These protein functions are related to cell morphogenesis, organogenesis, and development adjustment. This study presents a comprehensive analysis of phosphoproteome involved in protoplast regeneration and indicates that the mechanism of plant protoplast regeneration is similar to that of postembryonic development.

Introduction

Plant leaf mesophyll cells can be separated from their original tissue by cell-wall-degrading enzymes generating a large population of protoplasts that can then become totipotent and hence regenerate whole plants (Zhao et al., 2001). Becoming totipotent involves changes in DNA methylation pattern and increased transcription and reorganization of specific chromosomal subdomains (Zhao et al., 2001; Avivi et al., 2004), changes that are similar to those during embryo development, implying a similar mechanism. Protoplasts are also used to observe cellular processes and activities, such as cell-wall synthesis, cell cycle, and differentiation during regeneration, and hormone responses in various plant species (Sheen, 2001). These cellular processes might be similar to plant postembryonic development.

In plants, postembryonic development is organized by meristems, which both self-renew and produce daughter cells that differentiate and give rise to different organ structures. Mechanisms mediating postembryonic development have been mainly studied in seed plants. It has been established that the cell wall is responsible for organ shape and that the cytoskeleton plays an important role in cell division and expansion. Additionally, cell-cycle regulation is essential for development. Some cell-cycle regulators, such as cyclins and cyclin-dependent kinases, are particularly numerous in plants, reflecting the remarkable ability of plants to modulate their development (Inze and De Veylder, 2006).

Organogenesis is a postembryonic process that occurs in a continuous manner throughout the entire lifespan. During organogenesis, organ identity genes play a key role. In the course of shoot propagation, a number of regulators have been identified, including KNOTTED1, SHOOT MERISTEMLESS (STM), KNOTTED-like from Arabidopsis thaliana 2 (KNAT2), and CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and CUC2) (Vollbrecht et al., 1991; Long et al., 1996; Pautot et al., 2001; Cary et al., 2002; Gordon et al., 2007). In contrast, for root formation, certain other proteins appear to play dominant roles, including the PINFORMED (PIN) family, transport inhibitor response1 (TIR1), and the Aux/IAA family of transcription factors (Geldner et al., 2001; Rogg et al., 2001; Friml et al., 2002a, b). During organogenesis, DNA methylation, histone methylation and acetylation are important for the enormous variation in cell-type-specific and stage-specific gene expression (Fransz and de Jong, 2002). However, much less is known the molecular mechanism of protoplast regeneration.

The moss Physcomitrella patens has been established as a model system for the study of plant development (Cove et al., 1997; Sakakibara et al., 2003). As with all plants, the form of the moss plant is determined by the pattern of growth and division. Protoplast cultures provide an ideal system for the study of development because following protoplast formation intact plants are produced at high frequency and rapidly. In protoplasts formed from seed plants, the process of regeneration has been associated with numerous events, including dedifferentiation and the loss of photoautotrophic metabolism (Fleck et al., 1980; Vernet et al., 1982; Criqui et al., 1992; Nagata et al., 1994), cell-wall synthesis (Meyer and Abel, 1975), and activation of the cell-cycle machinery (Galbraith et al., 1981, 1983). The cell cycle is regulated by key developmental regulators, which are themselves phosphoregulated (Joubes et al., 2000).

Protein phosphorylation is among the most important post-translational modifications in cells. It underlies many regulatory functions, such as cell-cycle control, receptor-mediated signal transduction, differentiation, and metabolism. For these regulatory functions, eukaryotic cells rely extensively on phosphorylating the hydroxyl group of the side chains of serine, threonine, and tyrosine (Hunter, 1995; Schlessinger, 2000).

Here, phosphoproteomics has been used to increase understanding of the machinery of protoplast regeneration in P. patens. The work examined the global changes in the phosphoproteome following protoplast development using titanium dioxide (TiO2) phosphopeptide enrichment strategies coupled with LC-MS/MS. The study reveals the integration of protoplast regeneration mechanisms in P. patens.

Materials and methods

Plant material and harvesting

P. patens (Hedwig) ecotype ‘Gransden 2004’ was grown in BCDA medium as described (Khandelwal et al., 2010). Protonemata were cultured at 25 °C under a 16/8 light/dark cycle. To produce protoplasts, 7-d-old protonemata were treated for 1h with 0.5% driselase dissolved in 8% mannitol. After filtration and washing, the protoplasts are regenerated on liquid BCDA medium with 8% mannitol under the same culture conditions. Protoplast isolation was repeated three times. For analysis of the next experiment, freshly prepared protoplasts and those cultured for 2 and 4 d were harvested.

Fluorescence-activated cell sorter analysis

For fluorescence-activated cell sorter (FACS) analysis, nuclei were stained with 2.86 μM 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and analysed on a two-laser FACStar Plus platform (Becton Dickinson, Mountain View, CA, USA). An argon ion laser tuned to 488nm was used in the laser experiment and a detector with a 530 band pass filter was used for fluorescein isothiocyanate. Software compensation was applied to the collected data using CELLQUEST software (Becton Dickinson).

Protein extraction

Frozen plant material was suspended in 2ml extraction buffer containing 250mM sucrose, 20mM Tris-HCl (pH 7.5), 10mM EDTA, 1mM 1,4-dithiothreitol (DTT), and inhibitor cocktail for proteases (Sigma) and phosphatases (Sigma). Cell debris was removed by centrifugation at 8000 g for 10min at 4 °C. Supernatants were transferred to clean tubes and centrifuged at 120 000 g for 1h at 4 °C. The final supernatants were used for soluble protein extraction, as described previously (Wang et al., 2009). The pellets (membrane-associated proteins) were re-precipitated with acetone overnight at –20 °C. The precipitated membrane proteins were rinsed three times with ice-cold acetone containing 13mM DTT and subsequently lyophilized. Finally, both protein pellets were resuspended separately by adding 8M urea, 4% CHAPS, 65mM DTT, and 40mM Tris (pH 7.5). The protein concentration was determined according to Peterson (1977) using BSA as a standard. The supernatants were stored in aliquots at –80 °C or directly digested with trypsin.

Tryptic digestion and phosphopeptide purification

The tryptic digestion was processed as described previously (Dai et al., 2005). In brief, 500 μg of each protein sample (100 μl volume) were reduced with 20mM DTT at 37 °C for 2.5h and alkylated with 100mM iodoacetamide for 40min at room temperature in the dark. After that, the protein mixtures were spun and exchanged into 100mM ammonium bicarbonate buffer (pH 8.5), and then incubated at 37 °C for 16h with trypsin at an enzyme/substrate ratio of 1:50 (w/w) to produce a proteolytic digest. Finally, the digested peptide mixture was lyophilized and then diluted in a loading buffer containing 1M glycolic acid in 65% acetonitrile (ACN) and 2% trifluoroacetic acid (TFA).

Phosphopeptide purification was performed using TiO2 microcolumns (320 μm×50mm, Column Technology, Freemont, CA, USA; Thingholm and Larsen, 2009). The microcolumns were rinsed with 100 μl loading buffer and the samples (200 μl) were subsequently loaded by applying air pressure. After loading the sample onto the microcolumn, the columns were subsequently washed with 100 μl loading buffer, 100 μl washing buffer I (65% ACN and 0.5% TFA), and 100 μl washing buffer II (65% ACN and 0.1% TFA). The bound peptides were eluted with 100 μl elution buffer (300mM ammonium water and 50% ACN). The elution was acidified by adding 5 μl 100% formic acid prior to the desalting step.

Nano-LC/MS/MS analysis

Peptide separation was performed on a surveyor liquid chromatography system (Thermo Finnigan, San Jose, CA, USA), consisting of a degasser, MS pump, and autosampler and equipped with a C18 trap column (RP, 320 μm×20mm, Column Technology) and an analytical C18 column (RP, 75 μm×150mm, Column Technology). After sample loading, the column was washed for 30min with 98% mobile phase A (0.1% formic acid in water) to flush off remaining salt. Peptides were eluted using a linear gradient of increasing mobile phase B (0.1% formic acid in ACN) from 2 to 35% in 120min. A linear ion trap/Orbitrap hybrid mass spectrometer (Thermo Fisher, San Jose, CA, USA) equipped with a NSI nanospray source was used for the MS/MS experiment. Spray voltage applying to the Nano needle was 1.85kV and ion transfer capillary temperature was 160 °C. Normalized collision energy for collision-induced dissociation was 35%. The number of ions stored in the ion trap was regulated by the automatic gain control. The instrument method consisted of one full MS scan from 400 to 2000 m/z followed by data-dependent MS/MS scan of the 10 most-intense ions from the MS spectrum with the following dynamic exclusion settings: repeat count of 2, repeat duration 30 s, exclusion duration 1.5min. The resolution of the Orbitrap mass analyser was set at 100 000 (m/Δm 50% at m/z 400) for the precursor ion scans.

Protein assignment

The strategy for identifying phosphorylated proteins in P. patens was as follows. The MS/MS spectra files from each LC run were centroided and merged to a single file using the TurboSEQUEST program in the BioWorks 3.2 software suite (Thermo Electron), and then the MS/MS spectra were searched against the NCBI A. thaliana and P. patens combined protein database (including normal and reversed) with carbamidomethylcysteine as a fixed modification. Oxidized methionine and phosphorylation (serine, threonine, and tyrosine) were searched as variable modifications. The searches were performed with tryptic specificity allowing one missed cleavage and the precursor ion m/z tolerances of 50 ppm and fragment ion m/z tolerances of ±1Da. Cysteine residues were searched as a fixed modification by 57.02146Da because of carboxyamidomethylation. Oxidation was set as a variable modification on methionine (15.99492Da). Dynamic modifications were permitted to allow for the detection of phosphorylated serine, threonine, and tyrosine residues (+79.96633). The phosphoric acid neutral loss peaks of serine and threonine was about –18.01056Da.

To provide high-confidence phosphopeptide sequence assignments, an accepted SEQUEST result had to have a ΔC n score of at least 0.1 (regardless of charge state). Peptides with a +1 charge state were accepted if they were fully digested and had a cross correlation (Xcorr) of at least 1.9. Peptides with a +2 charge state were accepted if they had a Xcorr ≥2.2. Peptides with a +3 charge state were accepted if they had a Xcorr ≥3.3. All output results were filtered and combined together using BuildSummary software to delete the redundant data (Tabb et al., 2002). All identified proteins (whether phosphorylated or not) were calculated separately and filtered by precursor ion tolerance m/z of 10 ppm and 1.0% false-positive rate (Elias and Gygi, 2007). The false-positive rate (FPR) was calculated as: FPR = 2[N rev/(N rev + N for)], where N rev is the number of hits to the ‘reverse’ peptide and N for is the number of hits to the ‘forward’ peptide. The proteins were classified to a protein group if the same peptides were assigned to multiple proteins after false peptides were filtered. A further manual check removed phosphopeptides with unclear MS/MS spectra.

For phosphorylation site identification, first a stricter peptide identification criteria (FPR≤0.01) was set. Second, a modified site was considered to be unique only when the corresponding modified peptides had a ΔC n >0.1 because a ΔC n >0.1 is significant for discriminating the first (top) candidate peptide from the second candidate peptide (Deng et al., 2010). In addition, this work checked the phosphoric acid neutral loss peaks for phosphorylation site identification (Ballif et al., 2004).

Analysis of gene expression by real-time reverse-transcription PCR

Total RNA was extracted using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). After extraction, RNA samples were treated with DNase (Ambion, USA). First-strand cDNA was synthesized from total RNA using a iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. The P. patens actin3 cDNA gene was used as a standard to normalize the content of cDNA. Real-time reverse-transcription PCR was performed using gene-specific primers for phosphoproteins in the P. patens protein database and phosphoproteins in the A. thaliana protein database that had genes homologous to those in the P. patens database (Supplementary Tables S1 and S2, respectively, available at JXB online) on a Rotor Gene 3000 Real-Time Thermal Cycler (Corbet Research, Australia). SYBR Premix Ex Taq (Perfect Real Time) kit and reverse-transcription PCR reagents (Takara Bio) were used for quantification of differentially expressed gene sequences.

Results

Protoplast cell-cycle phase

To identify the phase of the cell cycle for cells in P. patens protonemata, the DNA content of protonemata cell nuclei was measured with FACS. The standard phase of cell cycle was determined using nuclei from A. thaliana leaves. The nuclei from A. thaliana had three peaks and two peaks from P. patens, and the second peak in A. thaliana has approximately the same relative fluorescence value as that of P. patens protonemata in the first peak (Fig. 1A and B). The A. thaliana genome size is 125Mb and the leaves are diploid. The P. patens protonemata are haploid and its genome size is 490Mb. The nuclei in the second peak of A. thaliana leaves are in G2 phase (4C, 500Mb; Fig. 1B). So, it was speculated that the nuclei from P. patens protonemata were in G1 phase (1C, 490Mb; Fig. 1A).

Fig. 1.

Fig. 1.

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Identification of cell-cycle phases. Nuclei were prepared from Physcomitrella patens protonemata (A) and Arabiposis thaliana leaves (B) or a mixture of nuclei from both species (C), stained with DAPI, and subjected to FACS analysis.

To investigate how P. patens protoplasts regenerate, 7-d-old protonemata were used to establish an efficient and reproducible ‘protoplast system’. FACS analysis showed that most protonemal nuclei (92%) had a DNA content corresponding to G1 phase and a small peak (8%) was present at a S/G2 level (Fig. 2A), whereas nearly 100% of the nuclei from freshly harvested protoplasts had a G1 level of DNA (Fig. 2B). This is consistent with previous report. Tobacco leaves were treated with cell-wall-degrading enzymes to produce a large population of protoplasts, which had a DNA content corresponding to G1 phase (Zhao et al., 2001). Fresh protoplasts appeared round and green. By 2 d of regeneration, the new polar axes were established and protoplasts with a S/G2 level of DNA were present (constituting about 8% of the population; Fig. 2C). By 4 d, asymmetric cell divisions were common and protoplasts with a S/G2 DNA content constituted about 13% of the population (Fig. 2D). Subsequently, the cultures were transferred to a regeneration medium (BCDA medium) for formation of protonemata.

Fig. 2.

Fig. 2.

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Protoplast regeneration and the cell cycle. (A) Protonemata. (B) Protoplasts. (C) Protoplasts after regeneration for 2 d. (D) Protoplasts after regeneration for 4 d. Nuclei were isolated from Physcomitrella patens protonemata or protoplasts at the indicated times, stained with DAPI, and subjected to FACS analysis. Bright-field images show representative cells. Bars, 20 μm.

Phosphopeptide enrichment and LC-MS/MS identification

To analyse the P. patens phosphoproteome, this work used a TiO2 phosphopeptide enrichment strategy in combination with LC-MS/MS for identification. The resulting data were analysed using the TurboSEQUEST program in the BioWorks 3.2 software suite. From the three treatments altogether, more than 2000 phosphoproteins were identified (data not shown). This work focused on phosphoproteins in protoplasts regenerated for 4 d. There were more than 300 of these expressed in protoplasts regenerated for 4 d which were not present in fresh protoplasts or those regenerated for 2 d. Of this group of unique phosphoproteins, 108 of them were functional annotation proteins and others are predicted proteins. These 108 phosphoproteins were chosen for further analysis (Table 1).

Table 1.

Phosphoproteins in Physcomitrella patens unique to protoplast regenerated 4 d* indicates methionine oxidation; # indicates phosphorylation sites.

Metabolic group Protein no. Theoretical mass (kDa) Theoretical pI Accession no. Description Subcellular localization Sequence
Metabolism (19.44%) C1 32.1 5.05 NP_194311 XTR6 probable xyloglucan endotransglucosylase/ hydrolase protein 23 Cell wall M*Y#S#SLWNAEEWATR
C2 61.9 9.08 NP_187947 Sks11 (SKU5 Similar 11); copper ion binding/oxidoreductase Membrane NCWQDGT#PGT#MCPIMPGT#NYTYHFQPK
C3 116.4 5.96 NP_172637 ATSS3 (starch synthase 3); starch synthase/transferase, transferring glycosyl groups Chloroplast DS#NATST#ATNEVSGISK
C4 42.2 5.5 NP_001031705 CORI3 (CORONATINE INDUCED 1); cystathionine beta-lyase/transaminase Apoplast FSSIVPVVT#LGSISK
C5 152.4 5.77 NP_193817 TPP2 (TRIPEPTIDYL PEPTIDASE II); tripeptidyl-peptidase Unknown VLDVIDCT#GSGDIDTST#VVK
C6 72.1 6.31 Q8RXN5 tRNA wybutosine-synthesizing protein 1 homologue Unknown CMET#T#PS#LACANK
C7 44.0 7.54 NP_568193 AtAGAL2 (Arabidopsis thaliana ALPHA-GALACTOSIDASE 2); alpha-galactosidase/catalytic/hydrolase, hydrolysing O-glycosyl compounds Cell wall FIAFT#LTITLT#QIADGFQS#R
C8 61.5 8.93 NP_196950 Pseudouridine synthase/transporter Chloroplast GET#VELS#PR
C9 7.04 6.05 Q9C6T3 Terpene synthase family protein, putative Membrane LIHLLVS#M*GISYHFDK
C10 66.6 6.63 NP_191966 Malate oxidoreductase, putative Mitochondrion MAGIS#ES#EATK
C11 87.6 6.37 Q9FZI2 Lupeol synthase 5 Unknown CCMLLS#T#M*PTDITGEK
C12 38.3 4.61 NP_849593 Lipid-binding serum glycoprotein family protein Membrane DQIGS#SVEST#IAK
C13 40.1 8.87 NP_196027 Short-chain dehydrogenase/reductase (SDR) family protein Unknown FNHS#HT#PVCVITGAT#SGLGK
C14 60.2 8.74 Q8W033 Aldehyde dehydrogenase family 3 member I1 Chloroplast LFS#EYLDNTT#IR
C15 79.9 6.12 NP_566041 Transketolase, putative Chloroplast S#IGIDT#FGASAPAGK
C16 45.8 5.12 NP_196694 Monooxygenase family protein Membrane S#SEPPVGSPT#GAGLGLDPQAR
C17 52.3 5.6 Q9C7Z9 Serine carboxypeptidase-like 18 Membrane T#EGYNGGLPSLVSTSYS#WTK
C18 39.7 5.27 NP_197540 Oxidoreductase, 2OG-Fe(II) oxygenase family protein Unknown SLELEENSFLDM*Y#GESATLDT#R
C19 25.8 8.79 Q8VY52 PsbP domain-containing protein 2 Chloroplast FFCFAQNPS#S#TVSINLSK
C20 134.7 7.39 NP_196999 GMII (GOLGI ALPHA-MANNOSIDASE II); alpha-mannosidase Golgi apparatus PLNDS#NS#GAVVDITT#K
C21 57.6 9.09 NP_193230 ATAO1 (ARABIDOPSIS THALIANA AMINE OXIDASE 1); amine oxidase/copper ion binding Cell wall VGVDLT#GVLEVK
Transcription (22.22%) C22 62.5 7.72 NP_192756 Light-mediated development protein DET1 Nucleus FGLFATSTAQIHDSS#SPS#NDAVPGVPS#IDK
C23 137.4 9.28 NP_568624 COP1-interacting protein-related Membrane VYT#LETEIIQIK
C24 102.4 8.35 EDQ54570.1 Trithorax-like protein, histone–lysine N-methyltransferase Nucleus TDCLTY#QPPVT#S#SGCAR
C25 70.3 4.44 EDQ52478.1 Paf1 complex protein Nucleus GLSPGY#LEDALEEEDEPDQYDR
C26 110.5 4.91 EDQ60631.1 Putative histone deacetylase complex, SIN3 component Nucleus FM*QVLY#GLLDGSVDNSK
C27 72.4 8.63 NP_181971 DNA-binding bromodomain-containing protein Nucleus SMNESNST#ATAGEEER
C28 35.6 4.91 P46640 KNAT2 homeobox protein knotted-1-like 2 Nucleus AEDNFS#LSVIK
C29 59.9 6.96 NP_199123 APUM13 (Arabidopsis Pumilio 13); RNA binding/binding Nucleus IMDAISSVALQLT#R
C30 29.5 8.75 EDQ59312.1 Type I MADS-domain protein PPTIM4 Nucleus MS#TT#TSPPALT#VK
C31 114.8 8.71 NP_173522 Squamosa promoter-binding-like protein 14 Nucleus GLDLNLGS#GLT#AVEETTTTTQNVR
C32 40.2 5.70 NP_198791 AGL81 (AGAMOUS-LIKE 81); transcription factor Nucleus CSSS#SSS#SSYSLASTSLSNR
C33 19.9 6.21 NP_001078798 AGL31 (AGAMOUS LIKE MADS-BOX PROTEIN 31); transcription factor Unknown TS#LEANSS#VDTQ
C34 64.9 5.02 NP_179283 AMS (ABORTED MICROSPORES); DNA binding/transcription factor Nucleus LMEALDS#LGLEVT#NANTTR
C35 176.3 5.65 EDQ61350.1 SNF2 family DNA-dependent ATPase Nucleus S#PENGS#PAQEYDSPS#R
C36 108.1 6.84 NP_564568 SNF2 domain-containing protein/helicase domain-containing protein/RING finger domain-containing protein Nucleus LDGT#MS#LIAR
C37 200.2 8.50 EDQ61497.1 SNF2 family chromodomain-helicase Nucleus NY#QEEGVTWLDHNFENR
C38 102.1 9.52 EDQ66080.1 Argonaute family member Unknown LMSPLEGGSTTS#SS#SS#R
C39 42.5 5.40 NP_179397 VND1 (VASCULAR RELATED NAC-DOMAIN PROTEIN 1); transcription factor Unknown FVASQLMS#QEDNGTS#SFAGHHIVNEDK
C40 34.6 5.47 NP_190248 PCL1 (PHYTOCLOCK 1); DNA binding/transcription factor Unknown FGSMAS#Y#PSVGGGS#ANEN
C41 263.8 5.34 NP_171710 Transcriptional regulator-related Membrane ETGT#SLQTLT#SAATM*ER
C42 67.5 8.98 NP_175971 Transcription factor-related Unknown HFWSSY#PITTT#Y#LHTK
C43 42.8 5.39 NP_566737 S1 RNA-binding domain-containing protein Chloroplast DT#S#DEASAAGPSDWK
C44 36.5 6.91 NP_188063 CID9 (CTC-interacting domain 9); RNA binding/protein binding Unknown NFFES#ACGEVTR
C45 38.2 8.81 NP_567159 DNA-binding storekeeper protein-related Nucleus T#LNS#PSAAVAVSDDSES#EK
C46 36.8 8.33 NP_174084 CYCT1;3 (CYCLIN T 1;3); cyclin-dependent protein kinase Nucleus NVSLFESPQCET#S#K
Signal transduction (12.96%) C47 71.7 6.43 Q8H0V1 CDK5RAP1-like protein Unknown AT#HAS#SS#SSSALLPR
C48 49.6 6.37 NP_568248 CYC3B (MITOTIC-LIKE CYCLIN 3B FROM ARABIDOPSIS); cyclin-dependent protein kinase regulator Nucleus WT#LDQTDHPWNPT#LQHYTR
C49 39.2 9.11 Q9C9M7 CAK4 cyclin-dependent kinase d-2 Cytoplasm T#IFPM*AS#DDALDLLAK
C50 134.2 6.53 NP_199264 TAO1 (TARGET OF AVRB OPERATION1); ATP binding/protein binding/transmembrane receptor Membrane QFLVDT#EDICEVLTDDT#GT#R
C51 20.3 6.59 EDQ82848.1 Arf6/ArfB family small GTPase Unknown NVNFT#VWDVGGQDK
C52 33.5 5.72 NP_200984 ATIPK2BETA; inositol or phosphatidylinositol kinase/inositol trisphosphate 6-kinase Nucleus LPHLVLDDVVSGY#ANPS#VM*DVK
C53 21.9 6.84 EDQ74392.1 Sar1 family small GTPase Unknown M*GY#GEGFK
C54 188.8 5.47 NP_195533 Guanine nucleotide exchange family protein Chloroplast T#ALGPPPGSS#T#ILSPVQDITFR
C55 52.5 6.11 NP_001031307 NHO1 (nonhost resistance to P. s. phaseolicola 1); carbohydrate kinase/glycerol kinase Unknown AVLES#M*CFQVK
C56 109.4 5.43 NP_849957 AtRLP19 (receptor-like protein 19); kinase/protein binding Membrane LTGTLPSNMSSLS#NLK
C57 75.5 9.02 NP_565976 TTL3 (TETRATRICOPETIDE-REPEAT THIOREDOXIN-LIKE 3); binding/protein binding Unknown GSASSSAAATPTS#SSGS#SGSAS#GK
C58 21.9 6.10 NP_172390 ATSARA1A (ARABIDOPSIS THALIANA SECRETION-ASSOCIATED RAS SUPER FAMILY 1); GTP binding Chloroplast M*GY#GEGFK
C59 22.6 9.41 NP_196631 Aminoacyl-tRNA hydrolase/protein tyrosine phosphatase Unknown FTTAS#PS#M*SQQTGEIDAVVDASSAEK
Transport (10.19%) C60 96.9 6.92 Q38998 Potassium channel AKT1 Membrane S#WFLLDLVSTIPSEAAM*R
C61 19.7 8.86 NP_175324 Plastocyanin-like domain-containing protein Membrane VMEVESTPQSPPPS#S#SLPAS#AHK
C62 50.2 5.65 NP_198698 Amino acid transporter-like protein Membrane IMLQVS#ILVSNIGVLIVYM*IIIGDVLAGK
C63 106.9 5.62 NP_201066 Transportin-SR-related Nucleus LDTVTYS#LLALTR
C64 46.5 9.7 NP_001078720 APE2 (ACCLIMATION OF PHOTOSYNTHESIS TO ENVIRONMENT 2); antiporter/triose-phosphate transmembrane transporter Chloroplast QFS#TAS#SSSFS#VK
C65 53.5 6.36 Q6AWX0 d-xylose-proton symporter-like 2 Membrane M*ALDPEQQQPISSVS#R
C66 25.6 5.24 Q9SP35 Mitochondrial import inner membrane translocase subunit Tim17 Membrane EDPWNS#IIAGAATGGFLSMR
C67 84.6 9.29 Q9S9N5 Putative cyclic nucleotide-gated ion channel 7 Membrane FVS#S#GETEWIK
C68 116.2 8.17 EDQ55253.1 Na+ P-type-ATPase Membrane INMVY#SS#T#TVAK
C69 98.2 5.34 NP_195035 APM1 (AMINOPEPTIDASE M1); aminopeptidase Membrane MLQSY#LGAEVFQK
C70 117.7 6.91 NP_566869 CEF (clone eighty-four); protein binding/transporter/zinc ion binding COPII vesicle coat T#FVGIATFDS#TIHFYNLK
Cell growth/division (7.41%) C71 137.2 6.45 EDQ50619.1 Condensin complex component SMC3 Nucleus AEMET#QLVDS#LTPEQR
C72 77.6 8.87 NP_850846 Kelch repeat-containing protein Cytoplasm S#SS#SAVLTSDES#VK
C73 102.7 6.19 NP_567236 PMS1 (POSTMEIOTIC SEGREGATION 1); ATP binding/mismatched DNA binding Nucleus MQGDSS#PSPTT#TS#SPLIR
C74 50.3 6.58 NP_199003 POLA3; DNA primase Unknown MDVDDDDS#DS#SLLGK
C75 58.0 9.61 P40602 Anter-specific proline-rich protein APG Unknown NDIDSYT#TIIADSAAS#FVLQLY#GYGAR
C76 82.7 7.60 NP_194589 NPGR2 (no pollen germination related 2); calmodulin binding Membrane DY#NGSSALST#AES#ENAK
C77 34.5 6.76 NP_564689 Embryo-abundant protein-related Unknown FDT#NCVVT#TLK
C78 16.3 9.38 NP_564332 SAUR68 (SMALL AUXIN UPREGULATED 68) Unknown S#NVFTS#SSS#TVEK
Cell structure (9.26%) C79 108.8 8.62 NP_198947 Kinesin-like protein Microtubule associated complex INVLETLASGT#TDENEVR
C80 98.7 9.34 Q9FJX6 Formin-like protein 6 Membrane EEVS#EALT#DGNPESLGAELLET#LVK
C81 80.1 8.41 NP_189792 Myosin heavy chain-related Unknown ET#TGSVPFPS#VGGSR
C82 106.3 5.60 NP_567048 VLN3 (VILLIN 3); actin binding Unknown LY#S#IADGQVESIDGDLSK
C83 56.8 11.32 NP_566340 Proline-rich family protein Unknown GTSPSPTVNS#LS#K
C84 20.0 6.16 Q9SIX3 Putative glycine-rich RNA-binding protein Unknown GGMVGGYGS#GGY#R
C85 128.9 8.51 NP_194467 VIIIB; motor Chloroplast Y#S#QDLIY#SK
C86 89.2 7.24 NP_192428 ATK5 (ARABIDOPSIS THALIANA KINESIN 5); microtubule motor Mitochondrion T#LM*FVNISPDPS#STGESLCSLR
C87 82.9 4.84 Q8L838 Conserved oligomeric Golgi complex subunit 4 Membrane SCLSELGELS#S#T#FK
C88 135.5 6.51 NP_174461 Actin binding Unknown S#GLS#TEMVYSMS#DK
Protein destination and storage (6.48%) C89 75.8 8.99 NP_187752 DNAJ heat shock N-terminal domain-containing protein/cell division protein-related Unknown EY#VPSFNS#Y#ANK
C90 105.5 5.55 Q9LV01 ETO1-like protein 2 Unknown LASS#LGHVYS#LAGVS#R
C91 28.3 9.39 NP_196816 Peptidyl-prolyl cis-trans isomerase CYP20–2 Chloroplast MAT#LS#M*TLSNPK
C92 74.0 7.57 NP_192255 Ankyrin repeat family protein Unknown TMWQHSNNGS#S#STS#TLASK
C93 46.9 9.73 NP_566289 PAPA-1-like family protein/zinc finger (HIT type) family protein Chloroplast T#LQT#NSEAGTYTK
C94 32.1 9.42 NP_199935 Ubiquinol-cytochrome C chaperone family protein Mitochondrion Y#GY#ATVAPAAADPPSQK
C95 67.4 4.47 NP_191403 Meprin and TRAF homology domain-containing protein/MATH domain-containing protein Unknown ET#VDINGFVVVSS#K
Defence (5.56%) C96 22.8 9.30 Q8LEQ3 Nectarin-like protein Secreted protein DS#TQIT#PEDFYFK
C97 29.7 9.54 NP_178377 WHY3 (WHIRLY 3); DNA binding Nucleus FGDS#S#S#SQNAEVSSPR
C98 145.4 5.64 NP_176378 Galactolipase/phospholipase Unknown IIVFS#GTFGPTQAVVK
C99 17.8 5.01 NP_189276 Major latex protein-related/MLP-related Unknown MAT#SGT#Y#VTQVPLK
C100 62.6 8.9 NP_001117315 Jacalin lectin family protein Unknown PSS#PHNAIVPHNNSGT#AQIENS#PWANK
C101 48.9 6.08 O80998 Myrosinase-binding protein-like Unknown VFGS#ES#SVIVMLK
Protein synthesis (3.70%) C102 55.6 9.10 NP_186876 eIF4-gamma/eIF5/eIF2-epsilon domain-containing protein Unknown IFT#HEIS#S#CYASR
C103 28.4 5.44 NP_194797 MA3 domain-containing protein Unknown FT#LSSSS#DLTNGGDAPS#FAVK
C104 50.9 5.35 NP_851097 HCF109 (HIGH CHLOROPHYLL FLUORESCENT 109); translation release factor/translation release factor, codon specific Chloroplast Y#TLAMAS#AVTN
C105 11.2 6.01 NP_001031319 LCR82 Putative defensin-like protein 86 Membrane CY#STPECNAT#CLHEGY#EEGK
Unknown (2.78%) C106 56.4 5.60 NP_201248 Octicosapeptide/Phox/Bem1p (PB1) domain-containing protein Chloroplast FS#Y#NS#YPDSTDSSPR
C107 21.4 5.44 NP_172268 LOB domain-containing protein 1 Mitochondrion S#DASVATTPIISSSSSPPPS#LS#PR
C108 30.1 9.32 NP_195061 Chloroplast inner membrane import protein Tic22, putative Chloroplast SGTPTTT#LSPS#LVAK
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Phosphorylation site identification

Protein phosphorylation in eukaryotes predominantly occurs on serine and threonine residues, whereas phosphorylation on tyrosine residues is less abundant. As expected, the serine and threonine phosphorylation sites were predominant among the phosphoproteins associated with the protoplasts regenerated for 4 d (Table 1). Tyrosine phosphorylation accounted for nearly 12.5% of all phosphorylation events, a proportion that is nearly identical to that reported previously for protonemata (13%) of P. patens (Heintz et al., 2006). In contrast, for A. thaliana, phosphorylation on tyrosine residues was reported to constitute only 4.3% of protein phosphorylation events (Sugiyama et al., 2008), and for animal cells (2–3%) and yeast (<1%), tyrosine phosphorylation is even less (de la Fuente van Bentem and Hirt, 2009). Evidently, the moss has a high ratio of tyrosine phosphorylation compared to other eukaryotes but the reason for this remains to be determined. Additionally, this work found tyrosine phosphorylation in multiple phosphorylated peptides, results that are similar to those for A. thaliana (Sugiyama et al., 2008).

Predicted localization and categorization of phosphoproteins

The 108 phosphoproteins identified from the 4-d culture were categorized by cellular location, based on annotation within the NCBI database, and by function, based on the EU A. thaliana genome project (Bevan et al., 1998). The majority of the proteins were predicted to be located in membrane, nucleus, and chloroplast (Table 1 and Fig. 3). As for putative function, proteins could be sorted into 10 categories (Table 1 and Fig. 4), with more than half of the proteins being involved in transcription, signal transduction, growth/division, and structure.

Fig. 3.

Fig. 3.

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Annotated subcellular localization of phosphoproteins, showing the percentage of the 108 phosphoproteins that were specific to 4 d of regeneration in each category. Numbers in parentheses indicate the number of phosphoproteins in each subcellular localization. Localization was based on the NCBI database.

Fig. 4.

Fig. 4.

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Annotated functional categorization of phosphoproteins, showing the percentage of the 108 phosphoproteins that were specific to 4 d of regeneration in each category. Numbers in parentheses indicate the number of phosphoproteins in each functional category. Function was based on the EU Arabiposis thaliana genome project.

Phosphoproteins involved in cell-wall metabolism and cytoskeleton structure

Among the collection of identified phosphoproteins were several involved in cell-wall metabolism, arguably one of the characteristic metabolic processes of protoplast regeneration. This work identified a xyloglucan endotransglucosylase/hydrolase (XTHs, C1) and a copper-binding oxidoreductase related to A. thaliana SKU5 (C2), proteins that have been previously implicated in cell-wall-loosening and expansion (Campbell and Braam, 1999; Sedbrook et al., 2002). This work also identified structural cell-wall proteins, including a proline-rich family protein (C83) and a glycine-rich protein (C84) although the NCBI database does not assign either to a cell-wall location.

The identified phosphoproteins included several that are cytoskeletal. These include two members of the kinesin superfamily (C79, C86), a formin-like protein (C80), and a myosin heavy chain (C81). Kinesins are microtubule-based motors, formin is involved in the organization of the actin cytoskeleton, and myosin is a motor protein that drives actin-dependent motility.

Phosphoproteins involved in signal transduction

At 4 d after protoplast formation, 14 proteins were categorized within the signal transduction group. Among these proteins, four are heterodimeric serine/threonine protein kinases. These kinases comprise a catalytic subunit, termed cyclin-dependent kinase (CDK, C46, C47), an activating subunit (CDK-activating kinase, CAK, C49), and a cyclin (C48). In addition, two proteins are putatively involved in signalling to the cytoskeleton. One of them is a putative homologue of TAO-1 (C50), a protein that, regulates microtubule dynamics and checkpoint signalling during mitotic progression (Draviam et al., 2007), and the other is annotated as a small GTPase of the Arf6/ArfB family (C51), which is well known to regulate actin cytoskeletal organization in animals and fungi (Myers and Casanova, 2008), although apparently not studied in plants.

Phosphoproteins associated with transcription

Among the 108 phosphoproteins, 24 of them were associated with transcription. Interestingly, the putative function of many of these transcription factors developmental. These identified transcription factors include putative homologues of the following proteins: A. thaliana DE-ETIOLATED1 (C22), a COP1-interacting protein (C23), a histone–lysine N-methyltransferase (C24), a paf1 complex subunit (C25), a bromodomain-containing protein (C27), KNOTTED OF ATHALIANA2 (KNAT2) (C28), a A. thaliana pumillio family member (C29), a squamosa promoter binding protein-like 14 (SPL, C31), a type I MADS-domain protein (C30), two AGAMOUS-LIKE (AGL) proteins (C32, C33), ABORTED MICROSPORES (C34), and three SNF2 family proteins (C35, C36, C37).

Phosphoproteins involved in growth and division

There are eight phosphoproteins involved in growth and division. These proteins include a putative condensin complex component SMC3 (C72), a Kelch repeat-containing protein (C72), a putative homologue of POSTMEIOTIC SEGREGATION 1 (C73), POLA3 (C74), a putative homologue of an anther-specific proline-rich protein (C75), a putative homologue of no pollen germination related 2 (C76), a protein related to embryo-abundant proteins (C77), and SMALL AUXIN UPREGULATED68 (C78).

Quantitative real-time PCR analysis of phosphoproteins

To correlate protein level with the corresponding mRNA level, this work performed quantitative real-time PCR to analyse the mRNA expression of 24 genes of the 108 proteins specifically phosphorylated on day 4 (Fig. 5). Expression of all of the tested genes was increased at 2 d but fell at 4 d, in many cases falling substantially below the level of the fresh protoplasts. This result indicates that the transcriptional response precedes the protein phosphorylation, a result that is consistent with cellular signal transduction.

Fig. 5.

Fig. 5.

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Real-time quantitative PCR, showing mRNA changed patterns of selected proteins searched for Physcomitrella patens protein database (A) and of the Physcomitrella patens homologous genes of the selected proteins searched in Arabiposis thaliana protein database (B). Message level is expressed as log2 of the ratio of the expression level at day 2 (or day 4) to day 0. All expression levels were measured relative to P. patens actin3 cDNA gene. Values are mean±SD of three replicate experiments.

Discussion

Two major processes that are involved in plant development are morphogenesis and organogenesis. Morphogenesis is the formation of shapes and structures, and this depends on aspects of cell behaviour such as cell-wall synthesis, cell division, and elongation. Organogenesis is the specification of organ identity. Plants are characterized by having continuous postembryonic development, where both meristematic maintenance and growth are coupled with organogenesis and reproduction (Bowman and Eshed, 2000; Gutierrez, 2005). This study shows that the mechanism of protoplast regeneration is similar to that of postembryonic development.

Cell morphogenesis in the process of protoplast regeneration

Protoplast regeneration is accomplished by cell-wall synthesis, cytoskeleton construction, and regulation of the cell cycle. The primary cell wall consists of three coextensive polymer networks: the cellulose–xyloglucan framework, pectin, and structural protein. During protoplast regeneration, xyloglucan endotransglucosylase/hydrolase (XTH, C1), SKU5 (C2), proline-rich family protein (C83), and glycine-rich protein (C84) are examples of phosphorylated proteins that have plausible roles in primary cell-wall synthesis. In addition, squamosa promoter binding protein-like 14 (C31) is involved in cell-wall regeneration from protoplasts (Yang et al., 2008).

Microtubules and actin filaments are essential components of the machinery required for nuclear division and cytokinesis. Cytokinesis in plant cells is achieved through the construction of a new cell wall between daughter nuclei after mitosis. This process is directed by the phragmoplast, a cytoskeletal structure that is made up in part by parallel microtubules and actin filaments. This work identified several phosphorylated proteins that are implicated in regulating the cytoskeleton, including TAO-1 (C49), a Arf6/ArfB family small GTPase (C50), two kinesins (C79, C86), and a Kelch repeat-containing protein (C72) that has been reported to influence cell shape through the actin cytoskeleton (Adams et al., 2000), and a formin-like protein (C80) and myosin heavy chain (C81) that have been implicated in tip growth in moss (Vidali and Bezanilla, 2012). It indicates that that these cytoskeletal proteins are involved in cell division during protoplast regeneration.

Additionally, this work found several cell-cycle-regulating proteins to be phosphorylated specifically at day 4, including two cyclin-dependent kinase catalytic subunits (CDK, C46, C47), a CDK-activating kinase (C49), and a cyclin (C48). The catalytic subunits do not act alone: their ability to trigger cell-cycle events depends completely on associated cyclin subunits. The timing of activation of the CDK is be controlled by the timing of expression of a particular cyclin subunit, which also contributes to substrate specificity (Harper and Adams, 2001), and by phosphorylation. CDK-activating kinase is such an enzyme that phosphorylates CDKs to activate them (Umeda et al., 2005). These versatile enzymes form the core of the cell-division cycle. Given that more than 90% of the protoplasts divide in the days after protoplast formation, it is not surprising to see evidence of protein phosphorylation among cell-cycle regulators.

Development adjustment in response to protoplast regeneration

In this study, there were several phosphoproteins associated with development and closely related to protoplast regeneration. DET1 (C22) and COP1 control the transcription of multiple genes involved in photomorphogenesis by regulating chromatin conformation (Lau and Deng, 2012). COP1-interacting protein-related (C23) together with COP1 mediates gene expression during photomorphogenesis. Histone–lysine N-methyltransferase (ATX1, C24) is a chromatin modifier that trimethylates lysine 4 of histone H3 of associated nucleosomes. Histone H3 methylation affects DNA methylation and chromatin structure in ways that are consequential for development (Tamaru and Selker, 2001; Jackson et al., 2002). Pumilio (C29) is a founder member of an evolutionarily conserved family of RNA-binding proteins that play an important role in embryo development, differentiation, and asymmetric division (Spassov and Jurecic, 2002).

Surprisingly, this study identified a number of transcription factors that are well known from studies of flowering in seed plants. FLOWERING LOCUS C (FLC) is a MADS-box transcriptional regulator controlling flowering time (Michaels and Amasino, 1999). FLC transcription is controlled in part by the PAF1 complex (C25), which mediates histone methylation of FLC chromatin (Yu and Michaels, 2010). AGAMOUS-LIKE (AGL, C32, C33) is also a MADS-box protein required for the normal development of the internal two whorls of the flower (Mizukami et al., 1996). The specific function of these flowering genes in P. patens should be further studied.

Plant organs are formed continuously during development from meristems. ATX1 (C24) is required for the expression of homeotic genes involved in flower organogenesis (Alvarez-Venegas et al., 2003). Within the meristem, the family of KNOX (KNOTTED homeobox) genes plays a crucial role in regulating organogenesis of meristematic cells (Reiser et al., 2000). In A. thaliana, KNAT2 (KNOTTED-like from A. thaliana 2; C28) homeobox gene is expressed in the vegetative apical meristem. It is also active during flower development and plays a role in carpel development (Pautot et al., 2001). In P. patens, KNOX2 acts to prevent the haploid-specific body plan from developing in the diploid plant body (Sakakibara et al., 2013).

Several proteins related to chromosome stability and DNA repair were also identified. The SNF2 family of proteins (C35, C36, C37) plays roles in processes such as transcriptional regulation, maintenance of chromosome stability during mitosis, and various aspects of repairing DNA damage (Eisen et al., 1995). The condensing-complex component SMC3 (C71) is the core component of the tetrameric complex cohesin, which is required for the establishment of sister chromatid cohesion during S phase, maintenance of cohesion, and segregation of chromosomes in mitosis. PMS1 (C73) is a protein involved in the mismatch repair process after DNA replication (Nicolaides et al., 1994). These findings suggest that these proteins play a role in protecting cell stability during protoplast regeneration.

In conclusion, this study is, as far as is known, the first reported assessment of the phosphoproteome during of protoplast regeneration. A comprehensive analysis of the phosphoproteome involved in protoplast regeneration is presented (Fig. 6). This study indicates that there are similar mechanisms for plant protoplast regeneration and postembryonic development. Further studies of how these proteins direct the specific processes will provide deeper insight into plant protoplast regeneration.

Fig. 6.

Fig. 6.

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Cell responses corresponding to phosphoproteins identified during protoplast regeneration in Physcomitrella patens.

Supplementary material

Supplementary data are available at JXB online.

Supplementary Table S1. Quantitative real-time PCR primer pairs for phosphoproteins in the P. patens protein database.

Supplementary Table S2. Quantitative real-time PCR primer pairs for phosphoproteins in the A. thaliana protein database homologous to genes in the P. patens database.

Supplementary Data
supp_65_8_2093__index.html (982B, html)

Acknowledgements

This work was supported by the Project of Ministry Science and Technology of China (2013CB967300, 2007CB948201 to Y.H.), by the National Natural Science Foundation of China (31371243 to X.W.), and Beijing University of Agriculture Improving Research Quality Foundation (GL2012001 to X.W.). The authors acknowledge editing from Dickinson Meadows.

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Associated Data

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