Effects of High Temperature on the Wheat Endosperm Proteome · 2006-04-26 · this poster, we...
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Introduction Envi ronment al condit ions dur ing gr ain-fi ll can af fect the dur ati on of storage protei n accumulation and starch deposition, and thus play an important role in determining grain yield and flour quality of wheat. To understand the genetic basis for the effects of environment on the complex program of grain-fill, high throughput transcript and protein profiling techniques must be utilized to identify the many genes and gene products integral to this developmental program. We are currently analyzing salt soluble wheat endosperm protein fractions using two- dimensional (2-D) gel electrophoresis and mass spectrometry to identify the proteins in these fractions and establish accumulation profiles during wheat grain development. One important outcome of these studies will be a better understanding of the timing of the metabolic events that take place during grain development and the effect of the environment on these events. In this poster, we report initial results of studies on the identification of endosperm proteins and their accumulation patterns during in grain development. We also demonstrate the effects of high temperatures initiated during an early and middle stage of grain development on these accumulation patterns. Effects of High Temperature on the Wheat Endosperm Proteome William J. Hurkman, Charlene Tanaka, William H. Vensel, Johanne H. Crawford US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan St. Albany, CA 94710; Nonlinear Dynamics Ltd, Tyne House, 26 Side, Newcastle-upon-Tyne NE1 3JA (JHC) Abstract High temperatures during wheat grain-fill decrease the duration of grain development, storage protein accumulation, and starch deposition. To understand the molecular basis for these changes, we are using a proteomics approach to identify endosperm proteins and determine the effects of high temperature on their accumulation profiles during wheat grain development. Wheat (Triticum aestivum L. cv. Butte 86) pl ants we re grown in a clima te- controlle d greenhouse that had an average daily maximum daytime temperature of 25 o C and nighttime temperature of 17 o C. At 10 and 20 days post anthesis, plants were transferred to a second greenhouse with identical growing conditions except that the average daily maximum daytime temperature was 37 o C and nighttime temperature was 28 o C. Developing grain from plants grown under the three regimens was harvested and endosperm collected. Salt soluble proteins were separated from the storage proteins and separated by 2-D gel electrophoresis. Qualitative and quantitative protein changes during grain development were characterized using computer- based image analysis. Many proteins were present throughout development, while some were only present early or late in development. The most noticeable difference in protein profiles was an increase in a number of proteins with low molecular weights and high pIs during the middle and late stages of development. Protein accumulation profiles reflected the shortened duration of grain development caused by high temperature. Of the 100 proteins identified in 2D gels by matrix-assisted laser desorption io niz ati on mass spe ctr ometr y an d electrospray ionization mass spectrometry, the majority function in protein synthesis, starch metabolism, and defense. Figure 1. Experiment tree of gel set analyzed as illustrated within the Progenesis‘ Workstation. Post-Analysis Procedures Set Experimental Structure Ÿ Select gels Ÿ Set area of interest for each gel Ÿ Create averaged gel groups Ÿ Set averaging criteria Ÿ Set Reference gel for Matching Automatic Analysis Procedures Ÿ Spot Detection Ÿ Background Subtraction Ÿ Warp and Match gel Ÿ Modify Reference gel Ÿ Normalization Ÿ Verify spot detection / matching Ÿ Data investigation Figure 2. A flow diagram illustrating the automatic workflow through the Progenesis‘ Workstation. Methods Protein Fractionation. Endos pe rm f rom de ve loping gr ain of plants grown under t hre e temperature regimens was suspended in cold (4 o C) KCl buffer (50 mM Tris-HCl, 100 mM KCl, 5 mM EDTA, pH 7.8; 200 :l of buffer per 50 mg of endosperm), incubated on ice for 5 min with intermittent mixing (Vortex), and centrifuged for 15 min at 4 o C and 14,000 rpm. The proteins in the supernatant were precipitated by the addition of 5 vol of 0.1 M ammonium acetate in methanol ammonium acetate in methanol and incubation overnight at _20 o C. The pellet rinsed with acetone and solubilized in a buffer containing 9 M urea, 4% NP-40, 1% DTT, and 2% ampholytes. Two-dimensional gel electrophoresis. The isoelectric focusing gels were run using the Mini Protean II tube cell (BioRad Laboratories). Gels were extruded into microcentrifuge tubes and equilibration buffer (2.3% SDS, 10% glycerol, 0.05% DTT, 62.5 mM Tris-Cl pH 6.8) added. Gels were immediately frozen on dry ice and stored at –70 o C. Gels were thawed, placed on top of NuPAGE Novex Bis-Tris 4-12% acrylamide gels with 2D well (Invitrogen), and overlaid with equilibration buffer. The second dimension gels were run using NuPAGE MES SDS running buffer and stained with Coomassie G-250. Image Analysis of 2D Gels. Dig itiz ed 2 D gel i ma ges wer e ana lyz ed u sin g the 2 D g el electrophoresis image analysis software Progenesis‘ Workstation version 2002.01 (Nonlinear Dynamics Limited). Triplicate samples were taken at each time point, resulting in a total of 69 2D gel images. The replicates were grouped together within Progenesis‘ Workstation to form averaged groups, as shown in Figure 1. Spot detection was performed using the fully automatic detection algorithm available within the software. No parameters are required thus ensuring that the detection process is as user independent and objective as possible. Post-analysis, some minor editing was carried out to rectify spot splitting of complex regions. Some editing to optimize matching was also performed. Spot volumes were automatically normalized and expressed as a percentage of the total spot volume for that gel, therefore allowing direct quantitative cross-gel comparisons to be made, regardless of loading and staining irregularities. The automatic analysis workflow is illustrated in Figure 2. All quantitative comparisons de sc rib ed her e wer e b as ed on no rmali zed sp ot vo lu me s. Aver age d ge l da ta wa s u se d t o investigate and identify expression changes across the different development stages. Mass Spectrometry and Spot Identification. A DigestPro xy robot was used to carry out alkylation and in-gel trypsin digestion of protein spots excised from 2-D gels. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF; Reflex II, Bruker Deltonics) mass spectrometry was used to obtain mass fingerprints of the peptide fragments. Mass spectra were obtained using using delayed extraction in the reflectron mode. Porcine trypsin fragments were used as internal calibrants. Electropray ionization mass spectrometry was carried out using a Finnigan LCQ Classic Ion-trap Mass Spectrometer (Finnigan MAT, San Jose, CA) using a New Objective nano-spray interface (New Objective Inc.,Woburn, MA). An LC Packings nano flow LC system (Dionex, Sunnyvale, CA) with autosampler, column switching device, loading pump, nano-flow solvent delivery system, and UV detector was used with the LCQ. The instrument was operated in the “triple play” mode with the LCQ set to acquire a full MS scan between 400 and 1400 m/z followed by a zoom scan of the most intense ion from the preceding scan followed by MS/MS fragmentation. Spectra were transferred to a PC and the batch identification was carried out using a suite of programs from ProteoMetrics. Amylase Inhibitors Alpha-Amylase Inhibitor 0.19 Alpha- Amylase Inhibitor 0.53 Alpha- Amylase/Subtilisin Inhibitor Alpha- Amylase/Trypsin Inhibitor Disease and Defense Ascorbate Peroxidase Catalase Chitinase GSH-dependent Dehydroascorbate reductase Peroxidase Serpin Trypsin Inhibitor (Cmx precursor) Xylanase Inhibitor Protein I Protein Destination 26S Proteosome Regulatory Particle Triple-A ATPase Alpha- Subunit of 20S Proteosome Beta-Subunit of 20S Proteosome Chaperonin 60 ClpC Protease Cyclophilin HSP70 HSP80 NLS Receptor Protein Disulfide Isomerase Metabolism Adenosine Kinase ADP-Glucose Pyrophosphorylase, Large Subunit ADP-Glucose Pyrophosphorylase, Small Subunit Alanine Aminotransferase Aldolase Enolase Formate Dehydrogenase Malate Dehydrogenase Methionine Synthase Nucleoside Diphosphate Kinase Phosphoglucomutase Pyrophosphate Dependent 6-Phosphofructokinase UDP-Glucose Phosphorylase S-adenosylmethionine Synthase Triosephosphate Isomerase Protein Synthesis 40S Ribosomal Protein S12 Elongation Factor 1alpha Elongation Factor 2 Eukaryotic Initiation Factor Transcription and Post-transcription Glycine-rich RNA Binding Protein Poly-A Binding Protein Storage Proteins Avenin Low Molecular Weight Glutenin Subunit Seed Globulin Triticin Cell Structure Actin Alpha-Amylase Alpha-Tubulin Transporters Nonspecific Lipid Transfer Protein Proteins Identified in the Wheat Endosperm Proteome Figure 3. Coomassie-stained, 2D gel of proteins present in endosperm at 36 dpa. Spot circled in red was identified as a peroxidase by mass spectrometry. Figure 4. Peroxidase levels in developing grain grown under moderate or high temperatures. Blue: Moderate temp. from10 to 40 dpa. Green: High temp. from 10 to 26 dpa. Red: High temp. from 20 to 28 dpa. Figure 5. Accumulation profiles of peroxidase during grain development under moderate temperatures and in response to high temperatures. 1-9: moderate temperatures from 10 dpa. 10-18: high temperatures from 10 dpa. 19-23: high temperatures from 20 dpa. Error bars indicate variation between triplicate gels. Accumulation profiles of endosperm proteins during development of grain grown under moderate or high temperatures. Moderate Temp. High Temp. from 10 dpa High Temp. from 20 dpa 1.6 0.8 0 Norm. Vol. Peroxidase 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 10 20 30 40 dpa norm vol Chitinase 0 0.5 1 1.5 2 10 20 30 40 dpa norm vol Serpin 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 10 20 30 40 dpa norm vol ADP-Glucose Phosphorylase 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 10 20 30 40 dpa norm vol Methionoine Synthase 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 10 20 30 40 dpa norm vol Nucleoside Diphosphate Kinase 0 0.5 1 1.5 2 10 20 30 40 dpa norm vol 26S Proteasome Regulatory Particle Triple-A ATPase Subunit 0 0.1 0.2 0.3 0.4 10 20 30 40 dpa norm vol Cyclophilin 0 0.2 0.4 0.6 0.8 1 1.2 10 20 30 40 dpa norm vol Phosphoglucomutase 0 0.05 0.1 0.15 0.2 0.25 0.3 10 20 30 40 dpa norm vol UDP-Glucosephosphorylase 0 0.1 0.2 0.3 0.4 0.5 10 20 30 40 dpa norm vol HSP70 0 0.05 0.1 0.15 0.2 0.25 0.3 10 20 30 40 dpa norm vol Eukaryotic Initiation Factor 0 0.1 0.2 0.3 0.4 10 20 30 40 dpa norm vol Alpha-Amylase/Subtilisin Inhibitor 0 0.5 1 1.5 2 2.5 3 10 20 30 40 dpa norm vol Alpha-Amylase Inhibitor 0.53 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 10 20 30 40 dpa norm vol Glycine-rich RNA Binding Protein 0 0.1 0.2 0.3 0.4 0.5 10 20 30 40 dpa norm vol Conclusions This proteomics approach is powerful for gaining insight into the complex processes of grain developme nt and the effect of the environment on these processes: Over 100 proteins were identified in the endosperm of developing wheat grain. Accumulation profiles of these proteins are being used to understand the timing of metabolic events that take place during grain development. Protein accumulation profiles also are being used to understand the effect of the environment on these events. The Progenesis‘ Workstation greatly simplified gel matching and protein quantification. 1453
Transcript of Effects of High Temperature on the Wheat Endosperm Proteome · 2006-04-26 · this poster, we...
IntroductionEnvi ronmental condit ions dur ing grain-fi ll can af fect the duration of storage proteinaccumulation and starch deposition, and thus play an important role in determining grain yieldand flour quality of wheat. To understand the genetic basis for the effects of environment on thecomplex program of grain-fill, high throughput transcript and protein profiling techniques mustbe utilized to identify the many genes and gene products integral to this developmentalprogram. We are currently analyzing salt soluble wheat endosperm protein fractions using two-dimensional (2-D) gel electrophoresis and mass spectrometry to identify the proteins in thesefractions and establish accumulation profiles during wheat grain development. One importantoutcome of these studies will be a better understanding of the timing of the metabolic eventsthat take place during grain development and the effect of the environment on these events. Inthis poster, we report initial results of studies on the identification of endosperm proteins andtheir accumulation patterns during in grain development. We also demonstrate the effects ofhigh temperatures initiated during an early and middle stage of grain development on theseaccumulation patterns.
Effects of High Temperature on the Wheat Endosperm Proteome
William J. Hurkman, Charlene Tanaka, William H. Vensel, Johanne H. Crawford
US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, 800 Buchanan St. Albany, CA 94710;Nonlinear Dynamics Ltd, Tyne House, 26 Side, Newcastle-upon-Tyne NE1 3JA (JHC)
AbstractHigh temperatures during wheat grain-fill decrease the duration of grain development, storageprotein accumulation, and starch deposition. To understand the molecular basis for thesechanges, we are using a proteomics approach to identify endosperm proteins and determine theeffects of high temperature on their accumulation profiles during wheat grain development.Wheat (Triticum aestivum L. cv. Butte 86) plants were grown in a climate-controlledgreenhouse that had an average daily maximum daytime temperature of 25oC and nighttimetemperature of 17oC. At 10 and 20 days post anthesis, plants were transferred to a secondgreenhouse with identical growing conditions except that the average daily maximum daytimetemperature was 37oC and nighttime temperature was 28oC. Developing grain from plantsgrown under the three regimens was harvested and endosperm collected. Salt soluble proteinswere separated from the storage proteins and separated by 2-D gel electrophoresis. Qualitativeand quantitative protein changes during grain development were characterized using computer-based image analysis. Many proteins were present throughout development, while some wereonly present early or late in development. The most noticeable difference in protein profileswas an increase in a number of proteins with low molecular weights and high pIs during themiddle and late stages of development. Protein accumulation profiles reflected the shortenedduration of grain development caused by high temperature. Of the 100 proteins identified in 2Dgels by matrix-assisted laser desorption ionization mass spectrometry and electrosprayionization mass spectrometry, the majority function in protein synthesis, starch metabolism,and defense. Figure 1. Experiment tree of
gel set analyzed as illustratedwithin the Progenesis‘Workstation.
Post-Analysis Procedures
Set Experimental Structure
Ÿ Select gelsŸ Set area of interest for each gelŸ Create averaged gel groupsŸ Set averaging criteriaŸ Set Reference gel for Matching
Automatic Analysis Procedures
Ÿ Spot DetectionŸ Background SubtractionŸ Warp and Match gelŸ Modify Reference gelŸ Normalization
Ÿ Verify spot detection / matchingŸ Data investigation
Figure 2. A flow diagramillustrating the automaticworkflow through theProgenesis‘ Workstation.
MethodsProtein Fractionation. Endosperm f rom developing grain of plants grown under threetemperature regimens was suspended in cold (4oC) KCl buffer (50 mM Tris-HCl, 100 mMKCl, 5 mM EDTA, pH 7.8; 200 :l of buffer per 50 mg of endosperm), incubated on ice for 5min with intermittent mixing (Vortex), and centrifuged for 15 min at 4oC and 14,000 rpm. Theproteins in the supernatant were precipitated by the addition of 5 vol of 0.1 M ammoniumacetate in methanol ammonium acetate in methanol and incubation overnight at _20oC. Thepellet rinsed with acetone and solubilized in a buffer containing 9 M urea, 4% NP-40, 1% DTT,and 2% ampholytes.Two-dimensional gel electrophoresis. The isoelectric focusing gels were run using the MiniProtean II tube cell (BioRad Laboratories). Gels were extruded into microcentrifuge tubes andequilibration buffer (2.3% SDS, 10% glycerol, 0.05% DTT, 62.5 mM Tris-Cl pH 6.8) added.Gels were immediately frozen on dry ice and stored at –70oC. Gels were thawed, placed on topof NuPAGE Novex Bis-Tris 4-12% acrylamide gels with 2D well (Invitrogen), and overlaidwith equilibration buffer. The second dimension gels were run using NuPAGE MES SDSrunning buffer and stained with Coomassie G-250.Image Analysis of 2D Gels. Digitized 2D gel images were analyzed using the 2D gelelectrophoresis image analysis software Progenesis‘ Workstation version 2002.01 (NonlinearDynamics Limited). Triplicate samples were taken at each time point, resulting in a total of 692D gel images. The replicates were grouped together within Progenesis‘ Workstation to formaveraged groups, as shown in Figure 1. Spot detection was performed using the fully automaticdetection algorithm available within the software. No parameters are required thus ensuring thatthe detection process is as user independent and objective as possible. Post-analysis, someminor editing was carried out to rectify spot splitting of complex regions. Some editing tooptimize matching was also performed. Spot volumes were automatically normalized andexpressed as a percentage of the total spot volume for that gel, therefore allowing directquantitative cross-gel comparisons to be made, regardless of loading and staining irregularities.The automatic analysis workflow is illustrated in Figure 2. All quantitative comparisonsdescribed here were based on normalized spot volumes. Averaged gel data was used toinvestigate and identify expression changes across the different development stages.Mass Spectrometry and Spot Identification. A DigestPro xy robot was used to carry outalkylation and in-gel trypsin digestion of protein spots excised from 2-D gels. Matrix assistedlaser desorption/ionization-time of flight (MALDI-TOF; Reflex II, Bruker Deltonics) massspectrometry was used to obtain mass fingerprints of the peptide fragments. Mass spectra wereobtained using using delayed extraction in the reflectron mode. Porcine trypsin fragments wereused as internal calibrants. Electropray ionization mass spectrometry was carried out using aFinnigan LCQ Classic Ion-trap Mass Spectrometer (Finnigan MAT, San Jose, CA) using a NewObjective nano-spray interface (New Objective Inc.,Woburn, MA). An LC Packings nano flowLC system (Dionex, Sunnyvale, CA) with autosampler, column switching device, loadingpump, nano-flow solvent delivery system, and UV detector was used with the LCQ. Theinstrument was operated in the “triple play” mode with the LCQ set to acquire a full MS scanbetween 400 and 1400 m/z followed by a zoom scan of the most intense ion from the precedingscan followed by MS/MS fragmentation. Spectra were transferred to a PC and the batchidentification was carried out using a suite of programs from ProteoMetrics.
Amylase InhibitorsAlpha-Amylase Inhibitor 0.19Alpha- Amylase Inhibitor 0.53Alpha- Amylase/Subtilisin InhibitorAlpha- Amylase/Trypsin Inhibitor
Disease and DefenseAscorbate PeroxidaseCatalaseChitinaseGSH-dependent DehydroascorbatereductasePeroxidaseSerpinTrypsin Inhibitor (Cmx precursor)Xylanase Inhibitor Protein I
Protein Destination26S Proteosome Regulatory Particle Triple-A ATPaseAlpha- Subunit of 20S ProteosomeBeta-Subunit of 20S ProteosomeChaperonin 60ClpC ProteaseCyclophilinHSP70HSP80NLS ReceptorProtein Disulfide Isomerase
MetabolismAdenosine KinaseADP-Glucose Pyrophosphorylase, Large SubunitADP-Glucose Pyrophosphorylase, Small SubunitAlanine AminotransferaseAldolaseEnolaseFormate DehydrogenaseMalate DehydrogenaseMethionine SynthaseNucleoside Diphosphate KinasePhosphoglucomutasePyrophosphate Dependent 6-PhosphofructokinaseUDP-Glucose PhosphorylaseS-adenosylmethionine SynthaseTriosephosphate Isomerase
Protein Synthesis40S Ribosomal Protein S12Elongation Factor 1alphaElongation Factor 2Eukaryotic Initiation Factor Transcription and Post-transcriptionGlycine-rich RNA Binding ProteinPoly-A Binding Protein
Storage ProteinsAveninLow Molecular Weight Glutenin SubunitSeed GlobulinTriticin
Cell StructureActinAlpha-AmylaseAlpha-Tubulin TransportersNonspecific Lipid Transfer Protein
Proteins Identified in the Wheat Endosperm Proteome
Figure 3. Coomassie-stained, 2D gel ofproteins present in endosperm at 36 dpa. Spotcircled in red was identified as a peroxidaseby mass spectrometry.
Figure 4. Peroxidase levels in developing grain grown under moderate orhigh temperatures. Blue: Moderate temp. from10 to 40 dpa. Green: Hightemp. from 10 to 26 dpa. Red: High temp. from 20 to 28 dpa.
Figure 5. Accumulation profiles ofperoxidase during grain developmentunder moderate temperatures and inresponse to high temperatures. 1-9:moderate temperatures from 10 dpa.10-18: high temperatures from 10 dpa.19-23: high temperatures from 20 dpa.Error bars indicate variation betweentriplicate gels.
Accumulation profiles of endosperm proteins during development of grain grown under moderate or high temperatures.
ModerateTemp.
High Temp.from 10 dpa
High Temp.from 20 dpa
1.6
0.8
0
Nor
m.
Vol
.
Peroxidase
00.20.40.60.8
11.21.41.6
10 20 30 40
dpa
no
rm v
ol
Chitinase
0
0.5
1
1.5
2
10 20 30 40
dpa
no
rm v
ol
Serpin
00.10.20.30.40.50.60.70.8
10 20 30 40
dpa
no
rm v
ol
ADP-Glucose Phosphorylase
00.050.1
0.150.2
0.250.3
0.350.4
10 20 30 40
dpa
no
rm v
ol
Methionoine Synthase
00.020.040.060.080.1
0.120.140.16
10 20 30 40
dpa
no
rm v
ol
Nucleoside Diphosphate Kinase
0
0.5
1
1.5
2
10 20 30 40
dpa
no
rm v
ol
26S Proteasome Regulatory Particle Triple-A ATPase Subunit
0
0.1
0.2
0.3
0.4
10 20 30 40
dpa
no
rm v
ol
Cyclophilin
0
0.2
0.4
0.6
0.8
1
1.2
10 20 30 40
dpa
no
rm v
ol
Phosphoglucomutase
0
0.05
0.1
0.15
0.2
0.25
0.3
10 20 30 40
dpa
no
rm v
ol
UDP-Glucosephosphorylase
0
0.1
0.2
0.3
0.4
0.5
10 20 30 40
dpa
no
rm v
ol
HSP70
0
0.05
0.1
0.15
0.2
0.25
0.3
10 20 30 40
dpa
no
rm v
ol
Eukaryotic Initiation Factor
0
0.1
0.2
0.3
0.4
10 20 30 40
dpa
no
rm v
ol
Alpha-Amylase/Subtilisin Inhibitor
0
0.5
1
1.5
2
2.5
3
10 20 30 40
dpa
no
rm v
ol
Alpha-Amylase Inhibitor 0.53
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
10 20 30 40
dpa
no
rm v
ol
Glycine-rich RNA Binding Protein
0
0.1
0.2
0.3
0.4
0.5
10 20 30 40
dpa
no
rm v
ol
Conclusions
This proteomics approach is powerful forgaining insight into the complex processes ofgrain development and the effect of theenvironment on these processes:
Over 100 proteins were identified in theendosperm of developing wheat grain.
Accumulation profiles of these proteinsare being used to understand the timingof metabolic events that take place
during grain development.
Protein accumulation profiles also arebeing used to understand the effect ofthe environment on these events.
The Progenesis‘ Workstation greatlysimplified gel matching and proteinquantification.
1453
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