Comparative Study of Early Cold-Regulated Proteins by Two ...cls.hebtu.edu.cn/resources/43/20180319112145513.pdf · Base for Zhejiang Sustainable Pest and Disease Control, Institute

Comparative Study of Early Cold-RegulatedProteins by Two-Dimensional Difference GelElectrophoresis Reveals a Key Role forPhospholipase D�1 in Mediating ColdAcclimation Signaling Pathway in Rice*□S

Chenmin Huo‡�**, Baowen Zhang‡**, Hui Wang‡, Fawei Wang§, Meng Liu‡,Yingjie Gao‡, Wenhua Zhang§, Zhiping Deng¶, Daye Sun‡, and Wenqiang Tang‡ ‡‡

To understand the early signaling steps that regulate coldresponses in rice, two-dimensional difference gel electro-phoresis (2-D DIGE)1 was used to study early cold-regu-lated proteins in rice seedlings. Using mass spectrometry,32 spots, which represent 26 unique proteins that showedan altered expression level within 5 min of cold treatmentwere identified. Among these proteins, Western blotanalyses confirmed that the cellular phospholipase D �1(OsPLD�1) protein level was increased as early as 1 minafter cold treatment. Genetic studies showed that reduc-ing the expression of OsPLD�1 makes rice plants moresensitive to chilling stress as well as cold acclimationincreased freezing tolerance. Correspondingly, cold-reg-

ulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive elementbinding 1 (OsDREB1) family of transcription factors wereinhibited in the pld�1 mutant. We also found thatthe expression of OsPLD�1 is directly regulated byOsDREB1A. This transcriptional regulation of OsPLD�1could provide positive feedback regulation of the coldsignal transduction pathway in rice. OsPLD�1 hydrolyzesphosphatidylcholine to produce the signal molecule phos-phatidic acid (PA). By lipid-overlay assay, we demon-strated that the rice cold signaling proteins, MAP kinase 6(OsMPK6) and OsSIZ1, bind directly to PA. Taken to-gether, our results suggest that OsPLD�1 plays a key rolein transducing cold signaling in rice by producing PA andregulating OsDREB1s’ expression by OsMPK6, OsSIZ1,and possibly other PA-binding proteins. Molecular &Cellular Proteomics 15: 10.1074/mcp.M115.049759, 1397–1411, 2016.

As one of the most important food crops, rice (Oryza sativa)provides food for more than half of the world’s population.Compared with other major crops like maize and wheat, thegrowth and yield of rice is more susceptible to environmentallow temperature stress. Therefore, revealing the cellular mech-anisms that could lead to the generation of low temperaturestress resistant rice cultivars is important for meeting theincreasing food demand due to increasing world population.

When exposed to a period of nonlethal low temperature,plants can develop a number of cellular changes, including anincrease in intracellular calcium concentration (1, 2), alterationof cold-regulated (COR) gene expression (3), changes in lipidcomposition (4, 5), scavenging of reactive oxygen species (6),accumulation of osmotic adjustment substances (7), and met-abolic reprogramming (8) to enhance tolerance to lethal freez-ing temperature. This process is called cold acclimation. Us-ing Arabidopsis thaliana as a model system, several coldacclimation regulating cold stress signal transduction path-ways have been identified (9). Among these pathways, the C

From the ‡Hebei Collaboration Innovation Center for Cell Signaling;Key Laboratory of Molecular and Cellular Biology of Ministry of Edu-cation; Hebei Key Laboratory of Molecular and Cellular Biology; Col-lege of Life Sciences, Hebei Normal University, Shijiazhuang, Hebei050024, China; §State Key Laboratory of Crop Genetics and Germ-plasm Enhancement, College of Life Sciences, Nanjing AgriculturalUniversity, Nanjing 210095, China; ¶State Key Laboratory BreedingBase for Zhejiang Sustainable Pest and Disease Control, Institute ofVirology and Biotechnology, Zhejiang Academy of Agricultural Sci-ences, Hangzhou 310021, China; �College of Biology Science & En-gineering, Hebei University of Economics & Business, Shijiazhuang,Hebei 050061, China

Received March 8, 2014, and in revised form, January 6, 2016Published, MCP Papers in Press, January 8, 2016, DOI 10.1074/

mcp.M115.049759Author contributions: D.S. and W.T. designed the research; C.H.,

B.Z., and M.L. performed the research; H.W., F.W., Y.G., W.Z., andZ.D. contributed new reagents or analytic tools; W.T. analyzed data;and C.H. and W.T. wrote the paper.

1 The abbreviations used are: 2D-DIGE, two-dimensional differencegel electrophoresis; CAMTA3, calmodulin binding transcription acti-vator 3; CBF/DREB, C repeat /dehydration-responsive element bind-ing protein; COR, cold-regulated; CRT/DRE, C-repeat/dehydrationresponsive element; EMSA, electrophoretic mobility shift assay; GST,glutathion S-transferase; GUS, �-glucuronidase; HOS1, high expres-sion of osmotically responsive gene1; ICE1, inducer of CBF expres-sion 1; MPK6, MAP Kinase 6; OST1, open stomata 1; PA, phospha-tidic acid; PC, phosphatidylcholine; PLD�1, phospholipase D �1;SIZ1, SAP and Miz1; UTR, untranslated region.

Research© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.This paper is available on line at http://www.mcponline.org

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repeat/dehydration-responsive element binding protein1 (CBF/DREB1)-dependent pathway is the best characterized and isconsidered to be the major cold acclimation regulatory path-way. In this pathway, CBF regulates the expression of anumber of COR genes (10, 11), and the expression of CBF iscontrolled by the inducer of CBF expression1 (ICE1) family oftranscription factors (12) and/or calmodulin-binding transcrip-tion activator 3 (CAMTA3) (13). ICE1 is phosphorylated byopen stomata 1 (OST1) kinase during cold acclimation (14).The phosphorylation of ICE1 activates and stabilizes ICE1 byE3 small ubiquitin-like modifier (SUMO)-protein ligase SIZ1(SAP and Miz1)-mediated sumoylation (15), which preventsICE1 ubiquitination mediated by an ubiquitin E3 ligase highexpression of osmotically responsive gene1 (HOS1) (16).

In comparison with what is known in Arabidopsis, themechanism for cold acclimation is not well studied in rice.Sequence homology searches have identified homologousOsHOS1, OsICE1, and OsDREB1s genes in the rice genome(17–19). Similar to what has been observed in Arabidopsis,OsHOS1 is able to regulate the protein abundance of OsICE1(19). Also, the expression of OsDREB1A and OsDREB1B isinduced by cold stress, and overexpression of these twogenes increases the cold stress tolerance of transgenic rice(20), which suggests that the CBF/DREB1 cold signaling path-way is conserved in rice. In addition to OsDREB1s andOsICE1, a number of other proteins such as OsCOIN (21),OsMYBS3 (22), OsPRP3 (23), and OsMAPK6 (24) have beenshown to regulate cold stress responses in rice. Whetherthese proteins function as COR genes or as primary regula-tors of early cold responses in rice needs to be furtherexplored.

Despite considerable progress in studying plant cold signaltransduction mechanisms, our understanding of the coldstress signal perception and early signaling transductionsteps leading to CBF/DREB1s activation is still very limited.With the development of mass spectrometry technologies,quantitative proteomic approaches have proven to be verypowerful tools, complementing traditional genetic ap-proaches, in finding new signaling components as well asrevealing new signaling mechanisms (25, 26). There has beena number of reports studying cold-regulated proteins in riceusing proteomic approaches (27). However, the rice plants inthese studies were cold-stress-treated for hours or days. Itwas reported that cold induces an increase in cytoplasmiccalcium levels as early as 40 s after cold treatment (1) and theexpression of CBFs in Arabidopsis is dramatically increasedwithin 15 min after cold treatment (10). These results suggestthat early cold stress signal transduction occurs within 15 minor less after a plant is exposed to cold temperature. Therefore,it is likely that cold-regulated proteins previously identified inrice using proteomic approaches were mostly late cold-reg-ulated proteins whose expression was regulated by DREB1sand other cold-regulated transcription factor-mediated tran-scriptional networks.

In order to help better understand early cold stress signal-ing, two-dimensional difference gel electrophoresis (2D DIGE)technology was applied to characterize rapid cold-regulatedproteomic changes and identify novel early cold-regulatedproteins in 1-week-old rice seedlings. A total of 32 spots,which represent 26 unique proteins whose abundancechanged in response to a five-minute cold stress treatment,were identified by mass spectrometry. Additional genetic,biochemical, and physiological evidence suggest one of theseearly cold-regulated proteins, OsPLD�1, plays a key role inthe early cold stress signal transduction pathway in rice.

EXPERIMENTAL PROCEDURES

Protein Extraction and Two-Dimensional Difference Gel Electro-phoresis—Because we were interested in finding proteins whoseabundance changed rapidly in response to cold stress, a water bathwas used for the cold treatment to make sure all the rice seedlingswere instantly exposed to a similar temperature at the same time.Wild-type (Oryza sativa japonica cv. Nipponbare) rice seedlings werefirst germinated in double distilled water in darkness for 3 days.Seedlings were then transferred to Hoagland’s liquid growth mediumto grow 1 more week at 28 °C, 50% humidity and long-day condi-tions. After being submerged in double distilled water at 28 °C or 0 °Cfor 5 min, the seedlings were quickly tap-dried with tissue paper andsnap frozen using liquid nitrogen. Cold-treated and untreated sam-ples were handled in parallel for protein extraction and separation toreduce variations.

To prepare different cellular fractions for proteomic studies, �6 gliquid nitrogen ground tissue powder were mixed with extractionbuffer (25 mM HEPES, pH 7.5, with NaOH, 0.33 M sucrose, 10%glycerol, 0.6% polyvinylpyrrolidone, 5 mM ascorbic acid, 5 mM Na2-EDTA, 50 mM NaF, 2 mM imidazole, 1 mM sodium molybdate, 1 mM

PMSF, 5 mM DTT, 1 mM sodium orthovanadate, 1 �M E-64, 1 �M

bestatin, 1 �M pepstatin, 2 �M leupeptin) on ice, filtered through twolayers of miracloth to remove cell debris, and centrifuged at 10,000 �g for 15 min to collect the pellet fraction. The supernatant was furthercentrifuged at 60,000 � g for 45 min to separate soluble and micro-somal fractions. The soluble, pellet, and microsomal fractions weremixed with SDS extraction buffer (100 mM Tris-HCl, pH 8.0, 2% SDS,1% �-mercaptoethanol, 5 mM EGTA, 10 mM EDTA, 1 �M E-64, 1 �M

bestatin, 1 �M pepstatin, 2 �M leupeptin) and incubated at 65 °C for10 min, followed by phenol extraction and methanol precipitation asdescribed previously (28). The proteins were resuspended in DIGEbuffer (7 M urea, 2 M thiourea, 4% CHAPS) at a concentration of 5–10�g/�l.

Cy3 and Cy5 labeling of the proteins and two-dimensional electro-phoresis were performed as described previously (29, 30). 2D DIGEimages were acquired using a Typhoon Trio scanner (GE Healthcare,Piscataway, NJ). The images were analyzed using DeCyder 6.5 soft-ware (GE Healthcare). Spots that showed consistent cold stressregulated changes in at least four biological repeat samples werepicked using a robotic spot picker (GE healthcare).

Mass Spectrometry and Protein Identification—Selected proteinspots were in-gel digested by trypsin as previously described (28, 29).The extracted peptides were dissolved in 10 �l 0.1% formic acid. Thepeptides were loaded onto a C18 reverse phase column (100 �m �150 mm, Thermo Fisher Scientific, Waltham, MA) coupled online to aLTQ-XL linear ion trap mass spectrometer (Thermo Fisher Scientific)at a flow rate of 350 nl/min. Peptides were separated using a gradientfrom 100% of A (0.1% formic acid) to 45% of B (0.1% formic acid,95% acetonitrile) for 60 min. MS1 spectra were acquired in a positivemode using the data dependent automatic survey MS scan in the m/z

OsPLD�1 Regulates Cold Acclimation Signaling in Rice

1398 Molecular & Cellular Proteomics 15.4

range between 400 and 2000. The 10 most intense ions that over 500counts were selected for MS2 acquisition using normalized collisioninduced dissociation (CID) with 35% collision energy, and activationQ value was set to 0.25. CID product ions were analyzed on the linearion trap in centroid mode. The dynamic exclusion activation time wasset to 30 s to prevent same m/z ions from being selected after itsacquisition.

LC-MS/MS peak lists were searched against database generatedfrom Oryza sativa subset of the NCBInr database (date 20/8/2010,84,086 entries searched), using the SEQUEST search algorithm. Pep-tides were selected in the mass range between 400–5000 amu. Thefollowing search parameters were applied: mass tolerance for pre-cursor ions and fragment ions were set to 2.00 amu and 1.00 amu,respectively; two incomplete cleavages were allowed; alkylation ofcysteine by carbamidomethylation and oxidation of methionine wereconsidered as possible modification. Search result option filter wasset as the following: Delta CN � 0.1; Xcorr (�1, 2, 3) � 1. 5, 2.0, 2.5;peptide probability �0.01.

Isolation of OsPLD�1 T-DNA Mutant—T-DNA insertional mutant forOsPLD�1 (PFG-1A-21508) was obtained from the Rice T-DNA Inser-tion Sequence Database (http://cbi.khu.ac.kr/RISD_DB.html), andwas generated in Oryza sativa japonica cv Dongjin background. Thegene-specific primers used for genotyping the mutant were: 5�-ttct-cccttctcgacgcccgcgaag-3� and 5�-taatccagaatttccagatcgtttc-3�.Primers used for semi-quantitative RT-PCR analysis of OsPLD�1 ex-pression level were: 5�-ggtggacctagagagccatggcatg-3� and 5�-gcca-tggatctggccacgggctggtt-3� for OsPLD�1; 5�-tcagatgcccagtgacagga-3� and 5�-ttggtgatctcggcaacaga-3� for OsTubulin (Os03g51600).

Molecular Cloning and Generation of Transgenic Rice—Oryza sa-tiva japonica cv Dongjin was used for all the rice transgenic studies.The genome sequence and coding sequence of OsPLD�1 were PCRamplified, cloned into pENTRY/S.D./D-TOPO vector (Thermo FisherScientific), and subcloned into pMDC107, pGWB3, or pMDC83 des-tination vectors by LR clonase (Thermo Fisher Scientific) to generatepOsPLD�1:OsPLD�1-GFP, pOsPLD�1:OsPLD�1-GUS, or p35S:OsPLD�1-GFP expressing binary vectors. To generate the RNAi vec-tors for OsPLD�1 and OsDREB1A, a conserved 420-bp (2042–2461)coding sequence of OsPLD�1 and a conserved 234-bp (117–350)coding sequence of OsDREB1A was amplified by RT-PCR using thefollowing primer sets: 5�-ggactagtagatctacgacgagtacatcatcgtcgggt-3� and 5�-ccgagctcggatccttggcgggaggtagtcggatttggc for cloning ofOsPLD�1; 5�-ggggtaccgcggaccaagttcagggagacgag-3� and 5�-cggg-atccgagtcggcgaagttgaggcagcat-3� for BamH1�KpnI cloning ofOsDREB1A; 5�-ggactagtgcggaccaagttcagggagacgag-3� and 5�-ccg-agctcgagtcggcgaagttgaggcagcat-3� for SpeI�SacI cloning ofOsDREB1A. The PCR products were sequentially subcloned into pT-CK303 RNAi binary vector by BamHI � KpnI followed by SpeI �SacI-mediated double digestion and ligation as described (31). Thebinary vectors were introduced into EHA105 strain of Agrobacteriumtumefaciens and transformed into rice plants using Agrobacterium-mediated callus transformation. T2 homozygous transgenic rice see-dlings were used for the cold stress phenotype analysis.

Cold Tolerance Assays—Cold tolerance assays of rice seedlingswere performed as described with modifications (21, 32, 33). Forgermination assays, a minimum of 40 wild-type or pld�1 mutantseeds were submerged in 25 ml double distilled water in a glass Petridish in darkness at 28 °C or 16 °C for up to 14 days. The seeds werechecked daily for their germination rate. Seed germination was de-termined by emergence of coleoptile tips. For cold sensitivity assays,seedlings were first germinated in water in darkness for 3 days at28 °C. A minimum of 30 seedlings with similar coleoptile length werethen transferred to Hoagland’s liquid growth medium at 28 °C or12 °C, with a daily photoperiodic cycle of 16 h light and 8 h dark (longday) with 50% humidity for 30 days before coleoptile length and

seedling weight measurements were made. Alternatively, the seed-lings were allowed to grow in Hoagland’s liquid growth medium at28 °C, long day with 50% humidity for 7 days before being subjectedto 4 °C, long day with 50% humidity and allowed to continue growingfor up to 7 days. The cold-treated seedlings were allowed to recoverat 28 °C, long day with 50% humidity conditions for 7 more daysbefore taking pictures and calculating the survival rates. All the ex-periments shown in this study have been performed at least threetimes with similar results. Representative data from one repetition areshown.

Freezing Tolerance Assays—Freezing tolerance assays of riceseedlings were performed according to Li et al. (34). Seedlings weregrown in soil at 28 °C, long-day conditions with 50% humidity for 6weeks. Freezing stress was performed using a programmable freez-ing chamber (model AR33L, Percival Scientific, Perry, IA). The plantswere first kept at �1 °C for 3 h. Temperature was decreased at �1 °Cper hour to �2 °C, �4 °C, or �6 °C. After holding at the designatedfreezing temperature for 3 h, the temperature was increased at 1 °Cper hour to �1 °C then incubated at 12 °C for 12 h before transferringto a greenhouse to recover for 7 days at 28 °C, long-day conditionswith 50% humidity. Ion leakage was determined immediately afterovernight incubation at 12 °C. Approximately 1 g of leaves was har-vested and shaken in a falcon tube with 20 ml double distilled waterat room temperature overnight. Leaves were then transferred to a newfalcon tube with 20 ml double distilled water and boiled for 15 min.Conductivity of the solution was determined using a Leici conductivitymeter (DDS-IIA, Shanghai, China). Relative ion leakage was calcu-lated by dividing the conductivity collected from room temperaturesamples by the sum of conductivities collected from room tempera-ture samples and boiled samples.

Quantitative Real-Time RT-PCR—Seven-day-old wild-type riceseedlings (Dongjin) grown in Hoagland’s liquid growth medium at28 °C, long-day conditions with 50% humidity were subjected to12 °C, long-day conditions with 50% humidity for different time inter-vals. Whole seedlings were collected and snap frozen using liquidnitrogen. Total RNA was isolated using TRIzol reagent (Invitrogen) andreverse-transcribed in a 20 �l reaction using PrimeScriptTM RT rea-gent Kit (TakaRa, Kyoto, Japan) according to the manufacture’s in-structions. Quantitative real-time RT-PCR was performed by standardprotocol using a PRISM 7500 system (ThermoFisher Scientific). Ex-pression of OsPLD�1 and OsDREB1s was normalized by OsACTIN1(Os03g50885). Relative expression of OsPLD�1 and OsDREB1s ispresented as the ratio to the expression level in nontreated wild-typecontrol plants. The primers used are: 5�-atggcatgatattcactcacgg-ctg-3� and 5�-accaccatcaatggatctaaatagc-3� for OsPLD�1; 5�-accg-acgacgacgaggagtc-3� and 5�-gccaagctcgcgtagtacaggt-3� forOsDREB1A; 5�-ggcgacgaagaagaagacaacaag-3� and 5�-gtagtacgac-ccggcgtccatc-3� for OsDREB1B; 5�-gaattcgaaatgcaggggta-3� and5�-ctcgcagtcgtagtcctcct-3� for OsDREB1E; 5�-aggacgccatcttcga-cat-3� and 5�-gtcgagagatctcccaatcg-3� for OsDREB1F; 5�-cccgtact-acgaggtcatgg-3� and 5�-gctacctacggcaggatcac-3� for OsDREB1G;5�-agcaactgggatgatatgga-3� and 5�-cagggcgatgtaggaaagc-3� forOsACTIN1.

GUS Histochemical Staining—Tissue from pOsPLD�1:OsPLD�1-GUS-expressing rice seedlings was vacuum infiltrated for 30 min instaining solution containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-�-D-glucuronide (X-Gluc), 50 mM NaH2PO4, 50 mM Na2HPO4, 10 mM ofEDTA�Na2, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, and 0.1% TritonX-100. The tissue was then stained for 1 h (radicle and coleoptile) or3 h (flower, stem, and mature leaves) at 37 °C and cleared with 70%ethanol.

Protein Purification and In Vitro Lipid Overlay Assay—Full-lengthcoding sequence of OsDREB1A, OsMPK6, AtSIZ1, OsSIZ1, OsOST1,and Arabidopsis RCN1 without stop codon were amplified by PCR,



http://cbi.khu.ac.kr/RISD_DB.html

cloned into pENTRY/S.D./D-TOPO vector (Thermo Fisher Scientific),and subcloned into Gateway-compatible vectors pGEX-4T-1 by LRclonase (Thermo Fisher Scientific). The GST-tagged proteins wereexpressed and purified from Escherichia coli using glutathione Sep-harose 4B beads by standard protocols. For in vitro lipid overlayassay, phospholipids (Avanti polar lipids, Alabaster, AL) dissolved inchloroform were spotted on a nitrocellulose membrane and dried atroom temperature. The membrane was incubated with blocking so-lution containing 3% (w/v) fatty-acid-free BSA (Sigma-Aldrich, St.Louis, MO), 10 mM Tris-HCl, pH 8.0, 140 mM NaCl, and 0.1%Tween-20 for 1 h. The membranes were incubated with 2 �g/mlGST-tagged proteins in blocking buffer for 12 h at 4 °C with gentleshaking. After washing three times with blocking buffer, the mem-brane was incubated with a rabbit HRP conjugated anti-GST antibody(Sigma-Aldrich 1:5000 in blocking buffer) for 1 h. Binding of proteinsto phospholipids was visualized with FluorChemQ MultiImage III (Al-pha Innotech, San Leandro, CA) using the SuperSignal West Durachemiluminescent reagent (Thermo Fisher Scientific).

Electrophoretic Mobility Shift Assay—Double-stranded oligonu-cleotide sequence for electrophoretic mobility shift assay (EMSA)was: 5�-cgccacttggaccgacctcgggacgacgac-3�. EMSA assay wasperformed using a modified 10 �l mixture, which contained 1.5 �l (0.5�g/�l) purified protein, 1 �l biotin-labeled oligonucleotides, 1 �l 10 �binding buffer (100 mM Tris, 500 mM KCl, and 10 mM DTT, pH 7.5), 0.5�l 50% glycerol, 0.5 �l 1% Nonidet P-40, 0.5 �l 1 M KCl, and 5 �lultrapure water. The reactions were incubated at 4 °C for 60 min andloaded onto a 6% native polyacrylamide gel in TBE buffer (45 mM Tris,45 mM boric acid, and 1 mM EDTA, pH 8.3). The gel was transferredto a nylon membrane (Millipore, Billerica, MA) using 0.5 � TBE bufferat 100 V for 60 min in cold room. Nylon membrane was cross-linkedat 120 mJ/cm2 for 45–60 s using a UV cross-linker. Biotin-labeledDNA was detected by using the Light Shift Chemiluminescent EMSAkit (Thermo Fisher Scientific) according to manufacturer’s instruction.

Transient Luciferase Expression Assay—To generate pOsPLD�1:firefly luciferase, a 2.5 kb promoter upstream of translation startcodon ATG of OsPLD�1 was PCR amplified, cloned into pENTRY/S.D./D-TOPO vector (Thermo Fisher Scientific), and subcloned intopHGWL7.0 vector by LR clonase (Thermo Fisher Scientific). A full-length coding sequence of OsDREB1A without stop codon was PCRamplified and inserted into a p35S:EGFP-HA expression vector byEcoRI � SalI to replace the enhanced green fluorescent protein(EGFP) sequence to generate a p35S:OsDREB1A-HA expression vec-tor. Transient luciferase expression assay was performed accordingto Yoo et al. (35). In brief, rice protoplasts were prepared from leavesof 7-day-old wild-type rice seedlings (Dongjin) plants grown at 28 °Cunder long-day conditions with 50% humidity. The expression vec-tors were transformed into rice protoplasts by polyethylene glycol(PEG) 4000 (Sigma-Aldrich) mediated transfection. An p35S-drivenrenilla luciferase expression vector was cotransformed to calibratethe transfection efficiency, and 16 h after transformation, luciferaseluminescence was recorded for 10 s using a microplate luminometer(Centro LB 960, Berthold technologies, Bad Wildbad, Germany).Quantitation of luminescent signal from each of the luciferase reporterenzymes was performed using a dual-luciferase® reporter assay kit(Promega, Fitchburg, WI) according to manufacturer’s instruction.

RESULTS

OsPLD�1 Is an Early Cold-Regulated Protein—To investi-gate the early proteomic response of rice to cold stress,1-week-old rice seedlings were treated with 0 °C water bathfor 5 min. Cellular proteins were then separated into soluble,microsomal, and pellet (which include most of the organelles)fractions to reduce the complexity of the protein samples, and

analyzed independently by two-dimensional difference in gelelectrophoresis (2D DIGE). Samples from six independentpreparations were analyzed. On average, 2683 � 120 spotswere detected in our 2D DIGE gels, proteins that showedconsistent cold-regulated changes (over 1.5 fold) in abun-dance in at least four independent experiments were chosenfor protein identification.

By LC-MS/MS, 32 spots, which represent 26 unique pro-teins, were identified. Interestingly most of these spots werefound in the soluble and pellet fractions of our samples (Figs.1 A-1C and Table I). Only six spots whose abundance wasconsistently regulated by cold treatment were identified fromthe microsomal fraction (Fig. 1B), suggesting a relative delayin the expression changes of membrane proteins to coldstress. Functional classification showed these proteins areinvolved in processes such as cellular metabolism, stressresponses, protein folding, cytoskeleton rearrangement, andlipid metabolism. A comparison of our results with previouscold stress proteomic studies in rice showed that 20 (76.9%)of our identified proteins had not been previously reported.For the proteins we identified that had also been identified byprevious cold stress proteomic studies, our results indicatethat these proteins are actually early cold-regulated proteinsand thus likely contribute to early cellular responses to coldstress treatment.

Of all the early cold-regulated proteins identified, OsPLD�1was chosen for further studies (Fig. 1D) because it was pre-viously reported that suppressing the expression of PLD�1 inArabidopsis made plants more resistant to freezing tempera-ture (5). We first validated the proteomic results by Westernblot analysis, using a polyclonal antibody raised specificallyagainst the conserved C-terminal 12-amino acid sequencefound in both AtPLD�1 and OsPLD�1 (36). Surprisingly, theincrease of the OsPLD�1 protein level inside the cell can beseen as early as 1 min after cold stress treatment (Figs. 1E and1F). This increase in OsPLD�1 protein level was sustained for5 min and then decreased to return to the basal level 10 minafter cold treatment. We also tested whether the abundanceof OsPLD�1 transcript is rapidly regulated by cold stress.Semi-quantitative RT-PCR results showed that the expres-sion of OsPLD�1 is decreased by cold treatment for 1 min,possibly due to sudden increase in protein translation, andthen continues to increase from 5 min to 30 min after coldtreatment (Figs. 1G and 1H).

Expression Pattern and Subcellular Localization of OsPLD�1—To examine the tissue-specific expression pattern of OsPLD�1,genomic sequence, without the stop codon and 3�untrans-lated region (UTR), of OsPLD�1, which includes 2537-bppromoter and 5� UTR sequence before ATG and two introns,was cloned, fused with a �-glucuronidase (GUS) reporterenzyme at the C terminus, and introduced into wild-type riceplants. T2 homozygous pOsPLD�1:OsPLD�1-GUS trans-genic plants were used for histochemical staining for GUSactivity. As shown in Fig. 2, GUS expression was mostly



detected at the tips of roots and coleoptiles of rice seedlingsthat had just germinated (Fig. 2A). When plants were 7 daysold, strong GUS activity was detected in whole roots. Exceptfor weak OsPLD�1-GUS expression at the tips of the leaves,no detectable GUS activity was found in coleoptiles andyoung leaves of 7-day-old rice seedlings grown under normalconditions. A 5 min treatment with 0 °C water bath induced asignificant increase in the expression of OsPLD�1-GUS in thecoleoptile and leaf, which further confirmed that the level of

OsPLD�1 protein is increased by cold treatment (Fig. 2B).During tillering stages, OsPLD�1-GUS is also expressed instem. A close examination of hand cross-dissected stemshowed that the expression of OsPLD�1 in rice stems is verystrong in vascular tissues (Fig. 2C). When plants enter theflowering stage, OsPLD�1 promoter activities can be de-tected in hulls but not in stamens and pistils (Fig. 2D).

We also generated pOsPLD�1:OsPLD�1-GFP-expressingtransgenic rice and observed the subcellular localization pat-

FIG. 1. 2D-DIGE analysis of rice proteins and identification of OsPLD�1 as an early cold-responsive protein. (A, B, and C), 2D-DIGEanalysis of early cold-regulated soluble (A) microsome (B), and pellet (C) proteins. Proteins from 7-day-old wild-type rice seedlings (Nipponbare)were submerged in 0 °C water bath for 5 min and proteins extracted and labeled with Cy5 (red). Protein from seedlings submerged in 28 °Cwater bath control were labeled with Cy3 (green). The proteins were separated using 24 cm, pH 4–7 IPG strips and a 10% SDS-PAGE gel. (D)The three-dimensional view of OsPLD�1 (spot 4 in A). (E-H), Western blot (E) and semi-quantitative RT-PCR quantitation (G) of theaccumulation of OsPLD�1 protein or transcripts in 1-week-old rice seedlings treated with 0 °C water bath for the indicated time. Theexperiments have been repeated for at least three times, and representative results are shown. Relative abundance of OsPLD�1 protein ortranscript was quantified and normalized from three biological repeats using Ponceau S. stained Rubisco large subunit (E,F) or level ofOsACTIN transcript (G,H), respectively. One-way ANOVA test was performed. Statistically significant differences are indicated by differentlowercase letters (p 0.05). Error bars represent �S.D.



tern of OsPLD�1 in vivo. Using confocal microscopy, wefound OsPLD�1-GFP protein is distributed in the cytoplasm,but excluded from the nucleus of cells in the primary rootelongation zone (Fig. 2E).

Suppressing the Expression of OsPLD�1 Impaired the Chill-ing Tolerance of Rice Plants—To investigate the possible roleof OsPLD�1 in rice cold stress responses, a transfer DNA(T-DNA) insertion mutant of OsPLD�1 (PFG-1A-21508) wasisolated. Although the T-DNA is inserted in the 5�UTR of theOsPLD�1 genome sequence (Fig. 3A), reverse transcriptionPCR (RT-PCR) analysis of the mutant showed that the ex-pression of OsPLD�1 was barely detectable (Fig. 3B). UsingPLD�1-specific antibody, we further confirmed that theOsPLD�1 protein is not detectable in the pld�1 mutant(Fig. 3C).

The isolated T-DNA mutant was backcrossed with wild-type (Dongjin cultivar) plants twice to clean up the back-ground. Segregated homozygous pld�1 mutant plants, whichcontained only one T-DNA insertion site (based on the seg-regation ratio of F2 hygromycin resistant seedlings) andshowed no obvious growth phenotype, were used for chillingtolerance studies. One-week-old wild-type and pld�1 mutantrice seedlings were exposed to chilling stress (4 °C) forvarious durations. After recovering at 28 °C for 7 days, theseedlings were photographed and their survival rate was cal-culated. Compared with slightly reduced survival rate of wild-type plants, about half of the pld�1 mutant seedlings diedafter growing at 4 °C for 5 days (Figs. 3D and 3E). Similarresults were also observed for seeds germinated at 16 °C orseedlings grown at 12 °C for 30 days. On average, the ger-

TABLE IEarly cold-regulated proteins identified by mass spectrometry. The cold-regulated abundance changes were the averages of at least fourbiological repeats. For MS/MS identification, number of unique peptides, sequence coverage of the identified proteins and the best matched

scores are listed. Refs indicate same protein was identified from previous cold stress proteomic studies in rice

Spot Gene locus Protein nameUniquepeptide

Coverage(%)

Score Fold change p value Ref.

Metabolism1 Os05g33570 Pyruvate, phosphate dikinase 14 19.20 130.28 –1.61 � 0.03 3.0E-032 Os05g33570 Pyruvate, phosphate dikinase 20 24.50 180.27 –1.59 � 0.01 1.0E-036 Os03g04410 Aconitate hydratase protein 3 3.65 30.24 –2.14 � 0.31 4.2E-02 50, 51, 527 Os02g53860 Putative subtilase 22 2 3.70 20.20 3.08 � 0.28 3.7E-0211 Os01g25484 Ferredoxin–nitrite reductase 12 17.34 120.22 2.57 � 0.30 4.7E-02 5112 Os01g25484 Ferredoxin–nitrite reductase 11 15.66 100.21 2.31 � 0.40 4.4E-02 5115 Os02g38210 Elongation factor Tu 16 40.90 150.24 1.66 � 0.13 1.1E-0217 Os08g41990 Aminotransferase 2 4.60 20.18 4.32 � 0.51 4.7E-0219 Os08g27840 Phosphoenolpyruvate carboxylase 3 3.32 30.19 1.62 � 0.06 2.7E-0221 Os08g27840 Phosphoenolpyruvate carboxylase 3 3.32 30.18 1.57 � 0.01 9.6E-0322 Os06g40940 Glycine dehydrogenase 4 3.48 40.17 –1.91 � 0.36 3.2E-02 50, 5123 Os09g32650 Leucyl-tRNA synthetase 2 1.90 20.17 1.71 � 0.31 4.3E-0224 Os02g32350 TUDOR protein 4 5.98 50.21 –1.91 � 0.30 2.7E-0225 Os03g28330 Sucrose synthase 17 20.80 170.21 –1.63 � 0.21 3.1E-02 5226 Os03g28330 Sucrose synthase 6 8.95 60.22 1.64 � 0.13 1.4E-02 5230 Os02g41630 Phenylalanine ammonia-lyase 6 10.56 60.19 –1.67 � 0.13 1.3E-0231 Os08g06100 O-methyltransferase 4 8.42 30.16 –2.27 � 0.25 1.6E-0232 Os10g21266 ATP synthase subunit beta 3 4.20 30.19 –3.74 � 0.19 1.3E-02 53

Protein folding8 Os04g01740 HSP90 7 9.10 70.20 1.67 � 0.24 3.9E-0210 Os08g41390 Peptidyl-prolyl isomerase 5 8.79 50.18 –1.80 � 0.38 3.6E-029 Os12g12850 ATP-dependent Clp protease 12 13.82 120.23 –2.11 � 0.18 1.5E-0213 Os04g32560 ATP-dependent Clp protease 13 13.60 120.23 2.66 � 0.20 3.5E-0214 Os04g32560 ATP-dependent Clp protease 2 3.38 20.23 2.44 � 0.39 4.3E-0227 Os03g04970 HSP60 BETA 2 4.16 20.21 –1.88 � 0.24 2.2E-02 51, 5229 Os02g01280 CHAPERONIN 60 3 6.02 30.23 1.60 � 0.01 1.6E-02

Cytoskeleton5 Os03g24220 Villin protein 7 7.30 80.1 2.31 � 0.07 2.2E-0216 Os03g61970 Actin 3 4 9.28 40.15 5.74 � 0.14 4.3E-0228 Os05g01600 Actin 7 3 6.47 30.15 –1.73 � 0.08 7.5E-03

Lipid metabolism3 Os01g07760 OsPLD�1 19 25.49 170.31 1.96 � 0.14 2.6E-024 Os01g07760 OsPLD�1 45 54.40 250.2 4.09 � 0.11 5.2E-0420 Os12g37260 Lipoxygenase-2 5 5.31 50.22 –1.97 � 0.05 4.3E-03

Other18 Os05g32220 Ribosomal L1P family proteins 3 10.31 30.21 1.70 � 0.05 5.0E-02



mination of pld�1 mutant seeds at 16 °C was delayed for 2days, and the pld�1 mutant seedlings grown at 12 °C showedreduced fresh weight and coleoptile length compared withwild-type control (Figs. 3F-3H).

To rule out the possibility that the reduced chilling toleranceof pld�1 mutant is due to additional mutation site in themutant background, we generated OsPLD�1 RNAi transgenicrice plants. Western blot analysis using PLD�1 antibodyshowed the expression of OsPLD�1 protein in our transgenicRNAi rice seedlings is either very weak or not detectable (Fig.3I). Based on the expression level, R4b and R14b RNAi lineswere chosen for chilling tolerance assays. As shown in Fig. 3J,on average, around 37.5% of wild-type seedlings survivedafter growing in 4 °C for 6 days. In comparison, the survivalrate of our RNAi transgenic seedlings was 25% for line R4band 9.7% for line R14b. Taken together, these results dem-onstrate that suppressing the expression of OsPLD�1 makesrice plants more sensitive to chilling stress.

Suppressing the Expression of OsPLD�1 Reduces the ColdAcclimation Increased Freezing Tolerance of Rice Plants—Wealso tested the freezing tolerance of the pld�1 mutant underacclimation and nonacclimation conditions. Six-week-old riceseedlings were subjected to various freezing temperatures for3 h and allowed to recover at 28 °C for 7 days. Because of thespace limitation in the growth chamber used for freezingtreatment, a maximum of eight plants (four mutants and fourwild type) could be treated at the same time. Thus, the ex-periment was independently repeated five times, and theaverage number of green leaves per plant from all of the

experiments after recovery was calculated and used to eval-uate the freezing tolerance of the mutant.

Suppressing the expression of OsPLD�1 in rice seedlingshad no significant effect to the development of leaves. Six-week-old wild-type and pld�1 mutant rice plants developed8.25 � 1.21 and 8.08 � 1.83 leaves on average, respectively.There was no obvious difference in the green leaves of freeze-treated wild-type and pld�1 mutant rice plants under nonac-climation conditions at all freezing temperatures tested (Figs.4A and 4B), suggesting the expression level of OsPLD�1 doesnot regulate the basal freezing tolerance of rice. Cold accli-mation at 12 °C under long-day conditions for 7 days signif-icantly increased the freezing tolerance of wild-type riceplants. However, compared with the wild-type control, theincreased freezing tolerance in pld�1 mutant was greatly re-duced (Figs. 4A and 4B). Correspondingly, freeze-inducedelectrolyte leakage, an indicator of plasma membrane dam-age, in the pld�1 mutant was dramatically increased undercold acclimation condition compared with wild-type plants.Based on the chilling and freezing tolerance assays of thepld�1 mutant, we conclude that OsPLD�1 is a positive regu-lator of the rice cold acclimation processes.

Seedlings Overexpressing OsPLD�1-GFP Are Hypersensi-tive to Cold and Freezing Stresses—To further assess the roleof OsPLD�1 in cold and freezing tolerance of rice plant,full-length coding sequence of OsPLD�1 with a C-terminalgreen fluorescent protein (OsPLD�1-GFP) was overexpressedunder the control of p35S cauliflower mosaic virus promoter.The expression of OsPLD�1-GFP was detected by GFP an-tibody (Fig. 5A). Rice seedlings were subjected to cold (4 °C)treatment or cold acclimation at 12 °C for 7 days followed byfreezing (�6 °C) treatment. Unexpectedly, after recovery at28 °C for 7 days, the survival rate of OsPLD�1-GFP overex-pressing rice seedlings was lower than wild-type controlseedlings but higher than the pld�1 mutant seedlings (Figs.5B and 5C). It appears that the protein level of PLD�1 has tobe carefully controlled and maintained at an appropriate level.Overexpressing OsPLD�1-GFP may cause overaccumulationof PA inside the cell and the corresponding decrease in phos-phatidylcholine (PC) concentration in the membrane, whichmight stimulate the formation of hexagonal phases of mem-brane and increase membrane damage when plant cells en-counter low temperature (37).

Cold-Stress-Regulated Proteomic Changes in pld�1 Mu-tant—Using 2D-DIGE, we compared the cold-stress-regu-lated proteomic changes in wild-type (Dongjin) and pld�1mutant seedlings. Because we are interested in finding cold-regulated proteins downstream of PLD�1, 1h cold treatmentin 0 °C water bath was chosen for the proteomic analyses. Weprepared and analyzed six independent biological repeats ofsoluble protein sample sets. Spots that showed consistentcold-regulated abundance changes in at least four independentexperiments were chosen for future protein identification. LC-MS/MS identified 25 spots, which represents 18 proteins,

FIG. 2. Expression and subcellular localization patterns ofOsPLD�1. (A to D): GUS staining pattern of OsPLD�1-GUS inpOsPLD�1:OsPLD�1-GUS transgenic rice plants. Images shown are:(A) germinated seeds and (B) 7-day-old seedlings treated with orwithout 0 °C water bath for 5 min. (C) Cross section of stem. Arrowsindicate the position of vascular tissue. Inserted box shows the stain-ing of stem. (D) Stamens and pistils. Inserted box shows spikelet. (E)Subcellular localization of OsPLD�1-GFP protein from root tips of4-day-old pOsPLD�1:OsPLD�1-GFP transgenic rice. Scale bar rep-resents 10 �m.



whose abundance was regulated by 1 h cold treatment fromwild-type seedlings (Fig. S1 and Table II). Compared with TableI, only eight spots, which represents seven proteins (38.9%)including a villin (Os03g24220), an actin 3 (Os03g61970), anactin 7 (Os05g01600), an aminotransferase (Os08g41990), anATP-dependent Clp protease (Os04g32560), a putative sub-tilisin (Os02g53860), and a sucrose synthase (Os03g28330),showed similar up- or down-regulated patterns. The differ-ences in cold-stress-regulated proteomic changes we ob-served might be due to using a different cultivar of rice or thedifference in the treatment duration. Of the 1 h cold-treat-ment-regulated proteins identified, the abundance changes ofnine spots, which represents eight proteins (44.4%) showed

significant differences between wild-type control and pld�1mutant seedlings (Table II and Fig. S2). These results furthersupport that cold regulates the abundance change of a groupof proteins through PLD�1 in rice.

Chilling-Induced Expression of OsDREB1s Is Impaired inOspld�1 Mutant—Cold acclimation has been shown to in-crease plant chilling/freezing tolerance by up-regulating theexpression of CBF/DREB family transcription factors. Thereare 10 CBF/DREB homologs (OsDREB1A to OsDREB1J)in the rice genome. It has been shown that the expressionof OsDREB1A, OsDREB1B, OsDREB1E, OsDREB1F, andOsDREB1G is regulated by cold acclimation (17, 38). To in-vestigate whether the hypersensitive phenotype of the pld�1

FIG. 3. Reducing the expression of OsPLD�1 impaired the chilling tolerance of rice plants. (A) A schematic diagram shows the T-DNAinsertion position in the pld�1 mutant. (B) RT-PCR analysis of OsPLD�1 expression levels in the pld�1 mutant. (C) Western blot analysis ofOsPLD�1 protein level in the pld�1 mutant. (D) 7-day-old rice seedlings were transferred to 4 °C, long-day condition and allowed to continueto grow for the indicated time period. Photographs were taken 7 days after recovery at 28 °C. (E) Quantification of survival rates shown in D.Data represent the mean value from three independent experiments. (F) Quantification of germination rates of rice seeds treated with 16 °C forvarious durations. Inserted box shows the wild-type and pld�1 mutant seeds germinated at 16 °C for 14 days. (G) Rice seedlings germinatedat 28 °C for 4 days were transferred to 12 °C and allowed to continue to grow for 30 days. (H) Quantification of fresh weight and coleoptilelength of seedlings shown in G. (I) Western blot analysis of OsPLD�1 protein level in 2-week-old OsPLD�1 RNAi transgenic rice seedlings. (J)One-week-old OsPLD�1 RNAi seedlings were grown in 4 °C, long-day condition for 6 days. Survival rate was calculated after recovery at 28 °Cfor 7 days. In E and J, one-way ANOVA test was performed. Statistically significant differences are indicated by different lowercase letters (p 0.05). Error bars represent �S.D.



http://www.mcponline.org/cgi/content/full/M115.049759/DC1


mutant in response to chilling/freezing stress is related to theendogenous level of OsDREBs, the chilling-induced expres-sion of OsDREBs was examined in the pld�1 mutant. Quan-titative real-time PCR analysis showed 12 °C chilling stressinduced a dramatic increase of OsDREBs’s expression inwild-type rice seedlings. In comparison, the increase in ex-pression of OsDREBs was greatly reduced in pld�1 mutant(Fig. 6), which suggests OsPLD�1 is required for the chilling-induced increase in OsDREBs expression during the coldacclimation process. We also found that the expression ofOsPLD�1 in wild-type plants increased by chilling treatmentbut remained at a basal level in the pld�1 mutant (Fig. 6). Asthe cold acclimation process is sustained for 6 h, the expres-sion of OsPLD�1 decreases, which could help prevent theoverhydrolysis of PC in the membrane.

PA Interacts with OsMPK6 and OsSIZ1 In Vitro—PLD�1 is aphospholipase that hydrolyzes phospholipids, such as phos-

phatidylcholine to produce the signaling molecule phospha-tidic acid (PA). PA is able to bind to a variety of signalingproteins, regulating their subcellular localization or their activ-ity to transduce signals to downstream components (39). Toinvestigate whether a similar mechanism exists in the ricecold-stress-signaling pathway, recombinant OsMPK6, At-SIZ1, a rice SIZ1 homologous protein (Os05g03430), and arice OST1 homologous protein (Os03g41460) were purifiedusing a GST tag and tested for their ability to interact directlywith PA. A previously demonstrated PA-binding protein, Ara-bidopsis RCN1, was used as a positive control. As shown,RCN1, OsMPK6, AtSIZ1, and OsSIZ1 all bound strongly toPA in a dose-dependent manner but not to other lipids suchas phosphotidylcholine (PC), phosphatidylethanolamine (PE),and phosphatidylglycerol (PG). In contrast, OsOST1 does notbind to any of the lipids we tested, similar to the GST proteincontrol (Fig. 7).

FIG. 4. Freezing tolerance of rice plants is reduced in the pld�1 mutant. (A) Freezing tolerance of 6-week-old rice plants under coldacclimation or nonacclimation conditions. Plants were treated with different freezing temperatures for 3 h. The photographs were taken 7 daysafter recovery at 28 °C. (B) Quantification of the green leaves in plants shown in A. (C) Relative ion leakage quantification of plants treated withvarious freezing temperatures for 3 h. Data represent the mean value from three independent experiments. One-way ANOVA test wasperformed. Statistically significant differences are indicated by different lowercase letters (p 0.05). Error bars represent �S.D.



Expression of OsPLD�1 Is Directly Regulated by OsDREB1A—CBF/DREB family transcription factors are able to bind to aCCGAC core sequence of C-repeat (CRT)/dehydration respon-

sive elements (DRE) and regulates COR gene expression (40).The finding that the expression of OsPLD�1 is up-regulated bychilling treatment motivated us to search the promoter of

FIG. 5. OsPLD�1 overexpressing transgenic rice seedlings are hypersensitive to cold and freezing stress. (A)Western blot analysis ofthe transgenic rice seedlings overexpressing OsPLD�1-GFP fusion protein. OX8 and OX12 are two independent transgenic lines. (B)One-week-old transgenic rice seedlings overexpressing OsPLD�1-GFP were transferred to 4 °C, long-day condition and allowed to continueto grow for 5 days. Survival of the seedlings was examined after recovery at 28 °C for 7 more days. (C) Six-week-old transgenic rice seedlingsoverexpressing OsPLD�1-GFP were cold acclimated and treated with freezing temperature (�6 °C) for 3 h. Seedlings were allowed to recoverat 28 °C for 7 days, and the green leaves per plants were calculated. Shown are average results from three independent experiment with atleast 24 plants per treatment. One-way ANOVA test was performed. Statistically significant differences are indicated by different lowercaseletters (p 0.05). Error bars represent �S.D.

TABLE IIEarly cold-regulated proteins in wild-type and pld�1 mutant seedlings identified by mass spectrometry. The cold-regulated abundance changeswere observed in at least four biological repeats. For MS/MS identification, number of unique peptides, sequence coverage of the identifiedproteins, and the best matched scores from wild type are listed. Refs indicate same protein was identified from previous cold stress proteomic

studies in rice. Proteins with abundance changes that are significantly different between wild type and the pld�1 mutant are underlined

Spot Gene locus Protein name Uniquepeptides

Coverage(%) Score

Foldchanges p value Ref.

WT pld�1

MetabolismU3 Os02g53860 Putative subtilisin 22 8 16.50 80.25 3.23 � 0.31 1.51 � 0.33 1.2E-02U4 Os02g07870 V-type ATPase alpha subunit 12 20.61 130.27 1.91 � 0.29 1.87 � 0.23 1.1E-01 52, 54U5 Os02g07870 V-type ATPase alpha subunit 5 9.50 40.23 1.53 � 0.24 1.44 � 0.11 3.2E-01 52, 54U6 Os02g07870 V-type ATPase alpha subunit 8 22.22 110.28 1.63 � 0.14 1.24 � 0.19 4.2E-02 52, 54U8 Os02g52700 Alpha-amylase precursor 4 9.81 40.21 2.73 � 0.22 1.10 � 0.04 4.3E-02U10 Os08g41990 Aminotransferase 5 17.36 50.23 1.82 � 0.43 1.11 � 0.10 1.1E-01U11 Os03g28330 Sucrose synthase 26 33.44 270.28 1.52 � 0.08 1.35 � 0.07 2.4E-01D5 Os02g46520 Hydrolase 3 9.93 30.19 –1.79 � 0.07 1.07 � 0.10 3.5E-02D7 Os03g28330 Sucrose synthase 16 19.61 150.22 –3.38 � 0.08 –3.03 � 0.17 9.5E-02 52D8 Os03g28330 Sucrose synthase 20 27.08 230.26 –2.19 � 0.08 –2.39 � 0.06 4.7E-01 52D9 Os03g28330 Sucrose synthase 24 31.25 240.23 –2.12 � 0.14 –2.33 � 0.02 3.0E-01 52D10 Os03g28330 Sucrose synthase 28 32.48 260.22 –3.11 � 0.22 –2.83 � 0.02 3.7E-01 52D11 Os01g71830 Glycosyl hydrolases 17 5 25.16 60.28 –1.59 � 0.10 –1.03 � 0.76 5.5E-02

Protein foldingU12 Os03g04970 HSP60 BETA 30 46.27 290.31 1.59 � 0.25 1.40 � 0.31 3.7E-01 51, 52U13 Os04g32560 ATP-dependent Clp protease 16 19.50 170.25 3.74 � 1.62 1.23 � 0.15 5.6E-02 52D1 Os03g60620 DnaK family protein 21 31.90 220.27 –2.00 � 0.11 –1.86 � 0.04 1.8E-01D2 Os03g64210 HSP60 ALPHA 14 26.54 130.28 –1.62 � 0.11 –1.60 � 0.07 4.8E-01 52D4 Os05g01310 Ankyrin repeat family protein 10 32.39 90.26 –2.27 � 0.03 –1.45 � 0.02 5.1E-03

CytoskeletonU7 Os03g24220 Villin protein 22 33.96 180.29 1.63 � 0.21 1.06 � 0.14 1.7E-02U9 Os03g61970 Actin-3 3 9.81 30.21 1.69 � 0.28 1.14 � 0.32 5.5E-02D6 Os05g01600 Actin-7 7 19.10 70.23 –1.57 � 0.18 1.16 � 0.11 8.7E-03

OtherU1 Os07g46370 WD domain family protein 21 18.19 100.21 2.16 � 0.09 1.33 � 0.12 3.1E-02U2 Os07g46370 WD domain family protein 17 19.50 170.27 2.46 � 0.06 1.25 � 0.15 4.4E-04U14 Os04g01540 Uncharacterized protein 5 21.51 50.22 1.53 � 0.69 1.23 � 0.12 6.6E-02D3 Os04g35570 Regulator of chromosome

condensation familyprotein

16 44.37 160.25 –1.95 � 0.12 –1.76 � 0.04 2.2E-01



OsPLD�1 for potential CRT/DRE consensus sequences. Usingan online analysis tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), we found three copies of a typicalCRT/DRE consensus sequence in the 1500-bp upstream ofthe ATG start codon of OsPLD�1 (Fig. 8A). In comparison, theCRT/DRE consensus sequence was not found in promotersequences 1500-bp upstream translation start codon ofPLD�1 in Arabidopsis (AtPLD�1), which explains the relativelystable expression level of AtPLD�1 in Arabidopsis in responseto cold treatment (41). This observation suggests that the roleof PLD�1 in regulating cold acclimation is different in rice andArabidopsis.

To investigate whether the expression of OsPLD�1 can bedirectly regulated by OsDREB1s, RNAi technology was usedto knock down the expression of OsDREB1A in rice plants.Quantitative real time PCR analysis showed that when endog-enous OsDREB1A transcript was knocked down by RNAi,chilling (12 °C)-induced expression of OsPLD�1 is inhibited(Fig. 8B). We also tested whether OsDREB1A activates theexpression of OsPLD�1 using a transient expression assay. A2537-bp promoter sequence upstream ATG of OsPLD�1 wasfused upstream of the firefly luciferase gene. The generatedpOsPLD�1:firefly luciferase reporter construct was cotrans-formed into protoplast generated from 1-week-old rice seed-lings with a p35S:OsDREB1A-HA or p35S:EGFP-HA expressionvector. As shown in Fig. 8C, expression of OsDREB1A-HA ledto a fivefold increase of firefly luciferase activity comparedwith the EGFP-HA control, indicating that OsDREB1A proteinactivates OsPLD�1 promoter in vivo. We also showed usinggel mobility shift assay (EMSA) that recombinant OsDREB1Aprotein purified from E. coli was able to bind specifically witha CRT/DRE containing 30-bp promoter sequence (-618 to-589 bp) of OsPLD�1 (Fig. 8D). Together, these results sug-gest that OsDREB1A activates the expression of OsPLD�1 bybinding directly to OsPLD�1 promoter.

DISCUSSION

The CBF/DREB1-dependent cold signaling pathway hasbeen shown to play an important role in regulating cold ac-climation in both Arabidopsis and rice. Many studies haveinvestigated the regulation of CBF/DREB1 by ICE1 and theirupstream regulators HOS1, SIZ1, and OST1, but the under-standing about the early cold signal perception and signalingmechanisms leading to activation of HOS1, SIZ1, and OST1 is

FIG. 6. The chilling-induced expres-sion of OsDREB1s is inhibited in thepld�1 mutant. Seven-day-old rice seed-lings grown at 28 °C were exposed to12 °C chilling stress for the indicatedtime. The expression of OsDREB1s andOsPLD�1 were quantified using quanti-tative real-time RT-PCR. Data representthe mean value from three independentexperiments. One-way ANOVA test wasperformed. Statistically significant differ-ences are indicated by different lower-case letters (p 0.05). Error bars repre-sent �S.D.

FIG. 7. OsMPK6, OsSIZ1, and AtSIZ1 proteins bind directly toPA in vitro. The phospholipids were dot blotted onto nitrocellulosemembrane. The membrane was blocked with fat-free BSA, probedwith recombinant GST-RCN1, GST-OsMPK6, GST-OsOST1, GST-AtSIZ1, GST-OsSIZ1, or GST proteins, followed by horseradish-per-oxidase labeled anti-GST antibody. Phospholipids used were: PA,phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethano-lamine; PG, phosphatidylglycerol.



http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

http://bioinformatics.psb.ugent.be/webtools/plantcare/html/

still very limited. Using 2D-DIGE, we identified 26 uniqueproteins from rice seedlings, whose abundance is regulatedrapidly by a 5 min chilling treatment. Of these proteins, ge-netic studies showed reducing the expression of OsPLD�1 byRNAi or T-DNA insertion impaired chilling tolerance and coldacclimation increased freezing tolerance of rice plants. Inaddition, chilling-induced expression of OsDREB1s is greatly

reduced in pld�1 mutant, suggesting OsPLD�1 plays an im-portant role in regulating the cold acclimation signaling path-way leading to activation of OsDREB1s in rice.

The activity of PLD inside a cell is regulated by intrinsicand extrinsic signals (42). Once activated, PLD�1 hydro-lyzes phospholipids such as phosphatidylcholine to producea lipid messenger phosphatidic acid (PA), which has been

FIG. 8. OsPLD�1 expression is directly regulated by OsDREB1A. (A) Diagram shows various stress related regulatory elements on 1.5 kbpromoter regions of OsPLD�1 and AtPLD�1. Numbers indicate the starting nuclear acid of the cis-element. (B) Quantitative real-time RT-PCRanalysis of chilling-induced expression of OsDREB1A and OsPLD�1 in wild-type and DREB1A RNAi transgenic plants. One-week-old seedlingswere treated with or without chilling stress (12 °C) for 1 h. (C) Relative luciferase activities in rice protoplasts transformed with a pPLD�1:luciferase reporter vector together with a p35S-OsDREB1A-HA or p35S-EGFP-HA vector. Data shown in B and C represent the mean valuefrom three independent experiments. Asterisks indicate values that differ significantly from control samples (p 0.05). (D) OsDREB1A proteinbinds directly to the CRT/DRE motif from OsPLD�1 promoter. Upper panel shows the sequence of probe and mutant probe (mProbe) for EMSAassay. Core OsDREB1A binding sequences are underlined, and the mutated bases are marked in red. Lower panel shows mobility shift ofrecombinant OsDREB1A-GST protein incubated with biotin-labeled probe in the absence or presence of unlabeled wild-type or mutatedprobes. This experiment has been repeated four times; representative result from one experiment is shown. (E) A model showing a proposedOsPLD�1-mediated cold signaling transduction pathway.



shown to regulate cellular processes by promoting membranefusion, altering protein-membrane interactions and modulat-ing protein activities (39). By lipid overlay assay, we demon-strated that OsMPK6 and OsSIZ1, which are cold-stress-signaling components upstream of OsDREB1s, are able tobind specifically to PA in a dose-dependent manner, thusproviding insight into the mechanism by which OsPLD�1regulates cold signaling pathway in rice. It will be interestingto investigate whether the cellular localization or protein ac-tivity of additional components in the rice cold signaling path-way are also regulated by PA.

There are 12 and 17 PLDs encoded in Arabidopsis and ricegenome, respectively. Based on differences in their biochem-ical and structural properties, these PLDs can be furthergrouped into two subfamilies: C2-PLDs (PLD�, �, �, � and )and PX/PH-PLDs (PLD) (39). Recent discoveries indicate thatPLDs play an important role in regulating growth, develop-ment, and abiotic stress responses in plants (43). There arealso studies showing the involvement of PLDs in plant coldstress responses. For example, the expression or proteinactivities of PLD in Arabidopsis (5), rice (44), cotton (45),Chorispora bungeana (46), and Jatropha curcas (47) havebeen shown to be regulated by cold stress. Suppressing theexpression of AtPLD� in Arabidopsis makes the mutant lesstolerant to freezing, whereas overexpression of AtPLD� in-creases freezing tolerance of the transgenic plants (34). Incontrast, suppressing the expression of AtPLD� in Arabidop-sis makes the plant more resistant to freezing stress (5). Itremains to be determined whether the substrate selection ortissue-specific localization of AtPLD� and AtPLD� is respon-sible for the distinct roles of these two PLDs in regulatingfreezing tolerance in plants. In this study, we found that cel-lular OsPLD�1 protein abundance is increased as early as 1min after chilling treatment, suggesting a role of OsPLD�1 inearly cold-regulated cellular responses. We also found thatchilling treatment increases the expression of OsPLD�1. Sup-pressing the expression of OsDREB1A in rice plants dimin-ished the chilling-induced increase of OsPLD�1 expression.Motif search of the 1500-bp promoter region upstream ATG ofOsPLD�1 identified three potential cold-responsive CRT/DREelements. By EMSA assay, we demonstrated OsDREB1A pro-tein binds directly with the CRT/DRE element (-609 to -601)that is closest to the ATG translation start site. Together theseresults suggest that OsPLD�1 is also a COR gene, and thechilling-induced expression of OsPLD�1 could provide posi-tive feedback regulation during the cold acclimation process.

It is interesting that OsPLD�1 and AtPLD�1 play oppositeroles in regulating cold stress responses in Arabidopsis andrice plants. An alignment indicates that OsPLD�1 andAtPLD�1 are 79% identical at the amino acid level. Aminoacid sequences in domains that are responsible for enzymaticactivity of OsPLD�1 and AtPLD�1 are even more conservative(Fig. S3). Thus, it is unlikely that differences in enzyme activityor substrate selection contribute to this functional discrep-

ancy between OsPLD�1 and AtPLD�1. The difference in re-sponses to cold and freezing stresses of transgenic plantswith knocking down expression level of OsPLDa1 or AtPLDa1suggested that the dynamics of phospholipids in responding tocold treatment is differently regulated in monocots and dicots.Meanwhile, examination of the 1500-bp promoter and the5�UTR sequence upstream of the translation start codon of theOsPLD�1 gene revealed the presence of various stress respon-sive cis-elements, including three copies of cold-responsiveCRT/DRE elements. This suggests OsPLD�1 might play impor-tant roles in regulating responses of rice plant to various envi-ronmental stresses. In contrast, no CRT/DRE elements and onlya few stress-responsive elements were found in AtPLD�1. Thedifference in transcription regulation suggests that tissue-spe-cific expression and/or cellular accumulation levels of OsPLD�1and AtPLD�1 protein are regulated differently, which might alsocontribute to the different roles of OsPLD�1 and AtPLD�1 inregulating cold responses in rice and Arabidopsis.

Given the important role of OsPLD�1 in regulating coldacclimation in rice plants, it is surprising to find that theexpression of OsPLD�1 is not ubiquitously distributedthroughout the whole plant. Histochemical staining of GUSactivity in pOsPLD�1:OsPLD�1-GUS transgenic rice plantsshowed the expression of OsPLD�1-GUS protein was mostlyin root, tip of young leaf, and stem and hulls. Very weak GUSactivity can be detected in young and old leaves grown undernormal conditions. This raises the question, where is the coldsignal sensed and how does locally expressed OsPLD�1regulate cold acclimation for the whole rice plant? Interest-ingly, a high level of GUS staining is detected in the vasculartissue of the stem. Recent studies have discovered the exist-ence of phospholipids, such as PC and PA, and lipid-bindingproteins in phloem exudates of Arabidopsis (48). Although thepresence of long distance transport of lipid signal moleculesstill needs to be tested, the accumulation of OsPLD�1 invascular tissue suggests that it is possible that cold stressincreases the local PA concentration in vascular tissue, whichcould then regulate the cold resistance of surrounding cellsand tissues in rice.

In conclusion, we found that the abundance of OsPLD�1protein is rapidly increased by cold stress treatment and thatOsPLD�1 is important for cold acclimation of rice plants. Ithas been demonstrated that the phospholipase activity ofPLD� is regulated by the intracellular calcium level and thatcold stress induces a rapid increase in the intracellular cal-cium level (49). Based on these observations and our currentresults, we propose a model for an OsPLD�1-mediated coldstress signal transduction pathway in rice (Fig. 7E). In thispathway, environmental chilling stress is sensed by a stillunknown mechanism, which activates the phospholipase activ-ity of OsPLD�1, possibly by increasing the intracellular calciumlevel. Activated OsPLD�1 uses PC as a substrate to producePA. Then PA binds to cold signaling components, such asOsMPK6 and OsSIZ1, altering their cellular localization and/or




protein activity and increasing the expression of OsDREB1s.The increased level of cellular OsDREB1s then can regulate theexpression of OsPLD�1, which could provide a positive feed-back enhancement of cold acclimation processes.

Acknowledgments—We thank Dr. Ying Sun at Hebei Normal Uni-versity for providing OsMPK6 entry vector.

* This work was supported by grant from the Ministry of Agricultureof China (2014ZX08009–003-002), Human Resource Department ofHebei province (E2012100004 to W.T.), the Science and TechnologyDepartment of Hebei Province (15966306D to W.T.), the ScienceFoundation from Hebei University of Economics and Business(2014KYZ07 to C.H.) and Natural Science Foundation of ZhejiangProvince (LR12C02002 to Z.D.).

□S This article contains supplemental material Supplemental Figs.S1-S3.

‡‡ To whom correspondence should be addressed: Tel.: 86 31180787594; E-mail: [email protected].

** These authors contributed equally to this work.

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