Proteomic Analysis of Rice Endosperm Cells in Response to

Expression of hGM-CSF

Junling Luo,† Tingting Ning,† Yunfang Sun,† Jinghua Zhu,‡ Yingguo Zhu,† Qishan Lin,‡ andDaichang Yang*,†

Center of Engineering Research of Plant Biotechnology and Germplasm Utilization, Ministry of Education,Department of Genetics, College of Life Sciences, Wuhan University, 430072, P.R. China, and UAlbany Proteomics

Facility, Center for Functional Genomics, University at Albany, 1 Discovery Drive, Rensselaer, New York 12144

Received April 17, 2008

The accumulation of significant levels of transgenic products in plant cells is required not only for cropimprovement, but also for molecular pharming. However, knowledge about the fate of transgenicproducts and endogenous proteins in grain cells is lacking. Here, we utilized a quantitative massspectrometry-based proteomic approach for comparative analysis of expression profiles of transgenicrice endosperm cells in response to expression of a recombinant pharmaceutical protein, humangranulocyte-macrophage colony stimulation factor (hGM-CSF). This study provided the first availableevidence concerning the fate of exogenous and endogenous proteins in grain cells. Among 1883identified proteins with a false positive rate of 5%, 103 displayed significant changes (p-value < 0.05)between the transgenic and the wild-type endosperm cells. Notably, endogenous storage proteins andmost carbohydrate metabolism-related proteins were down-regulated, while 26S proteasome-relatedproteins and chaperones were up-regulated in the transgenic rice endosperm. Furthermore, it wasobserved that expression of hGM-CSF induced endoplasmic reticulum stress and activated the ubiquitin/26S-proteasome pathway, which led to ubiquitination of this foreign gene product in the transgenicrice endosperm.

Keywords: Quantitative proteomics • Human granulocyte-macrophage colony stimulation factor •Transgenic rice endosperm • Storage protein • Endoplasmic reticulum stress • Ubiquitination

Introduction

Transgenic plants, seeds, and cultured plant cells are po-tentially the most economical systems for large-scale produc-tion of recombinant proteins for industrial and pharmaceuticaluses. Along with the development of biopharming using cerealgrain as a bioreactor, technical concerns regarding the fate ofpharmaceutical recombinant proteins and endogenous pro-teins have generated research interest. In addition to regulatoryconcerns and the need to develop more efficient methods fordownstream processing of recombinant proteins, there are atleast other two important issues: (1) Can we engineer plantcells to manufacture amounts that allow cost-effective produc-tion? (2) Can we engineer plant cells to post-translationallymodify recombinant proteins so that they are structurally andfunctionally similar to the native proteins? At present, only afew recombinant proteins expressed in plant cell are well-investigated, including phytase,1,2 human serum albumin,2

human lysozyme,3 the B subunit of Escherichia coli heat labileenterotoxin,4 human cytomegalovirus glycoprotein B,5,6 andrecombinant antibodies.7-10 These studies provide usefulinformation about the cellular localization and trafficking

mechanism of recombinant proteins, but the fate of endog-enous proteins has rarely been investigated. Batista et al. firstreported a global fate of the transcriptome of transgenic riceexpressing a recombinant antibody for cancer treatments.11

Recently, Van Droogenbroeck et al. reported that overproduc-tion of recombinant scFv-Fc disturbs normal endoplasmicreticulum (ER) retention and protein-sorting mechanisms inthe secretory pathway, with aberrant localization of the ERchaperone calreticulin and binding protein (BiP) and theendogenous seed storage protein cruciferin in the periplasmicspace.12 However, information regarding the effects on theproteome of transgenic cells is lacking.

The development of systems biology offers new opportuni-ties for appreciating the processes of gene transcription, mRNAstability, processing, translation initiation and efficiency, andprotein sorting and trafficking. Whereas microarray technolo-gies provide information about global gene expression withcells, complementary proteomics strategies monitor expressionof proteins and their post-translational modifications. Pro-teomics has been successfully applied to areas as diverse asdetermining the protein composition of organelles, systematicelucidation of protein-protein interactions and the large-scalemapping of protein phosphorylation in response to stimulus.Relative quantification proteomics using higher sensitivity massspectrometry techniques is becoming increasingly popular due

* To whom correspondence should be addressed. Tel: 86-27-6875-4680.Fax: 86-27-6875-4680. E-mail: dyang@whu.edu.cn.

† Wuhan University.‡ University at Albany.

10.1021/pr8002968 CCC: $40.75 XXXX American Chemical Society Journal of Proteome Research XXXX, xxx, 000 APublished on Web 09/09/2008

to its high-throughput, reproducibility13 and sensitivity. Cur-rently, an isobaric tag-based methodology for peptide relativequantification (iTRAQ) coupled with multidimensional liquidchromatography and tandem mass spectrometry enables theassessment of protein levels where 4 samples can be comparedfor their common effects.14

Human Granulocyte-Macrophage Colony Stimulation Factor(hGM-CSF) is a pharmaceutical protein widely used in clinicaltrials of chemotherapy and radiotherapy in various cancers.15

Sardana et al. expressed recombinant hGM-CSF in tobacco andrice seeds, but obtained relatively low expression levels.16,17 Wealso found that the expression level of hGM-CSF was muchlower than that of other proteins expressed in the same plantproduction system in our laboratory (unpublished data). Thisstimulated us to consider using a mass spectrometry-basedproteomic approach for the analysis of transgenic rice en-dosperm in response to the expression of recombinant hGM-CSF.

In the study described below, we report the use of a4-channel relative quantification mass spectrometry techniqueto compare the protein expression profiles of the transgenicand the wild type rice endosperm. We identified differentclasses of proteins that influence the processing machinery ofplant cells. For example, our results showed that the majorendogenous storage proteins, glutelin, globulin and prolamin,and a majority of carbohydrate metabolism-related proteinswere down-regulated, while 26S proteasome-related proteinsand molecular chaperones were up-regulated in the transgenicrice endosperm. Distorted protein body morphology wasobserved in the transgenic endosperm. Interestingly, higherlevels of protein ubiquitination were found in the transgenicendosperm cells, indicating that more proteins would bedegraded via ubiquitin/26S proteasome pathway.

Experimental Methods

Plant Material Preparation. A homozygous transgenic riceline 7-24-3 expressing recombinant hGM-CSF in rice en-dosperm was used for this study.18 The T3 seeds of line 7-24-3and seeds of nontransgenic rice variety TP309 were grownunder natural conditions in the summer season in Wuhan,China. The immature endosperms at 10 days after pollination(DAP) and mature endosperms were harvested, ground withliquid nitrogen, and stored at -80 °C until use.

Transmission Electron Microscopy. For microscopic obser-vation, immature endosperms were harvested at 10-14 DAP.The fixation, slice preparation and immunoelectronic micro-scopic observations were performed as described elsewhere.3

Western Blot Analysis. Antibodies against rice storageprotein glutelin and globulin were gifts from Dr. Ning Huang,Ventria Biosciences (Sacramento, CA). Ubiquitin and hGM-CSFantiserum were purchased from Bethyl Laboratories, Inc.(Montgomery, TA) and BioSource International, Inc. (Camarillo,CA), respectively. About 100 mg of immature endosperms and100 mg of mature endosperms were homogenized using 400µL of protein extraction buffer (66 mM Tris, pH 6.8, 2% SDS, 1mM DTT) followed by centrifugation at 10 000g for 20 min.Protein concentration of the supernatant was determined usinga DC Protein Assay Kit II (Bio-Rad, Hercules, CA). About 10 µgof protein in a 10 µL sample buffer (50 mM Tris, pH 6.8, 100mM dithiothreitol, 2% SDS, 0.1% bromophenol blue, and 10%glycerol) was loaded onto the gel and separated by 12%polyacrylamide gel electrophoresis. Samples were then trans-ferred to nitrocellulose membrane according to the manufac-

turer’s instructions (Bio-Rad, Hercules, CA). All procedures werefollowed as described,3 except for the image development with5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt and ni-tro-blue tetrazolium chloride.

Protein Extraction, Trypsin Digestion, and iTRAQ IsobaricLabeling. Total protein was extracted from 400-500 mg ofmature endosperm cell powder using extraction buffer (66 mMTris-HCl, pH 6.8, 2% SDS, 2% �-mercaptoethanol) at roomtemperature followed by centrifugation for 20 min at 40 000gto remove the insoluble fraction. One milliliter of the resultingsupernatant was mixed with the solutions in a 1:8:1 ratio inthe following order of 1 mL of supernatant, 8 mL of 100% ice-cold acetone, 1 mL of 100% trichloroacetic acid (100% TCA)and 10 µL of �-mercaptoethanol. The precipitation was fol-lowed by centrifugation at 18 000g for 15 min at 4 °C in amicrocentrifuge. The supernatant was discarded and theprotein pellet was washed with 1 mL of ice-cold acetone twice.The protein pellets were resuspended in 400 µL of samplepreparation buffer (100 mM Tris-HCl, 8 M urea, 0.4% SDS, 5mM tributylphosphine, pH 8.3) and placed on ice. Samplemixtures were sonicated with five 20-s pulses (50 W), with 1min intervals on ice, followed by iodoacetamide alkylation atroom temperature for 1 h. Protein concentrations were mea-sured using a MicroBCA protein assay kit (Pierce, Rockford,IL) as per the manufacturer’s instructions before reactions werequenched by the addition of dithiothreitol. The protein mix-tures were then diluted 8-fold in 50 mM Tris-HCl, pH 8.5.Modified trypsin (Sigma, Saint Louis, MO) was added to a finalsubstrate-to-enzyme ratio of 30:1 and the trypsin digests wereincubated overnight at 37 °C. The peptides from each digestsolution were acidified to pH 3.0 with formic acid (FA) andloaded onto a Discovery DSC-18 Cartridge (Sigma, Saint Louis,MO). The peptides were desalted (5 mL of 0.1% FA) and elutedwith 3 mL of a solution composed of 50% acetonitrile with 0.1%FA.

Equal amounts (100 µg) of sample were labeled with 4-plexiTRAQ reagent (Applied Biosystems, Framingham, MA) accord-ing to the manufacturer’s instructions. Briefly, after desaltingon a C18 cartridge, the peptide mixtures were lyophilized andresuspended in 30 µL of 0.5 M triethylammonium bicarbonate(TEAB), pH 8.5. The appropriate iTRAQ reagent (dissolved in70 µL of ethanol) was added, allowed to react for 1 h at roomtemperature, and then quenched with 10 µL of 1 M Tris, pH8.5. Replicates were used in the experiment to assess technicalvariation. The entire experiment (including the generation ofcell pellets) was performed twice. The general workflow of theiTRAQ experiment is shown in Figure 1.

Off-Line Strong Cation Exchange Chromatography. iTRAQlabeled peptides were then concentrated, mixed, and acidifiedto a total volume of 2.0 mL, followed by injection into an Agilent1100 HPLC system with a Zorbax 300-SCX column (4.6 i.d. ×250 mm) (Agilent, Waldbronn, Germany). Solvent A was 5 mMKH2PO4 and 25% acetonitrile (pH 3.0) and solvent B was 350mM KCl in solvent A. Peptides were eluted from the columnwith a 40 min mobile phase gradient of solvent B. A total of 30fractions were collected and samples were dried by a speed-vac prior to LC-MS/MS analysis.

Online Nano-LC ESI QqTOF MS Analysis. A nanobore LCsystem (Dionex, Sunnyvale, CA) which was interfaced to aQSTAR XL QqTOF mass spectrometer with a NanoSpray ionsource (Applied Biosystems, Foster City, CA) was used fortandem mass spectrometry. The Magic C18 100 A pore 75 µmi.d. × 150 mm (Michrom Bioresources, Inc. Auburn, CA) was

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packed in-house. Solvent A was 3% acetonitrile, 0.1% formicacid FA and 0.01% trifluoroacetic acid (TFA) and solvent B was85% acetonitrile, 10% isopropanol, 0.1% FA and 0.01% TFA.Peptide mixtures (reconstituted in 125 µL of 5% FA) wereinjected and eluted from the column with a 90 min mobilephase solvent B gradient (5-11% B in 8 min, 11-13% B in 7min, 13-14% B in 5 min, 14-36% B in 60 min, 36-92% B in0.5 min, and 92% for 5 min) at a flow rate of 250 nL/min. Themass spectrometer was operated in an information dependentacquisition (IDA) mode whereby, following the interrogationof MS data (m/z 350-1500) using a 1 s survey scan, ions wereselected for MS/MS analysis based on their intensity (>20 cpm)and charge state (+2, +3, and +4). A total of 3 product ionscans (2, 3, 3 s each) were set from each survey scan. Rollingcollision energies were chosen automatically based on the m/zand charge-state of the selected precursor ions. The IDAExtensions II script was set to one repetition before dynamicexclusion.

Data Analysis. Data was processed by a “through” searchagainst a TIGR Rice Genome Annotation Release 5 database(66 710 entries) using the Paragon algorithm19 within Protein-Pilot v2.01 software with trypsin as the digest agent, cysteinealkylation, an ID focus of biological modifications and otherdefault settings (Applied Biosystems, Framingham, MA). Thesoftware calculates a percent confidence which reflects theprobability that the hit is a false positive, so that at 95%confidence level, there is a false positive identification rate of5%.19 This software automatically accepts all peptides with aconfidence of identification >1%, so only proteins which hadat least one peptide with >95% confidence were initiallyrecorded. However, the software may also support the presenceof a protein identified using other peptides. Performing thesearch against a decoy (reversed) database allowed estimationof a false discovery level of 4%. Using the ProteinPilot 2.01software, quantification was based upon the signature peakareas (m/z: 114, 115, 116, 117) and corrected according to themanufacturer’s instructions to account for isotopic overlap.Relative quantification ratios of the identified proteins werecalculated, averaged, and corrected for any systematic errorsin the labeling of the iTRAQ peptides. The accuracy of eachprotein ratio is given by a calculated ‘error factor’ in thesoftware, and a p-value to assess whether the protein issignificantly expressed. Error factor expresses the 95% uncer-tainty range (95% confidence error) for a reported ratio, where

this 95% confidence error is the weighted standard deviationof the weighted average of log ratios multiplied by the Student’st-factor for n - 1 degrees of freedom, where n is the numberof peptides contributing to protein relative quantification.P-value is determined by calculating Student’s t-factor bydividing the Weighted Average of Log Ratios-log Bias by theWeighted Standard Deviation, allowing determination of the p-value, with n - 1 degrees of freedom, again where n is thenumber of peptides contributing to protein relative quantifica-tion. To be identified as being significantly differently ex-pressed, a protein had to be quantified in at least three spectra(allowing generation of a p-value), have a p-value < 0.05, andhave a ratio fold change >1.1 or <0.9 in both experimentalreplicates. These fold change limits were selected on the basisof our previous work with iTRAQ reagent.20

Co-Immunoprecipitation and Western Blotting. Matureendosperm powder (200 mg) from the transgenic plants andthe wild-type plants was, respectively, homogenized with 10mL of RIPA buffer (66 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.01%NP40 v/v) supplemented with a protease inhibitor tablet(Sigma, Saint Louis, MO). The resulting mixture was clarifiedby centrifugation at 13 000g for 10 min. Protein concentrationof the supernatant was determined using a DC Protein AssayKit II (Bio-Rad, Hercules, CA). Ten microliters of ubiquitinantibody (Bethyl Laboratories, Inc., Montgomery, TX) wasadded to the supernatant or RIPA buffer (as an empty control).The mixture was rotated at 4 °C for 1 h. Fifty microliters of50% Protein A (Sigma, Saint Louis, MO) was added to thesupernatant or RIPA buffer and rotated again at 4 °C for 1 h.The Co-IP complex was separated by microcentrifugation for30 s at 10 000 rpm. The supernatant was discarded and thepellet was washed five times with 500 µL of PBS buffer in thepresence of 3 µM MG132. Twenty microliters of 2× reducingsample buffer was added to the pellets. Western blotting wasperformed with hGM-CSF antibody (BioSource International,Inc., Camarillo, CA).

Results

Recombinant hGM-CSF Expressed in Rice EndospermShowed a Smear Pattern. The expression level of recombinanthGM-CSF in the transgenic endosperm was 13.67 µg/seed,18

which is much lower than the level of other recombinantproteins using the same expression system in our laboratory(unpublished data). Western blot analysis was done to explorepossible reasons for this. As shown in Figure 2, differentexpression patterns of hGM-CSF were observed in the im-mature endosperm compared to the mature endosperm of thetransgenic plants. In the transgenic immature endosperm,hGM-CSF was present in three different forms. One form withthe smallest size is similar to recombinant hGM-CSF derivedfrom E. coli; the other two forms could be the products of post-translational modification (Figure 2, lane 3). However, weobserved multiple bands with smear pattern ranging fromabout 18 to 120 kDa in the transgenic mature endosperm(Figure 2, lane 1). Interestingly, similar expression patterns werealso observed by others who found that recombinant hGM-CSF ranged in size from 18 to 50 kDa when it was overexpressedin yeast, mammalian and plant cells.16,17,21-23 There are twopotential N-glycosylation sites at Asn27 and Asn37 of hGM-CSF, and the molecular weight of unglycosylated hGM-CSF is15-18 kDa.24-26 Okamoto et al. observed 16-35 kDa hGM-CSF expressed in human lymphoblastoid Namalwa cells. Theyassumed that the different molecular masses of hGM-CSF are

Figure 1. Strategy for analysis of protein expression in thetransgenic and the wild-type endosperm cell lysates by 4-plexisobaric tagging. See Experimental Methods for details. Repli-cates were used to assess technical variation.

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due to full, partial or no glycosylation at two potential glyco-sylation sites.21 Sardana et al. suggested that higher molecularmass versions of the recombinant hGM-CSF could be causedby glycosylation and dimerization in plant cells.16,17 Unfortu-nately, those proposed mechanisms could not explain theextremely high molecular weight of hGM-CSF variants from50 to 120 kDa observed in this study. Possibly, the cause ofthis could be associated with our lower production and smearpattern of hGM-CSF in rice endosperm cells. With this in mind,we conducted proteomic analyses to elucidate the effect ofoverexpression of hGM-CSF in rice endosperm cells.

Proteomic Analysis of the Transgenic Endosperm Cells. Tostudy the effects of overexpressed hGM-CSF on rice endospermdevelopment, we analyzed the protein profile of the transgenicversus the wild-type endosperms at 10 DAP using iTRAQlabeling followed by LC-MS/MS analysis. A total of 1883 uniqueproteins (>95% confidence intervals) were identified in riceendosperm cell at 10 DAP. After two sets of comparative data(115 versus 114 and 117 versus 116) were processed, 117proteins were present with p-value < 0.05 and error factor <2(Supplementary Table 1 in Supporting Information). Amongthem, 7.69% were endogenous storage proteins, 5.13% weremolecular chaperones, 6.84% were translation related; 2.56%were from ubiquitin/26S proteasome-related protein degrada-tion pathways, 16.24% were related to carbohydrate metabolismand 61.54% were related to other miscellaneous pathways(Figure 3A). Overall, 103 proteins exhibited significant differ-ences (ratio >1.1 or <0.9, p-value <0.05) between the wild-type and the transgenic endosperm cells, composed of 46 up-

regulated proteins (39.32%) (ratio >1.1) and 57 down-regulatedproteins (48.72%) (<0.9). The molecular chaperones andproteins related to ubiquitin-proteasome degradation pathwayswere up-regulated, whereas the endogenous storage proteinsand a majority of carbohydrate metabolism related proteinswere down-regulated in the transgenic endosperm cells. Figure3B shows an example of MS/MS spectrum of a peptideAAPSVAVDNLNPK of glutelin A with a ratio of 0.5996 comparedto the wild-type.

Endogenous Storage Proteins Remarkably Reduced inthe Transgenic Endosperm. In rice endosperm, the majorstorage protein glutelin, which is encoded by a multigene family,accounts for 70-80% of the total protein.27,28 All glutelin proteinsidentified were expressed by eight of 12 known glutelinloci (Os02g15178.1, Os10g26060.1, Os02g16830.1, Os02g25640.1,Os03g31360.1, Os01g55690.1, Os02g15150.1, Os02g15070.1). Com-pared with the wild-type endosperm, the amounts of totalglutelin were reduced 30.69%, ranging from 22.22% to 40.01%(Figure 4A). In addition to the glutelin, another storage protein,cupin (Os03g21790.1), was significantly decreased to 80.91%of the content of the wild-type (Figure 4A). The remarkabledown-regulation of glutelin and cupin led us to trace changesin three other storage proteins: globulin, prolamin and albumin.We found that albumin levels were similar to wild-type, butthe levels of globulin (Os03g46100.1, Os05g41970.1, Os03g57960.1,Os03g46100.3)wereslightlydecreasedandprolamin(Os07g10570.1,

Figure 2. Expression of hGM-CSF in endosperm cells. Thesuccessful expression of hGM-CSF in endosperm cells wasconfirmed by Western blot analysis with anti-hGM-CSF antibody(A) and Coomassie Brilliant Blue (CBB) staining gel representsloading control responding to Western blotting (B). Lane 1 andlane 3 represent protein lysates from mature and immatureendosperm cells (10 DAP) of the transgenic plants, respectively.As controls, lane 2 and 4 are protein lysates from the wild-typemature and immature endosperm cells (10 DAP). Lane 5 repre-sents a protein lysate from E. coli cells expressing hGM-CSF. ThehGM-CSF displayed a heterogeneous protein expression patternin immature and mature transgenic rice endosperm. Similarresults were obtained in a second experiment.

Figure 3. Analysis of protein expression in the wild-type andtransgenic endosperm cells by 4-plex iTRAQ reagent. (A) Distri-bution pie of proteins found to change significantly (p-value <0.05) between the transgenic and the wild-type endosperm cellsbased on their functions. (B) An example of the data generatedis shown with the sequence determination by the pattern offragment ions (lower) and the quantification derived from theintensities of the 4 report ions (upper). The fragment ion patternrepresents a peptide AAPSVAVDNLNPK from a glutelin digestionmixture.

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Os07g10580.1) were significantly decreased in two independentsets of experiments even though the reliability of the ratios didnot meet the default significant criteria with the error factor<2 and p-value <0.05 (Figure 4B). To further confirm if theseproteins were down-regulated in the transgenic endosperm,Western blot analysis of the two major storage protein glutelinand globulin was performed. In both the immature and themature endosperm, the contents of glutelin and globulin weredecreased in the transgenic endosperm (Figure 4C), which isconsistent with our proteomic data.

ER Stress Found in the Transgenic Endosperm. Up-regula-tion of molecular chaperones such as heat shock proteins is ingeneral related to the ER stress.29 The iTRAQ analysis showed thatsix molecular chaperones were up-regulated in the transgenicendosperm, including heat shock protein 81-3 (Os09g30439.1),luminal-binding protein 3 precursor (Os02g02410.1), stromal70 kDa heat shock-related protein (Os12g14070.1), heat shockcognate 70 kDa protein 2 (Os11g47760.1), PDI-like protein(Os03g29240.1) and heat shock 70 kDa protein 4 (Os01g08560.2).The contents of those proteins were significantly augmented

from 11.25% to 61.65% in the transgenic endosperm with anaverage of 31.03% compared to the wild-type endosperm(Figure 5A). Up-regulation of the chaperone proteins, especiallythe luminal binding protein,30 might suggest that the ER stresscould occur in the transgenic endosperm. From our previousobservation of morphologically different protein bodies in thetransgenic rice endosperm overexpressing a human lysozyme,3

we postulated that morphological changes of the protein bodiescould probably occur in the transgenic endosperm cellsexpressing hGM-CSF. To test this possibility, we used transmis-sion electron microscopy to observe the morphology of thetransgenic endosperm cells. As expected, the morphology ofthe protein bodies was remarkably distorted. A large numberof nascent protein bodies with a smaller size were observed inthe cordial area of the transgenic endosperm, but not in thewild-type endosperm (Figure 5B). Most of the protein bodieswere surrounded by a clear halo-like structure (Figure 5B-a andB-c, arrow). Some were found to attach to the ER lumen toform structure similar to a multivesicular body (MVB)31 (Figure5B-a and B-c, arrowhead). These structures were not observed

Figure 4. Comparison of expression level of endogenous storage proteins between the transgenic and wild-type rice endosperm. PanelA, relative quantification of glutelin from eight loci and one cupin protein. The relative ratios of the transgenic versus the wild-type areshown on the bar chart. The bars shown represent the mean ( SD. Similar to panel A, panel B shows that the expression of globulinfrom four loci was decreased slightly and prolamin from two loci was decreased significantly. The upper image in panel C, the down-regulation of glutelin and globulin was confirmed by Western blot analysis with anti-glutelin and anti-globulin, respectively. Lanes 1and 3 represent protein extracts from mature and immature endosperm cells (10 DAP) from the transgenic plants, respectively; lanes2 and 4 represent protein extracts from mature and immature endosperm cells (10 DAP) of the wild-type plants. The lower image inpanel C shows Coomassie Brilliant Blue (CBB) staining gel as a loading control for Western blotting. Similar results were obtained ina second experiment.

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in the wild-type endosperm cells (Figure 5 B-b and B-d).Distortion of protein body II was less than that of protein bodyI. These results suggested that serious ER stress takes place inthe transgenic endosperm cells.

More Proteins Were Degraded by Ubiquitin-ProteasomePathway in the Transgenic Endosperm. The ubiquitin/26Sproteasome complex consists of a 19S regulatory particle (RP)for directing ubiquitinated polypeptide into the proteasomelumen and a 20S protease core particle (CP) for breaking downthe polypeptide in the lumen. Recognition and degradation ofubiquitinated substrates by the 26S proteasome is tightlyregulated to maintain normal cell growth.32,33 From iTRAQanalysis, three proteins related to the 26S proteasome pathway,proteasome subunit beta type 2 (Os03g48930.1), proteasome

subunit beta type 4 precursor (Os09g33986.2) and 26S proteaseregulatory subunit 4 (Os07g49150.1), were significantly up-regulated in the transgenic endosperm. Proteasome subunitbeta type 2 and type 4, which belong to subunits of the 20Sprotease core particle, were up-regulated 61.28% and 16.82%,respectively. 26S protease regulatory subunit 4, which belongsto the 19S regulatory particle, was up-regulated 28.38% (Figure6A). The up-regulation of those proteins suggested that moreubiquitin-dependent protein degradation events occur in thetransgenic endosperm. To evaluate this possibility, we per-formed Western blot analysis with a plant specific anti-ubiquitin antibody. No obvious difference was observed at 10DAP between the transgenic and the wild-type immatureendosperm. However, obvious smear patterns were found inthe mature transgenic endosperm but not in the wild-type(Figure 6B). To verify whether the unusual electrophoresispattern of hGM-CSF resulted from ubiquitination, we carriedout a co-immunoprecipitation assay. As shown in Figure 6C,proteins immunoprecipitated with ubiquitin contained hGM-CSF immunoreactivity, indicating hGM-CSF was indeed ubiq-uitinated. The molecular weight of ubiquitinated hGM-CSFranged from about 50 kDa to more than 100 kDa, which fitswith our previous observation. This evidence demonstrates thathGM-CSF was ubiquitinated in the transgenic endosperm,suggesting hGM-CSF could be degraded through the ubiquitin-proteasome pathway during the endosperm development. Thisphenomenon was not found in other transgenic rice en-dosperm expressing other pharmaceutical proteins (unpub-lished data). It does, however, explain why hGM-CSF exhibiteda lower expression level than other recombinant proteinsexpressed in rice endosperm using the same system. This isthe first report to reveal recombinant protein ubiquitinationin the field of pharmaceutical protein production. This findingsuggested to us that preventing the degradation of hGM-CSFthrough the ubiquitin/26S proteasome pathway could be aneffective approach to improve the accumulation of hGM-CSFin the rice endosperm.

Discussion

Overexpression of Recombinant Protein in EndospermCould Promote Unfolded Protein Response (UPR). A cellularunfolded protein response (UPR) will be elicited when defectiveproteins accumulate and aggregate in the ER. UPR inducesseveral mechanism to rescue cells from the ER stress, includingup-regulation of molecular chaperones to facilitate proteinfolding and an increase of the ER-associated degradation(ERAD) activity to remove unfolded proteins.34 In our study,these two mechanisms could clearly be traced. First, manymolecular chaperones were up-regulated in transgenic en-dosperm cells; these are assumed to play a role in facilitatingprotein folding and preventing aggregation. This result wasconsistent with the report of Gasser and his co-worker,35 whodiscovered that several molecular chaperons were up-regulatedin a yeast strain that overexpressed a recombinant proteincompared with a wild-type strain. Similarly, the up-regulationof molecular chaperones was also observed when the ERsuffered stress in transgenic plant cells.36 The second mecha-nism of increased ERAD activity was observed in transgenicendosperm cell. Under normal conditions, basal ERAD activityis sufficient to remove unfolded proteins. However, the UPRis required to increase ERAD activity when the ER is stressed.37

ERAD is an important response to alleviate the ER stress, sothat unwanted proteins that are misfolded, unfolded, or

Figure 5. Comparison of expression level of molecular chaper-ones and the ultrastructure between the transgenic and the wild-type rice endosperm. Panel A shows that the expression ofmolecular chaperones in the transgenic endosperm was up-regulated. The relative ratios are shown on the figure. The barsshown represent the mean ( SD. Panel B shows the comparisonof ultrastructure of the protein bodies from the transgenic andthe wild-type endosperms. Panels B-a (7500×) and B-c (11 000×)indicate the morphology of the protein bodies in the transgenicendosperm cells. A halo-like structure surrounding both nascentprotein bodies is clearly seen (arrow). Multiple nascent proteinbodies remained in the ER lumen (arrowheads). Panel B-b(7500×) and B-d (11 000×) represent the morphology of theprotein bodies in the wild-type endosperm cells. They clearlyshow two types of the protein bodies with regular morphology.PB I and PB II stand for Protein body I and Protein body II,respectively.

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metabolically unnecessary are recognized and returned to thecytosol by a process called retrotranslocation or dislocation,then degraded by the ubiquitin/26S proteasome pathway.38-42

In this process, a series of proteins including molecularchaperones, ubiquitin, components of the 26S proteasome andother cofactors are involved.43,44 The ER luminal chaperones,such as BiP, play important roles in facilitating protein foldingas well as in the recognition of the ERAD substrates byrecognizing the exposed hydrophobic patch of unfolded pro-teins. Polyubiquitin is covalently conjugated with the ERADsubstrates and leads the substrate through the degradationpathway, while the 26S proteasomes breakdown the substrates.Our data showed the molecular chaperones and the compo-nents of 26S proteasome were up-regulated, and proteinubiquitination was enhanced, suggesting overexpression ofhGM-CSF could promote the ERAD response in the transgenicrice endosperm. In conjunction with previous reports aboutproteasome-enriched nuclear bodies called “clastosomes”(doughnut shaped nuclear bodies),45 it is possible that theprotein bodies surrounded by a clear halo-like structure inthe transgenic endosperm cells might be the consequence ofthe protein degradation by the ubiquitin/26S proteasome

pathway. This concept needs to be confirmed in the future. Inaddition, we hypothesized that the down-regulation of storageproteins (glutelin and globulin) was induced by the ubiquitin/26S-proteasome degradation pathway, but further study indi-cated that there was no ubiquitination of those proteins (datanot shown), suggesting other mechanisms could be involved.Taken together, the observation that ubiquitinated proteinsaccumulate in the mature endosperms rather than immatureendosperms suggests that the ubiquitin/26S proteasome-mediated ERAD pathway could be promoted at the later stagesof endosperm development. Ning et al. reported that hGM-CSF was biologically active and soluble,18 suggesting that atleast some hGM-CSF in endosperm could be properly folded.But the high level of polyubiquitination of hGM-CSF goingthrough the ERAD pathway suggests that there was a great dealof unfolded or misfolded hGM-CSF accumulating in the ER.Mattanovich et al. have reviewed that different kinds of stress,including not only the ER stress, but also osmotic stress andpH stress, would be induced when overexpressing a recombi-nant protein in yeast.46 This report suggested that the ERADand other changes in the transgenic endosperm cells are dueto the cascades of overexpression of the foreign protein hGM-

Figure 6. Comparison of protein expression of 26S proteasome and ubiquitination between the transgenic and the wild-type riceendosperm, and evidence of ubiquitination of hGM-CSF. Panel A shows the up-regulation of 26S proteasome proteins in the transgenicrice endosperm. The relative ratios are shown on the figure. The bars shown represent the mean ( SD. The upper image in panel Bis a Western blot analysis with anti-ubiquitin displaying a high level of ubiquitination in mature endosperm cells. The lower image inpanel B shows Coomassie Brilliant Blue (CBB) staining gel as a loading control corresponding to Western blotting. Lanes 1 and 3represent protein extracts from mature and immature endosperm cells (10 DAP) of the transgenic plants, respectively; lanes 2 and 4represent protein extracts from the wild-type mature and immature endosperm cells (10 DAP). Panel C shows ubiquitination of hGM-CSF. The cell lysates were subjected to immunoprecipitation (IP) with anti-ubiquitin and the IPs were analyzed by Western blotting forhGM-CSF and ubiquitinated hGM-CSF. The input sample is 5% of total protein used in each immunoprecipitation. The result showsthat hGM-CSF was ubiquitinated and its size was shifted to a higher molecular weight range.

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CSF. However, there is still a lack of direct evidence todetermine whether the unfolded or misfolded hGM-CSF wasthe original signal that triggered the ERAD pathway in thetransgenic endosperm cells.

A Crowded and Disordered Environment of ER AffectsNormal Protein Trafficking in Rice Endosperm. Goossens etal. and Tada et al. reported that repression of endogenousstorage protein genes could improve exogenous gene expres-sion in endosperm cells.47,48 In our study, the protein levels ofendogenous storage proteins in the transgenic endosperm cellare remarkably decreased. Taken together, this suggests thatthe competition within the ER subdomains or translationalmachinery could occur between recombinant proteins andendogenous storage proteins. Overexpressing an exogenousprotein in endosperm cells could cause a crowded environmenton the ER surface and in the ER lumen that could activateintracellular signal transduction pathway and initiate signalintegration in the ER for the unfolded protein response.49

Because storage protein mRNA is translated on the ER throughspecifically bound subdomains on the ER surface,50,51 themRNAs of recombinant proteins and endogenous proteinscould deplete the subdomains on the ER surface and thetranslational machineries of endogenous proteins. Further-more, this crowded environment on the ER surface or in theER lumen could promote and enhance the ER stress thatinduces up-regulation of a number of the molecular chaper-ones, disturbs the normal protein trafficking route, and sub-sequently affects the biogenesis of the protein bodies. Hunteret al. analyzed the transcriptomic profiles of several maizeopaque endosperm mutations with altered protein bodies,discovering that the increased expression of genes associatedwith physiological stress and unfolded protein response arecommon features of protein body distortion and opaquemutants.52 Published data3 and our unpublished data on theprotein body distortion in the transgenic endosperm cellindicate that normal vacuolar biogenesis is largely affected.However, the mechanisms affecting native protein traffickingroutes and vacuolar biogenesis remain to be elucidated in thefuture.

Acknowledgment. We would like to thank Dr. NingHuang for kindly providing the antibodies against the ricestorage proteins, glutelin and globulin. This work is supportedby the Project of the National “863” high technology Programin China (No. 2007AA100505); the Project of the NationalNatural Science Foundation of China (No. 30671286) and theKey grant Project of the Chinese Ministry of Education (No.307018).

Supporting Information Available: SupplementaryTable 1 contains proteins identified in the endosperm cells byiTRAQ analysis. This material is available free of charge via theInternet at http://pubs.acs.org.

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