Download Proteome of amyloplasts isolated from

Journal of Experimental Botany, Vol. 57, No. 7, pp. 1591–1602, 2006
Plant Proteomics Special Issue
doi:10.1093/jxb/erj156 Advance Access publication 4 April, 2006
Proteome of amyloplasts isolated from developing wheat
endosperm presents evidence of broad metabolic
capability*
Yves Balmer1,2, William H. Vensel2, Frances M. DuPont2, Bob B. Buchanan1,† and William J. Hurkman2
1
Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA
2
Received 10 April 2005; Accepted 7 February 2006
Abstract
Introduction
By contrast to chloroplasts, our knowledge of
amyloplasts—organelles that synthesize and store
starch in heterotrophic plant tissues—is in a formative
stage. While our understanding of what is considered
their primary function, i.e. the biosynthesis and degradation of starch, has increased dramatically in recent
years, relatively little is known about other biochemical
processes taking place in these organelles. To help fill
this gap, a proteomic analysis of amyloplasts isolated
from the starchy endosperm of wheat seeds (10 d postanthesis) has been conducted. The study has led to
the identification of 289 proteins that function in a
range of processes, including carbohydrate metabolism, cytoskeleton/plastid division, energetics, nitrogen
and sulphur metabolism, nucleic acid-related reactions, synthesis of various building blocks, proteinrelated reactions, transport, signalling, stress, and
a variety of other activities grouped under ‘miscellaneous’. The function of 12% of the proteins was unknown. The results highlight the role of the amyloplast
as a starch-storing organelle that fulfills a spectrum of
biosynthetic needs of the parent tissue. When compared with a recent proteomic analysis of whole endosperm, the current study demonstrates the advantage
of using isolated organelles in proteomic studies.
Although known for many years, our understanding of
amyloplasts, plant organelles functional in the synthesis
and storage of starch in heterotrophic plant tissues, remains
in its infancy. Aside from pathways leading to the synthesis
and breakdown of starch, relatively little is known about
the biochemistry of this organelle (Neuhaus and Emes,
2000). In their recent proteomic identification of 171
proteins in amyloplasts isolated from wheat starchy
endosperm, Andon et al. (2002) provided a foundation
for understanding processes taking place in the plastid.
According to their work, however, the biochemical activities of amyloplasts seem relatively restricted; 85% of the
proteins fell under the protein destination/storage, energy
metabolism, and unknown categories. Thinking that, by
analogy with chloroplasts (Kleffmann et al., 2004; van
Wijk, 2004) and their etioplast relatives (von Zychlinski
et al., 2005), amyloplasts should have broad metabolic
capability, the question of the nature of their resident
proteins was reopened. Using amyloplasts, also isolated
from developing wheat endosperm, 289 proteins were
identified. Of these, less than one-third fall in the protein
destination/storage, energy metabolism, and unknown
categories and more than half function in metabolism and
response to stress. Particularly prominent are enzymes of
amino acid, nucleic acid, and sulphur metabolism. In
demonstrating the versatility of amyloplasts, the results
add to our understanding of plastid function, and, by
building on findings obtained with whole endosperm
(Vensel et al., 2005), show the advantage of using isolated
organelles for proteomic analysis.
Key words: Amyloplast proteins, amyloplast proteome, dithiothreitol, endosperm, Global Proteome Machine, GPM, isolated
amyloplasts, membranes, wheat.
* Disclaimer: The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department
of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
y
To whom correspondence should be addressed. E-mail: [email protected]
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: [email protected]
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
US Department of Agriculture, Agricultural Research Service, Western Regional Research Center, Albany,
CA 94710, USA
1592 Balmer et al.
Materials and methods
Materials
Wheat (Triticum aestivum L. cv. Butte) was grown in a climatecontrolled greenhouse under a 16 h light (supplemented with 100 W
sodium lamps)/8 h dark, day/night regimen that had a maximum
daytime temperature of 24 8C and a minimum night-time temperature
of 17 8C (Altenbach et al., 2003). Water and fertilizer (Plantex 20-2020, 500 ml of 0.6 g lÿ1 potÿ1 dÿ1) were applied by drip irrigation.
Heads were harvested 10 d post-anthesis (dpa) and used immediately
for amyloplast preparation.
Isolation of amyloplast proteins
The pellet containing intact amyloplasts was suspended in a small
volume of plasmolysis buffer without sorbitol and supplemented
with a protease inhibitor cocktail (Complete Mini; Roche, Basel,
Switzerland). The suspension was frozen in liquid nitrogen and
thawed three times to break the intact organelles. The lysate was
centrifuged (10 000 g, 20 min, 4 8C) to collect the soluble proteins
(supernatant fraction). The pellet, containing starch granules and
other insoluble materials, was extracted with the same buffer plus
2% Triton X-100 and centrifuged to collect the membrane proteins (supernatant fraction). The supernatant solution of this second
extraction yielded a fraction enriched in membrane proteins. Four
volumes of cold acetone were added to each fraction, and following
incubation overnight (–20 8C), precipitated proteins were recovered
by centrifugation (Wong et al., 2004).
Two-dimensional electrophoresis (2-DE)
Isoelectric focusing and SDS/PAGE were performed using the
systems of Invitrogen Corp. (Carlsbad, CA, USA) and Bio-Rad
Laboratories (Hercules, CA, USA), according to the manufacturers’
instructions. Proteins were solubilized in a solution containing 7 M
urea, 2 M thiourea, 0.5% ampholytes, 2% b-dodecyl maltoside, and
10 mM dithiothreitol (Luche et al., 2003). Isoelectric focusing was
Fig. 1. Amyloplast isolation procedure.
carried out using an IPG strip with a non-linear pH range of 3–10
(Invitrogen Corp. or Bio-Rad Laboratories, depending on the 2-D
system used). The second dimension was developed with a NuPage
4–12% BIS-TRIS Zoom gel (Invitrogen Corp.) or a Criterion Precast
gel (Bio-Rad Laboratories). Gels were stained with Coomassie
brilliant blue G-250 (Kasarda et al., 1998).
Protein spot excision and digestion
Gels were scanned (Powerlook III, Umax) and the spots detected with
the Progenesis software package (Nonlinear Dynamics Ltd,
Newcastle upon Tyne, UK). Gels were transferred to a ProPic gel
spot picker (Genomic Solutions, Ann Arbor, MI, USA) that excised
and placed spots into 96-well reaction plates for subsequent in-gel
tryptic digestion with an automated protein digester (DigestPro,
Intavis, Langenfeld, Germany). The DigestPro was programmed to
destain the gel piece and carry out reduction with dithiothreitol,
alkylation with iodoacetamide, enzymatic digestion with trypsin,
and elution of the generated peptides into a 96 well plate that was
subsequently inserted into the autosampler of the mass spectrometer.
LC/MS/MS of tryptic peptides of proteins
A QSTAR PULSAR i quadrupole time-of-flight (TOF) mass
spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada)
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Amyloplast preparation
Intact amyloplasts were isolated using the procedure of Tetlow et al.
(1993) as summarized in Fig. 1. Twenty heads were harvested and
used within a 2 h period for each preparation. Embryos were cut from
the grain, and endosperm was squeezed through the opening created
and collected in ice-cold buffer (0.5 M sorbitol, 50 mM HEPES pH
7.5). The endosperm was transferred to plasmolysis buffer (0.8 M
sorbitol, 50 mM HEPES pH 7.5, 1 mM EDTA, 1 mM KCl, 2 mM
MgCl2) and incubated for 1 h on ice. Plasmolysed endosperm was
chopped twice for 30 s with an electric knife, the blades of which
were replaced with holders fitted with single-edge razor blades. The
resulting homogenate was filtered through two layers of Miracloth
and gently pipetted onto a 4 ml cushion of 2% Nycodenz (Nycomed,
Oslo, Norway) in plasmolysis buffer in a 15 ml conical tube
containing a 2 ml 1% agar pad at the bottom. Following centrifugation (30 g, 10 min, 4 8C) (Centrifuge 5810 R, Eppendorf, Westbury,
NY, USA), the supernatant fraction was removed by aspiration and
discarded. The pellet containing the amyloplasts was gently
suspended in plasmolysis buffer and the Nycodenz procedure
repeated once more. The purity of the amyloplast preparation was
assessed previously by the group who devised the protocol (Tetlow
et al., 1998). They found minimal contamination of amyloplast
preparations by other cell components. Based on an assay of marker
enzymes, mitochondria (<0.2%) were the major source of contamination. This conclusion was confirmed in the present study as small
amounts of cytochrome oxidase (Table 1), one of the four classical
mitochondrial marker proteins (Quail, 1979) were found, but the
other three (fumarase, succinate, and cytochrome c reductase) were
not detected.
Wheat amyloplast proteome
Table 1. Function of proteins identified in amyloplasts isolated
from 10 dpa wheat endosperm
Function
Sz
Q8LI30
M
S
B
Q6UZD6
Q8H277
P12299
ÿ282,7
ÿ3,9
ÿ228,6
B
P55238
ÿ113
M
M
B
B
B
Q9SYU0
Q8L5G8
Q8VX48
Q9FUU7
Q8H1Y9
ÿ169
ÿ9,9
ÿ117,7
ÿ141,9
ÿ136,3
B
Q6ZKB1
ÿ43,9
B
B
S
S
Q9C9C4
ÿ76,9
Q94JJ0
ÿ86
Q6YXI1 ÿ165,5
Q8VXD9 ÿ25,7
B
S
S
B
Q94CN9 ÿ162,6
P12782
ÿ130,9
Q7XYD2 ÿ13
Q7XTJ3 ÿ240,9
B
O65087
ÿ44,9
B
B
Q8SA22
P46225
ÿ9,6
ÿ127,5
B
O81237
ÿ32,8
S
B
S
B
O24357
Q8S1Y0
Q6ZEZ1
Q9FPB6
ÿ3,3
ÿ73,1
ÿ7,2
ÿ222,4
S
S
B
Q6YZX6
Q9FI53
Q6K9N6
ÿ75,7
ÿ28,2
ÿ87,5
S
Q6ZL94
ÿ7,8
B
S
Q94JA2
Q9LDH7
ÿ58,9
ÿ202
ÿ9,6
Cytoskeleton/division
*ACTIN 42
FtsZ protein
Kinetochore protein, putative
(SKP1/ASK1-like protein)
Plastid division protein [Arc6]
*Tubulin alpha chain
B
S
M
P93587
Q9SDW5
Q9M3X1
ÿ15,6
ÿ11,9
ÿ35,5
B
S
Q7PC78
Q9ZRB7
ÿ7,4
ÿ7,6
Energetics
ATP/PP synthesis/transformation
Adenylate kinase A
ATP synthase 24 kDa subunit, putative
y
ATP synthase alpha chain
ATP synthase beta subunit
ATPase
F0 ATP synthase, D chain, putative
S
M
B
M
B
M
Q08479
Q6IY71
P12112
Q41534
Q9FS11
Q7XXS0
ÿ43,9
ÿ167,9
ÿ129,2
ÿ275,9
ÿ128,9
ÿ123,4
Table 1. Continued
Function
Fraction SwissProt log(e)
no.
y
B
S
Q6ZGJ8
Q762A0
S
M
M
M
M
S
S
S
P14619
ÿ6,3
Q943W1 ÿ149,9
Q9SDM1 ÿ36,7
Q6Y0E5
ÿ7
Q94DH6
ÿ74,4
P27788
ÿ8,7
Q41736
ÿ104,1
P80680
ÿ10,4
B
M
M
Q9FLX7
Q00434
P36213
ÿ80
ÿ142,8
ÿ7
M
Q8W0B2
ÿ6
B
Q6ZDY8
ÿ40,5
B
B
O04973
Q6Z702
ÿ188,9
ÿ62,1
B
Q6URQ0 ÿ111,2
B
B
Q7Y096
Q93VK6
B
S
S
S
B
S
S
B
Q84U07 ÿ264,6
Q949B4
ÿ20,5
Q688Q8
ÿ26,6
P52894
ÿ4,4
Q6VMN8 ÿ62,7
Q7XUS2
ÿ4,9
Q9LEU8
ÿ74,6
Q9SZX3
ÿ68,9
S
Q69LG7
ÿ3,8
B
Q93Y73
ÿ113,2
S
S
Q8H8D3
Q8H7T7
ÿ20
ÿ11,7
S
S
S
Q9LWJ5
Q67UZ0
O65917
ÿ6,8
ÿ40
ÿ19,3
B
S
B
Q69JT8
Q6ZG77
Q9LFG2
ÿ9,2
ÿ59,1
ÿ47,4
B
S
Q67W29
P24846
ÿ56,1
ÿ22,6
B
S
Q6YZH8
Q08258
ÿ152,8
ÿ15,8
S
S
Q9LGY8
Q9SZ30
ÿ126,9
ÿ28,4
B
S
Q8RZF3
Q6AV34
ÿ276,8
ÿ47,3
S
Q6YVI0
ÿ22,8
Inorganic pyrophosphatase
*Nucleoside diphosphate kinase
Electron transport
Apocytochrome f precursor
33 kDa oxygen-evolving protein
Chlorophyll a/b-binding protein
Chlorophyll a/b-binding protein
Cytochrome B5, putative
Ferredoxin III
y
Ferredoxin-NADP reductase
Ferredoxin-thioredoxin reductase,
variable subunit
y
NADH-ubiquinone oxidoreductase 18
Oxygen-evolving enhancer protein 2
Photosystem I reaction centre
subunit II
Putative cytochrome P-450LXXIA1
(Cyp71A1) family
Succinate dehydrogenase flavoprotein
alpha subunit
Nitrogen/sulphur metabolism
Amino acid
*2-Isopropylmalate synthase A
3-Isopropylmalate dehydratase,
large subunit
3-Isopropylmalate dehydratase,
small subunit
3-Isopropylmalate dehydrogenase
3-Phosphoshikimate
1-carboxyvinyltransferase
*Acetohydroxyacid synthase
Acetylglutamate kinase-like protein
Acetylornithine aminotransferase
*Alanine aminotransferase 2
Aminotransferase AGD2, putative
Anthranilate synthase, beta subunit
Argininosuccinate lyase
*,yArgininosuccinate synthase,
chloroplast
Aspartate kinase-homoserine
dehydrogenase
Aspartate-semialdehyde
dehydrogenase, putative
ATP phosphoribosyl transferase
Branched-chain amino acid
aminotransferase
Cystathionine beta-lyase, putative
Cysteine conjugate beta-lyase
Dehydroquinate dehydratase/
shikimate:NADP oxidoreductase
Dehydroquinate synthase, putative
Diaminopimelate decarboxylase
Diaminopimelate epimerase-like
protein
Dihydrodipicolinate reductase-like
Dihydrodipicolinate synthase 1,
chloroplast
Dihydroxy-acid dehydratase
Ferredoxin-dependent glutamate
synthase (FD-GOGAT)
Histidinol dehydrogenase
Imidazole glycerol phosphate
synthase hisHF
*Ketol-acid reductoisomerase
N-acetyl-gamma-glutamyl-phosphate
reductase
Ornithine carbamoyltransferase
ÿ27,9
ÿ56
ÿ99,5
ÿ65,2
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Carbohydrate metabolism
Starch
4-Alpha-glucanotransferase
(amylomaltase)
Alpha 1,4-glucan phosphorylase
Fructokinase
*,yGlucose-1-phosphate
adenylyltransferase, large subunit
*Glucose-1-phosphate
adenylyltransferase, small subunit
Granule-bound starch synthase
Hexokinase
*Phosphoglucomutase
y
Starch branching enzyme 2
y
Starch synthase II
Glycolysis
Dihydrolipoamide
acetyltransferase, putative
*,yEnolase
*Fructose-bisphosphate aldolase
Glucose-6-phosphate isomerase
*,yGlyceraldehyde-3-phosphate
dehydrogenase
y
Lipoamide dehydrogenase
*Phosphoglycerate kinase
*Phosphoglycerate mutase
Pyruvate dehydrogenase E1,
alpha subunit
Pyruvate dehydrogenase E1,
beta subunit
Pyruvate kinase, putative
*Triosephosphate isomerase
Pentose phosphate cycle
*6-Phosphogluconate dehydrogenase,
putative
Glucose-6-phosphate 1-dehydrogenase
Putative transaldolase
Ribose-5-phosphate isomerase
Transketolase
Citric acid cycle
*Aconitate hydratase, putative
Fumarate hydratase 2
Succinyl-CoA ligase (GDP-forming),
beta-chain
Succinyl-CoA ligase, alpha subunit,
putative
Malate valve
*,yMalate dehydrogenase
Malic enzyme (NADP-dependent),
chloroplast
Fraction SwissProt log(e)
no.
1593
1594 Balmer et al.
Table 1. Continued
Table 1. Continued
Fraction SwissProt log(e)
no.
Phosphoglycerate dehydrogenase,
putative
Phosphoribosylanthranilate isomerase 1
Phosphoribosyl-ATP
pyrophosphohydrolase, putative
Phosphoribosylformimino-5aminoimidazole carboxamide
ribotide isomerase
y
Threonine synthase
Tryptophan synthase, alpha chain
Tryptophan synthase, beta subunit
Sulphate assimilation
ATP sulphurylase
Cysteine synthase 1
Phosphoadenylyl-sulphate reductase
Thiosulphate sulphurtransferase
Nucleic acid-related
DNA/RNA
29 kDa ribonucleoprotein A
y
40S ribosomal protein S13
40S ribosomal protein S19
40S ribosomal protein S2, putative
*40S ribosomal protein S20
y
40S ribosomal protein S5
40S ribosomal protein SA (p40)
*60S acidic ribosomal protein P0
*60S ribosomal protein L12, putative
Cp31BHv (31 kDa ribonucleoprotein,
chloroplast)
DNA-binding protein
*Elongation factor 1-alpha
*Elongation factor 1-beta
*Glycine-rich RNA-binding
protein 2, putative
Histone H2A.1
Histone deacetylase 2 isoform b
Nucleolin, putative
Nucleosome/chromatin assembly factor
Putative fibrillarin
Ribosomal protein L7Ae-like
Ribosome recycling factor
Translational elongation factor Tu
Translational inhibitor protein,
putative
Nucleotide biosynthesis
59-Phosphoribosyl-5-aminoimidazole
synthetase
Adenylosuccinate synthetase,
chloroplast
Aminoimidazolecarboximide
ribonucleotide transformylase
Aspartate carbamoyltransferase
Carbamoyl phosphate synthase,
small subunit
*Carbamoyl phosphate synthetase,
large subunit
Dihydroorotate dehydrogenase,
putative
y
Serine hydroxymethyltransferase
Succinoaminoimidazolecarboximide
ribonucleotide synthetase
Building blocks, other
Isoprenoid
2-C-methyl-D-erythritol 4-phosphate
cytidylyltransferase
GCPE protein (hydroxymethylbutenyl
4-diphosphate) synthase)
Geranylgeranyl diphosphate synthase
B
Q7XMP6 ÿ106,5
S
S
Q6ETX4
Q9SDG1
ÿ3,5
ÿ23
S
O82782
ÿ66,9
S
S
S
Q6L492
Q6ZL61
Q8W0T4
ÿ39,8
ÿ78,5
ÿ43,5
S
M
S
S
Q84MN8 ÿ116,5
Q84SE4
ÿ8,6
Q6Z4A7
ÿ21
Q9ZPK0
ÿ17,4
B
M
M
M
M
M
M
M
M
S
Q8LHN4
Q05761
P40978
Q84M35
P35686
O24111
Q8H3I3
O24573
Q6Z8E0
O81988
ÿ75
ÿ12,4
ÿ35,5
ÿ13,8
ÿ17,6
ÿ9,3
ÿ9,5
ÿ11,6
ÿ56,6
ÿ9,5
S
B
S
B
Q7XZF8
Q03033
P29546
Q7F2X8
ÿ5,8
ÿ29,9
ÿ13,4
ÿ11,4
S
M
M
S
M
M
S
S
B
P02275
ÿ9
Q9M4U5 ÿ173
Q68Q07
ÿ11,1
Q8L8G4
ÿ5,9
Q6K701
ÿ19,7
Q8LBE4
ÿ45,7
Q6YTV7
ÿ26,1
Q8W2C3 ÿ33,2
Q8H4B9 ÿ102,7
B
Q850Z8
ÿ105,1
B
O24396
ÿ181
S
Q6ZKK5
ÿ35,8
S
S
Q9S983
Q8L6J8
ÿ24,1
ÿ19,1
S
Q8S1A5
ÿ42,1
S
Q8S3J6
ÿ9,3
B
B
O23254
ÿ108,7
Q6YXG8 ÿ27,8
S
Q9RR90
ÿ7
S
Q6K8J4
ÿ10,1
B
Q6ET88
ÿ64,6
Function
Fraction SwissProt log(e)
no.
Isopentenyl pyrophosphate isomerase
Tetrapyrrole
Coproporphyrinogen III oxidase
Delta-aminolevulinic acid dehydratase
Ferrochelatase II
Glutamate-1-semialdehyde
2,1-aminomutase
Haem oxygenase 1
Porphobilinogen deaminase
Uroporphyrinogen decarboxylase
Vitamin/cofactor
*10-Formyltetrahydrofolate synthetase
Delta-24-sterol methyltransferase
Gamma-tocopherol methyltransferase
y
Phytoene dehydrogenase, chloroplast
Riboflavin synthase, alpha chain,
putative
Thiamine biosynthesis protein ThiC
Tocopherol cyclase, chloroplast
Lipid metabolism
3-Oxoacyl-[acyl-carrier-protein]
reductase, putative
y
3-Oxoacyl-[acyl-carrier-protein]
synthase I
Acetyl-coenzyme A carboxylase
Acyl-[acyl-carrier protein] thioesterase
Beta-hydroxyacyl-ACP dehydratase
Enoyl-ACP reductase, putative
Malonyl-CoA:Acyl carrier protein
transacylase
*Stearoyl-acyl-carrier protein desaturase
S
Q71RX2
ÿ37,2
B
S
M
S
Q42840
Q42836
P42045
P18492
ÿ57
ÿ53,6
ÿ67
ÿ85,1
S
S
S
Q94FW9
Q8RYB1
Q9AXB0
ÿ9
ÿ17,8
ÿ4,2
B
M
B
S
M
Q9SPK5
Q41587
Q6ZIK0
Q9ZTN9
Q9SKU8
ÿ145,9
ÿ113,3
ÿ171,9
ÿ6
ÿ6,3
B
Q9AXS1
Q6K7V6
ÿ184,9
ÿ16,4
M
Q7XMI8
ÿ113,4
S
Q69YA2
ÿ31,2
S
S
B
B
B
O48959
Q8L6B1
Q6I5L0
Q6H5J0
Q8RU07
ÿ19
ÿ54,5
ÿ56,6
ÿ92,1
ÿ52
B
Q8S059
ÿ58,6
B
S
S
B
S
S
S
Q69Y99
Q6K635
Q7X7E8
Q6XPZ6
P36183
P22954
Q43638
ÿ182,8
ÿ85,6
ÿ20,7
ÿ108,7
ÿ117
ÿ67
ÿ167,3
B
S
M
S
Q40058
ÿ187,9
Q75GT3
ÿ66,5
Q70YJ6
ÿ72,6
Q69WA8 ÿ25,5
B
B
Q9FE55
Q7X9A7
ÿ289,7
ÿ268,9
B
Q43831
ÿ484,9
S
S
S
B
Q9FER4
Q851D9
Q6H852
P31542
ÿ15,6
ÿ27,9
ÿ71,7
ÿ287,8
S
Q93X33
ÿ12,5
S
M
S
B
M
Q03106
O80983
Q6K669
Q6K9T1
P42210
ÿ12,2
ÿ8,4
ÿ71,6
ÿ95,1
ÿ321,5
S
Q69Y12
ÿ3,5
S
Q40164
ÿ5,3
Protein related
Assembly/folding
20 kDa chaperonin, chloroplast
Co-chaperone CGE1 isoform b, putative
*Cyclophilin
Cyclophilin-like protein
Endoplasmin homolog
Heat shock cognate 70 kDa protein 2
Heat shock cognate 90 kDa protein,
putative
*Heat shock protein 70 kDa
Heat shock protein ClpB, putative
Immunophilin FKBP type
y
Peptidyl-prolyl cis-trans
isomerase-like protein
*,yProtein disulphide isomerase
Rubisco subunit-binding-protein,
alpha subunit
*Rubisco subunit-binding-protein,
beta subunit
Turnover
*20S proteasome alpha subunit
*20S proteasome, beta 4 subunit
Alpha 2 subunit of 20S proteasome
ATP-dependent clp protease,
ATP-binding subunit clpA
Beta 3 proteasome subunit (20S),
putative
Cathepsin B-like cysteine proteinase
FtsH protease, putative
*Leucine aminopeptidase
Oligopeptidase A-like
y
Phytepsin precursor (Aspartic
proteinase)
Putative aminopeptidase C
(Acyl-peptide hydrolase-like)
Ubiquitin
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Function
Wheat amyloplast proteome
Table 1. Continued
1595
Table 1. Continued
Fraction SwissProt log(e)
no.
Zinc metalloprotease, putative
Storage
*,yAlpha/beta-gliadin precursor
(Prolamin)
*Alpha-amylase inhibitor 0.19
Alpha-amylase/trypsin inhibitor
CM16
Alpha-amylase/trypsin inhibitor
CM2 precursor
*,yAlpha-amylase/trypsin
inhibitor CM3
Chymotrypsin inhibitor WCI
y
Gamma 3 hordein
Gamma-gliadin
Puroindoline-B
Purothionin A-I
Storage protein
Transport
Membrane
ABC transporter ATPase, putative
ABC transporter, putative
y
ADP-glucose transporter, plastidial
Amino acid selective channel protein
Chloroplast inner envelope
protein, 110 kDa
Coated vesicle membrane
protein-like
Import receptor subunit TOM40
homologue, probable
Import-associated channel
protein homologue
Inner membrane protein
(translocase), putative
Outer mitochondrial membrane
protein porin
Pore protein of 24 kDa (OEP24)
y
Porin-like protein
Processing peptidase alpha-chain
Processing peptidase, beta subunit
Translocase Toc34-1
Translocon-associated protein,
alpha subunit
Translocon Tic40-like protein
TOC75, protein translocase
Vesicle transport-related protein,
putative
Ion
Calnexin homologue
y
Calreticulin
Ferritin
Signalling
Hormone
Ent-kaurene oxidase 1
Steroid membrane-binding protein
Phosphorylation
Serine/threonine protein kinase,
putative
GTP-linked
GTP-binding protein (Ras-related
protein RIC2)
GTP-binding protein Rab6
*Guanine nucleotide-binding protein,
beta subunit
RAB1-like
RAN GTPase activating protein 2,
hypothetical
Ras-related protein Rab11B
S
Q8RUN6
M
P02863
ÿ49,6
B
B
P01085
P16159
ÿ59,1
ÿ55,1
B
P16851
ÿ50,2
B
P17314
ÿ111,2
M
M
M
M
M
B
P83207
Q6EEY6
Q94G97
Q10464
P01543
Q38794
ÿ14,6
ÿ124,9
ÿ49,6
ÿ105,9
ÿ15,6
ÿ22,3
ÿ8,2
S
S
B
M
B
Q9CAF5
Q941V2
Q6E5A5
O82688
O24293
ÿ100,5
ÿ3,8
ÿ318,2
ÿ25,7
ÿ18,3
M
Q6YZD2
ÿ47,4
M
Q9LHE5
ÿ38,5
B
Q84Q83
ÿ82
M
Q9FWD5
ÿ9
M
P46274
M
M
M
M
M
M
Q75IQ4
ÿ99,6
Q84P97
ÿ88,8
Q9FNU9
ÿ48,1
Q9AXQ2 ÿ36,3
Q9SBX0 ÿ105,9
P45434
ÿ22,1
B
B
M
Q7XQG5
Q9STE8
Q851W1
M
B
S
Q7XV86
ÿ80,4
Q40041
ÿ218,2
Q6DQK1 ÿ91,3
M
M
Q673G1
Q9FVZ9
ÿ12,4
ÿ80,4
M
Q69ML2
ÿ31,9
M
Q9ATK6
ÿ52,7
M
B
Q8H4Q9
P49027
ÿ15,3
ÿ148
M
M
Q68V18
Q9XEN1
ÿ41,8
ÿ54,2
B
Q40521
ÿ16,1
ÿ43,1
ÿ22,7
ÿ34,7
ÿ4,9
Function
Fraction SwissProt log(e)
no.
Ras-related protein Rab7
Ras-related protein RIC1
Ras-related protein RGP2
Ras-related protein YPT3
Other
*14-3-3-like protein B
TGB12K interacting protein 3,
possible
Prohibitin, putative
Stress related
Thiol-linked
*2-Cys peroxiredoxin BAS1
Gamma-glutamylcysteine synthetase
Glutaredoxin protein family-like
Lactoylglutathione lyase
Thioredoxin peroxidase (Type 2)
Ascorbate-linked
*Ascorbate peroxidase
Monodehydroascorbate reductase
S
M
S
M
Q40787
P40392
Q40723
Q01111
ÿ14
ÿ41,3
ÿ7,4
ÿ7,6
S
M
Q43470
Q40785
ÿ5,7
ÿ25,6
M
Q6AVQ4
ÿ52,5
S
B
B
S
S
O81480
Q8GU95
Q84Z96
O49818
Q7F8S5
ÿ143,2
ÿ78,4
ÿ49,7
ÿ98,1
ÿ11,5
S
S
Q7XJ02
Q84PW3
ÿ21,1
ÿ5,7
S
S
S
P55313
O24400
Q6H660
ÿ8,5
ÿ33,4
ÿ3,6
S
Q8LQS5
ÿ18,5
M
S
M
S
Q84PD0
Q6UA21
P40621
Q6Z4I1
ÿ10,6
ÿ26,3
ÿ9,8
ÿ54,2
M
M
B
B
B
Q8W3I2
Q9FTY4
Q6Z4N6
Q9ZR33
O20243
ÿ47,1
ÿ12,2
ÿ236,4
ÿ69,4
ÿ20,4
B
Q6ZL16
ÿ16,2
M
S
S
S
M
S
M
S
M
B
S
M
S
S
S
S
B
M
S
Q7EZD2
Q500U9
Q8LAE4
Q8LB85
Q8LDD3
Q93ZV1
Q8H124
O80564
Q8L722
Q93VT6
Q6NM26
Q8H0Y1
Q9FGS4
Q9CAC8
Q9M1J1
Q94F00
Q7XQG8
Q6L5C9
Q69SQ6
ÿ78,5
ÿ4,8
ÿ30,7
ÿ51,2
ÿ7,7
ÿ73,7
ÿ5,7
ÿ24,3
ÿ43,8
ÿ36,7
ÿ5,5
ÿ17,4
ÿ3,8
ÿ10,7
ÿ40,4
ÿ12,9
ÿ44,1
ÿ5,9
ÿ4,3
M
Q8W3H8
ÿ7
M
Q5QMU3
ÿ6,7
M
Q8H7W5
ÿ12,1
Other
*Catalase
*,yCu/Zn superoxide dismutase
*Stress-induced protein sti1, putative
Miscellaneous
Carboxymethylenebutenolidase,
putative
DNAJ-like protein
Fibre protein Fb4
HMG1/2-like protein
N-amidino-scyllo-inosamine-4phosphate phosphatase putative
Plastid-lipid associated protein
y
r40c1 protein
y
R40g2 protein
*Reversibly glycosylated polypeptide
y
Ribulose 1,5-bisphosphate
carboxylase, large subunit
Root border cell-specific protein,
putative
Unknown
Band 7 protein, putative
Expressed protein At1g03030
Hypothetical protein
Hypothetical protein
Hypothetical protein
Hypothetical protein At1g26160
Hypothetical protein At2g34460
Hypothetical protein At2g43940
Hypothetical protein At3g55760
Hypothetical protein At5g08540
Hypothetical protein At5g26990
Hypothetical protein At5g42960
Hypothetical protein At5g50210
Hypothetical protein F24D7.19
Hypothetical protein F24I3.170
Hypothetical protein F28K20.15
Hypothetical protein OJ000114_01.9
Hypothetical protein OJ1007_H05.1
Hypothetical protein
OSJNBa0016O19.25
Hypothetical protein
OSJNBa0027L23.4
Hypothetical protein
OSJNBa0054L14.15
Hypothetical protein
OSJNBa0064E16.12
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Function
1596 Balmer et al.
Table 1. Continued
Function
Fraction SwissProt log(e)
no.
Hypothetical protein
OSJNBa0071K18.7
Hypothetical protein P0450E05.20
Hypothetical protein P0534A03.117
OJ000315_02.14 protein
OJ000315_02.15 protein
OSJNBa0038O10.22 protein
OSJNBa0068L06.10 protein
OSJNBa0095E20.4 protein
OSJNBb0026L04.9 protein
OSJNBb0116K07.9 protein
S
Q8H916
ÿ25,5
S
M
M
M
S
M
M
M
S
Q69IM6
Q84ZC8
Q7XL03
Q7XL02
Q7XKI6
Q7XTB3
Q7XRN1
Q7XTG7
Q7F8Y3
ÿ16,8
ÿ12,5
ÿ10,9
ÿ33,8
ÿ43,4
ÿ3,8
ÿ63,6
ÿ6,6
ÿ15,4
equipped with a Proxeon Biosystems (Odense, Denmark) nanoelectrospray source was used to perform ESI-MS of the tryptic
peptides. HPLC peptide separation was carried out using a nano-flow
liquid chromatograph (LC Packings/Dionex, Sunnyvale, CA, USA).
The 96 well plate from the DigestPro was covered with a self-sealing
silicone compression mat (catalogue no. CM-96-EXP; Axygen
Scientific, Union City, CA, USA) and cooled to 10 8C to prevent
sample evaporation. Aliquots (15 ll) of sample from the DigestPro 96
well receiving tray were loaded into a loop using an autosampler
(FAMOS, LC Packings/Dionex) and pumped at a flow rate of 20 ll
minÿ1 onto a C18, 5 lm, 300 A, Nano-Precolumn (300 lm i.d. 3
1 mm, P/N 160459, LC Packings/Dionex) with a loading pump
(SWITCHOS, LC Packings/Dionex). The loading pump eluent was
0.5% acetic acid, 0.02% heptafluorobutyric acid in HPLC-grade
water. After 2 min, the loading valve was switched to place the trap
in-line with the LC pump (Ultimate, LC Packings/DIONEX).
Samples were eluted from the trap onto the C-18 monomeric,
Vydac EVEREST column (catalogue number 238EV5.07515;
W.R. Grace & Co.) at a rate of 200–250 nl minÿ1. The spray tip was
an 8 lm i.d. PicoTip emitter (catalogue no. FS360-75-8-CE-20, New
Objective, Woburn, MA, USA). Spray voltage was maintained at
1800 V. A co-axial counter-current flow of ultra-high purity nitrogen
(curtain gas) was used to shield the orifice of the mass spectrometer
and contributed to charged droplet desolvation. Mobile phase A was
0.5% glacial acetic acid (Fisher Scientific, reagent grade) diluted in
HPLC-grade water (Burdick and Jackson, Muskegon, MI, USA).
Analytical column elution solvents were labelled A (0.5% acetic
acid) and B (80% acetonitrile, 0.5% acetic acid). Samples were
eluted with the following gradient profile: 8% B at 0 min to 80%
B by 12 min through 13 min to 8% B by 14 min continuing at 8% B
to 28 min.
The TOF mass analyser of the instrument was calibrated using the
187.0713 and 1245.5444 m/z fragment ions from the CID of the +2
charge state (m/z 785.8) of glufibrogen. Data were acquired using
the IDA acquisition mode of Analyst QS software; from an initial
survey scan of mass range m/z 400–1500, the most abundant double or
triple charged ion above a threshold of 20 counts was selected for
fragmentation. The quadrupole mass filter (Q1) was adjusted so
that only ions of the selected m/z entered the collision cell of Q2. CID
of the mass-selected ion in the collision cell of Q2 was carried out
using ultra-high purity nitrogen as the collision gas. Analysis of the
fragment ions by the TOF mass analyser was set to range of 70–2000
m/z. The precursor ion was precluded from further MS/MS experiments. An IDA script was used to determine the optimum collision
energy for each precursor ion. Following the 3 s MS/MS fragmen-
Database analysis and identification of proteins
The WIFF acquisition files created by Analyst QS were converted to
DTA files using a WIFF-to-DTA converter (Genomic Solutions). The
software used to analyse the DTA files was obtained from the Global
Proteome Machine (GPM) organization (http://www.thegpm.org/). A
local installation of the GPM open-source software was used for
visualization and analysis of the data. The spectrum modeler X!
TANDEM (Fenyo and Beavis, 2003; Craig and Beavis, 2004) that is
a part of the GPM software was used to match MS/MS fragmentation
data to peptide sequences. The DTA files from each fraction were
joined together into a single file and submitted to the locally installed
copy of X! Tandem (version 2005.06.01.2) using the JAVA program
XtandemAppend (authored by Jayson Falkner and posted to the
LiveCD Project at http://www.thegpm.org/). The scripts were
modified to process DTA files and recompiled on Windows XP.
The resulting single DTA file for each fraction was searched against
a flat file containing amino acid sequences of all plant proteins in the
HarvEST:Wheat version 1.04 (http://harvest.ucr.edu/), NCBI nonredundant green plant database, NCBI Triticum aestivum: UniGene
Build No. 37, and wEST Database (http://wheat.pw.usda.gov/wEST)
(Lazo et al., 2004). The translation of the nucleotide sequences into
amino acid sequences in all six reading frames was carried out using
the Knexus suite of software (Genomics Solutions).
Results and discussion
Identification of proteins
Amyloplasts were resolved into soluble and membrane
fractions, proteins separated by 2-DE, and gel spots
analysed by LC/MS/MS. Three gels, including one overloaded to visualize proteins of low abundance, were
developed for the soluble and insoluble protein fractions.
In this way, >600 protein spots were analysed and 85 218
usable spectra obtained. The mass spectra fragment ion data
were searched against two sets of databases—NCBI nonredundant and a wheat EST database combining HarvEST,
Unigene, and wEST. The search against the NCBI database
assigned 7037 spectra yielding 1001 identifications with an
expectation value of <10ÿ3; the search against the EST
database assigned 9162 spectra for 1053 accepted identifications. These 2054 identifications were reduced to 289
unique proteins after exclusion of redundant and closely
related homologues. The peptide sequences identified for
each of the 289 proteins listed in Table 1 are included in the
supplementary Table 1 (see www.jxb.oxfordjournals.org).
Surprisingly, out of the 171 proteins identified in amyloplasts by Andon et al. (2002), only 31 were identified in the
present study. The limited overlap between these two
similar preparations could stem from several factors. First,
the endosperm for the preparations in the present study
was collected at 10 dpa and that for Andon et al. (2002) at
12 dpa. As the endosperm is a rapidly developing tissue,
collection time, as well as growth regimen, could account
for part of the difference observed. Secondly, the isolation
procedures were slightly different. In the present study, the
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
* Previously identified in wheat endosperm extract (Vensel et al., 2005).
Previously identified in amyloplasts (Andon et al., 2002).
z
S = amyloplast soluble fraction; M = amyloplast membrane-bound
fraction; B = both fractions.
y
tation period, the MS survey scan was repeated until another MS/MS
period was triggered.
Wheat amyloplast proteome
procedure followed was that described for wheat amyloplasts (Tetlow et al., 1993), whereas Andon et al. (2002)
used a protocol developed for amyloplasts from maize
(Neuhaus et al., 1993). Finally, since 2002, the wheat
sequence database has been greatly increased, therefore
enhancing the efficiency of protein identification by MS
[see http://harvest.ucr.edu/ and Lazo et al. (2004) for more
information].
Purity of preparation
Soluble versus membrane-enriched fractions
The isolated amyloplasts were subfractionated by centrifugation into soluble and insoluble fractions. The insoluble
fraction was extracted with buffer containing the non-ionic
detergent, Triton X-100, yielding a fraction enriched in
membrane proteins. Analysis of the data showed that, of the
289 proteins identified, 87 were present in both fractions,
119 were unique to the soluble fraction, and 83 to the
membrane-enriched fraction (Table 1). Fractionation of the
amyloplast proteins into soluble and membrane-enriched
fractions allowed the identification of low-abundance
proteins that were not identified previously in the endosperm. The majority of these were soluble proteins that
function in carbohydrate metabolism, nitrogen/sulphur
metabolism, nucleotide biosynthesis, and protein assembly/
folding and turnover. There were also many integral or
membrane-bound proteins unique to the membrane-enriched
fraction, including proteins that function in energetics
(ATPase subunits), electron transport (oxygen-evolving
enhancer), transport (porins), and signalling (GTP-binding
proteins). However, the membrane-enriched fraction also
contained a number of proteins that are not known to be
associated with membranes. As would be expected,
granule-bound starch synthase was recovered exclusively
in the membrane-enriched fraction because of the insolubility of the starch granules. Storage proteins were also
found in this fraction owing to limited solubility in the
extraction buffer. Other non-membrane proteins may be
present in this fraction as they bind non-specifically to the
starch granules during amyloplast breakage (see above).
Origin of the amyloplast proteins
To estimate the relative proportion of proteins of plastid and
nuclear origin, the 289 proteins identified in the present
study were compared with the list of known plastidencoded proteins in plants published on the HAMAP
proteome website (http://www.expasy.org/sprot/hamap/
plastid.html). Seventy-six genes are described in the wheat
plastid genome, whereas rice plastids encode 94 polypeptides. Most of these proteins are participants in the
photosynthetic electron chain and the biogenesis/assembly
of its components (40 in wheat) or in plastid protein
translation (22 in wheat). As amyloplasts are organelles
without photosynthetic activity, it is not surprising that few
plastid-encoded proteins were identified. Indeed, only three
such proteins were detected in the amyloplast preparation
(ATP synthase alpha and beta chains and Rubisco large
subunit). It is of interest that no subunits of the prokaryotictype ribosome were identified, suggesting that in organello
protein synthesis is limited in amyloplasts. This observation
agrees with early evidence showing that only a small
number of plastid-encoded genes are required in nongreen plastids (Harris et al., 1994).
Function of proteins
Table 1 lists the 289 unique proteins categorized according to function, SwissProt accession number, and log of
the expectation value. The proteins are grouped in 12
major categories: carbohydrate metabolism, cytoskeleton/
division, energetics, nitrogen/sulphur metabolism, nucleic
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Identified proteins were determined to be of plastid origin
on the basis either of their designation in the SwissProt
database or as a result of a search on PubMed. On this basis,
about 10% of the proteins discussed below appear not to be
of plastid origin—the most obvious being the storage
proteins (e.g. gliadins and alpha-amylase/trypsin inhibitors)
housed in protein bodies that may co-purify with amyloplasts. In this connection, a subsequent extraction of the
insoluble fraction of the amyloplast preparation with the
detergent Triton X-100 yielded a membrane-enriched
fraction (M fraction in Table 1) which contained a high
level of non-plastidial proteins such as storage proteins.
Further, certain proteins in the preparations in the present
study, such as those associated with ribosomes, possibly
adhered to starch granules released from broken amyloplasts that co-purify with intact amyloplasts. Indeed, there
is evidence that starch granules bind endosperm proteins in
a non-specific manner when the tissue is homogenized (F
DuPont, W Hurkman, D Kasarda, unpublished observations). The presence of members of the citric acid cycle
raises the question of mitochondrial contamination. While
possible for certain entries, one of these, fumarate hydratase
2, is annotated as plastid targeted in the SwissProt database.
Several proteins functional in the synthesis and transformation of ATP, for example, ATP synthase subunits,
and electron transport were also identified. Whereas certain
of these could be mitochondrial contaminants, most are
typical of chloroplasts. Accordingly, low levels of these
proteins may be present in amyloplasts owing to a lack of
strict control over their expression such as seems to occur
for a number of photosynthetic proteins (e.g. 33 kDa
oxygen-evolving enhancer protein, chlorophyll a/b-binding
protein, and Rubisco large subunit) (Table 1). In short,
while certain of the proteins in question are likely to
be contaminants, the relevance of others to amyloplasts
remains unclear.
1597
1598 Balmer et al.
Carbohydrate metabolism
Among all the biochemical reactions of the amyloplast,
the biosynthesis of starch is generally considered the major
activity, since the newly formed starch granule will
ultimately occupy the bulk of the organelle. Accordingly
(Table 1), most of the enzymes of starch biosynthesis
(Smith, 2001; James et al., 2003) were identified in the
preparation described here. However, somewhat surprisingly, counterparts catalysing the degradation or rearrangement of starch, such as a-1,4-glucan phosphorylase
(Smith et al., 2005) were also found along with those
associated with glycolysis, the oxidative pentose phosphate
pathway, and malate valve. The presence of these components suggests that a significant part of the carbon imported
by amyloplasts is diverted to the production of reducing
power and ATP (Neuhaus and Emes, 2000) to support
myriad biosynthetic reactions taking place within the
organelle (more below). The overall distribution of enzymes of carbohydrate metabolism is in accord with the
conclusion that, at this early stage of development (10 dpa),
the plastid actively catalyses processes in addition to starch
synthesis (Vensel et al., 2005).
Cytoskeleton/division
Five of the proteins identified were assigned a role in
determining plastid ultrastructure and division. Of these,
two (actin 42 and tubulin) are problematic and, while
assumed to be of plastid origin, could originate from the
cytosol (for a discussion of this point, see Kleffmann et al.,
2004). On the other hand, two of plastid origin, Arc6 and
FtsZ, provide evidence that endosperm amyloplasts are
actively dividing at 10 dpa (Marrison et al., 1999; Vitha
et al., 2001).
Energetics
A number of proteins found were involved in electron
transport and in the synthesis and transformation of ATP.
The function of many of these is unclear, i.e. components
associated with photosynthetic electron transport of chloroplasts such as the 33 kDa oxygen-evolving enhancer
and chlorophyll a/b-binding proteins. By contrast, the
role of others is obvious, for example, the ferredoxin
and ferredoxin/NADP reductase isoforms specific to nonphotosynthetic plastids catalyse the reverse transfer of
reducing equivalents from NADPH to ferredoxin in order
to support processes that in chloroplasts are driven by light
(Onda et al., 2000). The finding of ferredoxin-thioredoxin
reductase suggests the presence of the ferredoxin/thioredoxin
system described for chloroplasts. However, the inability
to detect thioredoxin in wheat amyloplast preparations in
the present study shows the need for further work.
Nitrogen/sulphur metabolism
Fig. 2. Comparison of the functional distribution of proteins identified
in amyloplast and endosperm extracts.
The plastid is the site of nitrogen and sulphur assimilation
as well as most of the carbon skeletons of the amino acids
(Neuhaus and Emes, 2000; Hofgen et al., 2001). The
current finding of a large number of proteins (39) functional
in the synthesis of amino acids is consistent with these
activities and with the large quantity of these building
blocks required for the synthesis of enzymes and storage
proteins. At 10 dpa the endosperm is entering an extended
phase of starch accumulation and storage protein biosynthesis (Altenbach et al., 2003). It appears that the
plastids play an essential role in both processes.
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
acid-related, building blocks (other than specified), proteinrelated, transport, signalling, stress-related, miscellaneous,
and unknown. In certain cases, subcategories were devised
for clarity: carbohydrate metabolism (starch, glycolysis,
pentose phosphate pathway, citric acid cycle, malate valve),
energetics (ATP/PP synthesis/transformation, electron
transport), nitrogen/sulphur metabolism (amino acid,
sulphate assimilation), nucleic acid-related (DNA/RNA,
nucleotide biosynthesis), building blocks (isoprenoid,
tetrapyrrole, vitamin/cofactor, lipid metabolism), proteinrelated (assembly/folding, turnover, storage), transport
(membrane, ion), signalling (hormone, phosphorylation,
GTP-linked, other), stress-related (thiol-linked, ascorbatelinked, other).
A graphical view of the functional distribution of the
amyloplast proteins from 10 dpa wheat endosperm highlights four biochemical processes—carbohydrate and nitrogen/sulphur metabolism, nucleic acid- and protein-related
reactions—that together comprised ;50% of the identifications (Fig. 2). Twelve per cent of the proteins have no
known function, and, although another 4% have defined
activity, they do not fit into one of the present categories
and are thus placed in the miscellaneous group. In the
following section, certain processes are highlighted, especially with respect to relevance to developing cereal
endosperm.
Wheat amyloplast proteome
Nucleic acid-related proteins
Other building blocks
The metabolic versatility of amyloplasts is reflected in the
large number of enzymes active in the biosynthesis of
building blocks not discussed above: isoprenoids (four
members), tetrapyrroles (seven), vitamins and cofactors
(seven), and lipids (eight). Plants have two pathways for
the synthesis of isoprenoids—one cytosolic and the other
plastidic (Lichtenthaler, 1999). As expected, each of the
four enzymes listed in Table 1 is a member of the plastid
pathway (also called the glyceraldehyde 3-phosphate/
pyruvate pathway). The synthesis of tetrapyrroles via the
C5 pathway is also a process that takes place entirely in
plastids. Six members of this pathway were identified: glutamate semialdehyde aminomutase, delta-aminolevulinic
acid dehydratase, porphobilinogen deaminase, uroporphyrinogen decarboxylase, coproporphyrinogen oxidase,
and ferrochelatase. One enzyme of haem degradation,
haemoxygenase, was also found. Enzymes linked to the
synthesis of chlorophyll were not detected, in agreement
with the fact that, unlike chloroplasts, amyloplasts do
not require photosynthetic pigments. Further, by contrast
to the chloroplast electron carriers discussed above, the
formation of enzymes active in chlorophyll biosynthesis
appear to be under strict control by light.
In another category, enzymes needed for the biosynthesis
of several vitamins and cofactors were found in the
preparations, confirming the need of plastids for their
biosynthesis. Finally, eight members functional in the
de novo synthesis of fatty acids—an energy demanding
pathway—were also identified (Rawsthorne, 2002). This
finding is consistent with the need of plastids to satisfy the
fatty acid requirement for the cell. The identification of
members of these different biosynthetic pathways is in
keeping with the idea that, at this early stage of development, the amyloplast functions as a factory that
supplies essential metabolites and building blocks to the
parent tissue.
Protein-related
This category, which includes components active in the
folding, assembly, turnover, and storage of proteins is one
of the major groups found in amyloplasts. Interestingly, the
number of proteases (13) is equivalent to the number of
chaperones (14), thus suggesting a rapid turnover of the
plastid protein components—a feature in agreement with
the dynamics of grain development (Vensel et al., 2005).
As discussed above, the presence of several storage
proteins residing in protein bodies (e.g. gliadins, amylasetrypsin/chymotrypsin inhibitors) is probably due to trace
contaminants in the amyloplast preparations. Similar likely
contaminants were reported by Andon et al. (2002).
Transport
Membrane transporters are critical not only for the import of
metabolites, but also for the export of amyloplast products
(Fischer and Weber, 2002). In wheat endosperm, one of the
major amyloplast transporters is the ADP-glucose translocator that imports ADP-glucose formed in the cytosol
(Shannon et al., 1998). In addition, 18 proteins essential for
various pores, channels, and translocators were detected,
reflecting the complexity of the transport machinery of the
plastid envelope. Several proteins active in the sequestration of ions were also identified, in support of a role for
amyloplasts in ion storage (e.g. ferritin and calreticulin).
Signalling
Proteins linked to hormones, phosphorylation, GTP, and
other signalling pathways are well represented in 10 dpa
amyloplasts. These proteins (16 in all) are likely to function in the regulation of plastid development, as well as in
the partitioning of resources such as carbon between different pathways. The amyloplast is obviously connected
to a number of complex signalling networks in cereals as
a result of its central role in endosperm development.
Stress-related
Relatively few stress-related proteins were detected in
the preparations, probably due to the early stage of development (10 dpa) that precedes the onset of grain maturation and drying. Those proteins identified were linked
either to thiols (peroxiredoxins and enzymes of glutathione
biosynthesis) or ascobate (enzymes detoxifying reactive
oxygen species). Also catalase and superoxide dismutase—
enzymes functional in reactive oxygen species removal—
were identified. Of the stress-related proteins identified,
only one, sti1, is not directly redox linked owing to its role
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
Two different groups of proteins linked to nucleic acids
were identified in amyloplast extracts. Most are involved in
the synthesis of DNA and RNA and in transcription and
translation. Surprisingly, the eight ribosome subunits
identified are of eukaryotic origin (Table 1). While the
source of contamination is unclear, it is noted that Andon
et al. (2002) obtained similar results. The inability in the
present study to detect subunits of the 70S ribosome
indicates that most proteins needed by amyloplasts are
encoded in the nucleus (Leister, 2003; Jarvis and Robinson,
2004). The second subcategory related to nucleic acids comprises enzymes of nucleotide—purine and pyrimidine—
biosynthesis. While the de novo synthesis of nucleotides is
still poorly characterized in plants, the evidence suggests
that most of the reactions are localized to plastids (Boldt
and Zrenner, 2003). In the present study, the presence of
nine enzymes of nucleic acid biosynthesis was confirmed:
six members of the purine and three of the pyrimidine
biosynthesis pathways.
1599
1600 Balmer et al.
as a component of a chaperone multicomplex (Hernandez
Torres et al., 1995; Wegele et al., 2003).
Miscellaneous and unknown
Eleven proteins that had either limited functional description or did not fit into the classification categories are
included under ‘Miscellaneous’. The characterization of
these proteins, together with those in the ‘Unknown’
category, awaits further study.
Comparison with whole endosperm proteome
Concluding remarks
The present findings show that amyloplasts, starch-storing
organelles of heterotrophic plant tissues, have broad bio-
Fig. 3. Functional distribution of proteins identified in plastids: (A)
amyloplasts from wheat endosperm; (B) chloroplasts from Arabidopsis
thaliana and maize (Zea mays). Data taken from (Friso et al., 2004) and
the Cornell Plastid Proteome Database website http://ppdb.tc.cornell.edu/.
Downloaded from http://jxb.oxfordjournals.org/ at Pennsylvania State University on February 21, 2013
In a recent study, Vensel et al. (2005) identified ;250
proteins in the salt-soluble (metabolic protein) fraction of
whole wheat endosperm by using a proteomic approach
similar to that of the current study. That fraction would
contain the soluble proteins present in amyloplasts. A
comparison of those results with the ones currently described can, therefore, be used to illustrate the effect of
fractionation. As seen in Table 1, 46 proteins distributed
throughout the various functional categories are common to
the two studies (these are identified with an asterisk in
Table 1). Among these, ;40 reside in amyloplasts (six
appear to be extraplastidic). Based on this comparison, the
use of isolated amyloplasts enhanced the identification of
organellar proteins by ;7-fold, thus illustrating the advantage of organelle isolation in proteomic analysis. In this
case, the isolation step eliminated highly abundant extraplastidic proteins, thereby allowing the detection of organellar polypeptides not detected within the parent tissue. A
case in point relates to enzymes of methionine biosynthesis.
The current study led to the identification of a plastidic
enzyme (cystathionine b-lyase), while the earlier total
endosperm work revealed a member of the pathway
that could be either cytosolic or plastidic (Ravanel et al.,
2004). Analysis of the isolated organelle also gives information on functional processes that is not obvious when
examining the parent tissue (Fig. 2).
The endosperm and amyloplast studies also differ in the
nature of the proteins identified (Fig. 2). Thus, with whole
endosperm, a greater percentage of proteins was linked to
quantitatively prominent processes such as carbohydrate
metabolism, protein-related functions (including storage
proteins), and stress response. By contrast, with amyloplasts, more proteins were affiliated with nitrogen/sulphur
metabolism, nucleic acids, transport, and signalling. Also
there was a much greater percentage of proteins of unknown function with amyloplasts. The observed distributions emphasize the importance of amyloplasts in fulfilling
biosynthetic functions essential for the parent cell.
synthetic capability that is presumably required not only
for their own growth and development, but also for
that of parent cells. The results indicate, for example, that
amyloplasts are endowed with enzymes catalysing the
synthesis of amino acids, isoprenoids, fatty acids, and
tetrapyrroles via pathways that, in leaves, are known to take
place in chloroplasts. However, despite this general similarity, there appear to be quantitative differences in the
distribution of enzymes in these organelles (compare A
and B in Fig. 3). Notably, chloroplasts have, relative to
amyloplasts (i) a larger number of unknown (33% versus
12%), and nucleic acid-related proteins (17% versus 11%),
and (ii) a lower number of proteins related to other
processes, for example, carbohydrate metabolism (2%
versus 12%), energetics (2% versus 7%), transport (4%
versus 8%), and nitrogen/sulphur metabolism (2% versus
15%). It remains to be seen whether these percentages
are linked to species or developmental stage, or whether
they reflect fundamental differences in the organelles. Nonetheless, the results suggest that overall plastids display
Wheat amyloplast proteome
unity of biosynthetic function in supplying essential
building blocks, whether the parent cell is photosynthetic
or heterotrophic. The question that emerges is how the
biosynthetic processes are regulated in amyloplasts. There
is extensive knowledge of the mechanisms operative in
chloroplasts where light interfaces with several elements to
modulate biochemical processes (Buchanan and Balmer,
2005). The corresponding knowledge for amyloplasts is,
however, fragmentary. In filling this heterotrophic gap,
future efforts should focus on amyloplast processes whose
chloroplast counterparts are regulated by light.
Supplementary data
Note added in proof
While this manuscript was in process, our work demonstrating the presence of a complete ferredoxin/thioredoxin
system in isolated amyloplasts was published together with
evidence on the identification of thioredoxin-lined proteins
present in the organelle (Y. Balmer et al., 2006, A complete
ferredoxin-thioredoxin system regulates fundamental processes in amyloplasts. Proc. Natl. Acad. Sci. USA 103,
2988–2993).
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