AJCS 4(3):196-208 (2010)

ISSN:1835-2707

Abiotic stress responsive proteins of wheat grain determined using proteomics
technique
Abu Hena Mostafa Kamal1, Ki-Hyun Kim1, Kwang-Hyun Shin1, Jong-Soon Choi2, Byung-Kee Baik3,
Hisashi Tsujimoto4, Hwa Young Heo5, Chul–Soo Park6, Sun–Hee Woo1*
1

Dept. of Crop Science, Chungbuk National University, Cheong-ju, 361-763, Korea
Proteome Research Team, Korea Basic Science Institute, Daejeon 305-333, Korea
3
Dept. of Crop Soil and Sciences, Washington State University, Pullman, WA 99164-6420, USA
4
Laboratory of Plant Genetics and Breeding Science, Tottori University, Tottori 680-8553, Japan
5
Breeding Resource Developments, National Institute of Crop Science, Suwon, 441-857, Korea
6
Winter Crop & Upland Crop, National Institute of Crop Science, Iksan 570-080, Korea
2*Corresponding author: shwoo@chungbuk.ac.kr

Abstract
The analysis of stress-responsiveness in plants is an important route to the discovery of genes conferring stress tolerance.
Evaluation of two-dimensional electrophoresis gels revealed many proteins to be differentially expressed as a result of abiotic
stress among cultivars. More than 140 protein spots were detected by 2-DE, and some selected protein spots analyzed by
MALDI-TOF mass spectrometry. 22.58 % of abiotic stress responsive proteins were identified in cv. China-108, 32.25 % in
cv. Yeonnon-78, 70.96 % in cv. Norin-61 and 69.35 % in cv. Kantou-107. Of the total identified proteins, 124 proteins wererecognized as abiotic stress responsive unique proteins, of which 31.56 % are induced by heat, 26.61 % by drought, 23.38 %by salt, 21.77 % by cold and 22.58 % by other environmental stress. Furthermore, elucidating the function of proteins
expressed by genes in stress tolerant and susceptible plants will not only advance our understanding of plant adaptation but
also tolerance to environmental stresses. Genes that have been identified by proteomics can be used for marker-assisted
breeding or gene transformation programs to improve the architecture of crop plants and resistance or tolerance to abiotic
stresses.
Key words: Abiotic stress; Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF);
Proteomics; Two-dimensional electrophoresis (2-DE); Wheat grain
Introduction
most wide-ranging difficulty among abiotic stresses for
production of wheat in the world environment. Strategy is
to obtain plants with higher performance under water
stress conditions by identifying and modifying themolecular mechanisms that take place when the water
availability becomes limiting. Drought and heat are thetwo major abiotic constraints affecting the yield andquality of wheat. Plants, as sessile organisms, rely on
proteomic plasticity to remodel themselves during
periods of developmental change, to endure varying
environmental conditions, and to respond to biotic andabiotic stresses. To investigate these facts, morecomprehensive approaches that include quantitative andqualitative analyses of gene expression products arenecessary at the transcriptome, proteome, and metabolome levels. Environmental stresses that result in cellulardehydration, such as freezing, salt and water stress, often
lead to similar changes in plant gene expression andmetabolism (Cook et al., 2004; Kreps et al., 2002). The
proteome is not a static entity, being it affected bymultiple modifications such as cell cycle, changes ofexternal conditions, kind of tissue examined, andfastidious physiological states. Proteomics is becoming a
powerful tool to analyze biochemical pathways and thecomplex response of plants to environmental stimuli. In

Any living organism has to survive conditions.
Specifically for plants, the possibilities to escape fromstress are limited because plants are motionless (Kuiper,
1998). As a general rule, emblematic response toenvironmental stress conditions is established by the
induction of a set of stress proteins that protects theorganism from cellular damage. Abiotic stresses such asheat, cold, drought, salinity, nutrient deficiency, ozone,
heavy metals, UV-B radiation, visible light, chemical
toxicity and oxidative stress are serious intimidation toagriculture. Abiotic stresses usually cause protein
dysfunction. The yield and quality of cereals are severely
affected by heat stress in many countries (Treglia et al.,1999). Heat stress affects the grain yield and quality ofwheat through affecting sink strength and source
capacity. Wheat genotypes express a differential responseto chronic heat as well as a heat shock (Yang et al.,2002). Drought and soil salinity significantly affect plant
growth, development and productivity, thus posing asevere threat to agriculture throughout the world. It isprojected that up to 50% of agricultural yield will be lostdue environmental stresses such as heat, cold, drought,
salinity, nutrient, heavy metals and so on, compared toabout 10 to 20% crop loss caused by biotic stress such as
pathogens and diseases (Kreps et al., 2002). Water is the196were loaded on to the acidic side of the IEF (Iso-electric
focusing) gel with pH range of 3-10 for the first
dimension. SDS-PAGE in the second dimension (Nihon
Eido, Tokyo, Japan) was performed with 12% separationand 5% stacking gels. Protein spots in 2-DE gels werevisualized by Coomassie Brilliant Blue (CBB) R-250
staining (Woo et al., 2002). Each sample was run threetimes and the best visualized gels were selected.

particular, comparative proteomic investigations of plants
before and after specific or interactive stresses allow us toobtain information on how defensive mechanisms are
adopted from plants. Proteomics also makes an essential
bridge between the transcriptome and metabolome (Wanget al., 2004; Gray and Heath, 2005), complementing
genomics research. Only by grouping all this information
together is it possible to achieve a comprehensive andexhaustive analysis of the mechanism of plant defense
against abiotic and biotic stress. Upon several stressresponses protein, protein-protein interaction and posttranslation modification have been also identified(Salekdeh et al., 2002). In the last decade, methodological improvements have allowed comparative
proteomic investigations of plants under stress whichhave allowed us to analyses biochemical pathways andthe complex response of plants to environmental stimuli(Qureshi et al., 2007). In this study, we determined
specific proteins induced by each abiotic stress, particularemphasis will be placed on the heat shock, drought, cold,
salt and others environmental stress by proteomic
approaches.

In-gel Digestion and Mass Spectrometry Analysis
Selected protein spots were excised from preparative
loaded gels, and then washed with 100 µl distilled water.
Each gel piece was dehydrated by 25 mM ammonium
bicarbonate (ABC) / 50% acetonitrile (ACN) and washedwith 10 mM DTT /0.1 M ABC. Gel pieces were dried
under vacuum centrifugation, rehydrated with 55 mM
iodoacetamide (IAA) / 0.1 M ABC for 30 min in dark.
After removing the solution, the gels pieces werevortexed with 100 mM ammonium bicarbonate for 5 min
and soaked in ACN for dehydration. The gel pieces werethen dried under vacuum centrifugation. Trypsin solution(4µl) was added in rehydrated gel particles and incubatedfor 45 min at 40 C and overlaid with 30 μL of 25 mM
ABC (pH 8.0) to keep them immersed. The gel pieces
were then incubated overnight at 37ºC. After incubation,
the solution was spin down and transferred to a 500 µl
siliconized tube. The gel particles were suspended in 40µl ACN / DDW / TFA (660 µl:330 µl:10 µl) three times
and 100% ACN, then vortexed for 30 min, respectively.
The supernatant was dried under vacuum centrifugationfor 2 hr. The digests were desalted with C18 Zip Tip(Millipore, Boston) and subjected to the analysis by
MALDI-TOF Mass spectrometry.

Materials and methods
Plant Materials
Wheat grain of four cultivars (two Chinese cvs. China108, Yennon-78 and two Japanese cvs. Norin-61,
Kantou-107) were used in this study for identification of
abiotic stress responsive proteins by proteomic analysis.
Wheat were grown in field at the National Institute of
Crop Science, Suwon, Korea. Wheat were grown in fieldunder low temperature (-20º ~ -10º C) for four months,
then slowly increase temperature and naturally exposed
up to 28º C until harvesting. The harvested grains werestored at -20º C until used.

Bioinformatics Analysis
According to Kamal et al. (2009), by using peptide
fragmentation methods, the proteins were identified bysearching NCBI non-redundant database using the
MASCOT program (http://www.matrixscience.com). The
search parameters allowed for modifications of acetyl(K), carbamidomethyl (C), oxidation (M), propionamide(C) with peptide tolerance (±100 ppm). For MS/MS
searches, the fragmentation of a selected peptide
molecular ion peak is used to identify with a probabilityof less than 5%. When more than one peptide sequence
was assigned to a spectrum with significant score, thespectra were manually examined. Sequence length, genename and also protein functions (categorized such asheat, drought, salt, cold and other environmental stress)
were identified by searching Swiss-Prot / TrEMBL
database using UniProtKB (http://www.uniprot.org).

Sample preparation by KCl solubility method
Osborne's (1924) solubility method was used to
fractionate wheat endosperm with some modifications(Hurkman and Tanaka, 2007; Kamal et al., 2009). Wheat
flour (50 mg) was suspended in 200 μl of cold (4 °C) KClbuffer (50 mM Tris-HCl, 100 mM KCl, 5 mM ethylenediaminetetraacetic acid (EDTA) (pH 7.8). The suspension
was incubated on ice for 5 min with intermittent mixing
by vortex including sonication (Sonics and Materials Inc.,
USA) and centrifuged at 16,000 × g for 15 min at 4 °C
(Hanil Science Industrial Co. Ltd. Korea). The pellet wassuspended in 800 μl of SDS buffer (2% SDS, 10%
glycerol, 50 mM DL-dithiothreitol (DTT), 40 mM Tris–
Cl, pH 6.8), incubated for 1 hr at room temperature, andinsoluble material removed by centrifugation at
16,000 × g for 10 min at room temperature. The proteins
were precipitated by the addition of 4 vol. of cold (20 °C) acetone and incubation overnight at -20 °C.
Following centrifugation, the pellet was rinsed with coldacetone, then dried by vacuum centrifugation (BIOTRON
Inc., Korea) and solublized in urea buffer (9 M urea, 4%
Triton X-114, 1% DTT, and 2% ampholytes) at 250 µl.

Results and Discussion
2D-PAGE based protein comparison among four wheat
cultivars
We identified more than 140 protein spots among four
cultivars using the gels of pH 3-10. There were analyzed
by MALDI-TOF/MS about 38 protein spots in China108, 40 in Yeonnon-78, 26 in Norin-61 and 36 in Kantou107. The protein spots patterns were highly reproducible
for at least three self-determining protein extractions.
Using 2-DE gels of pH 3-10, we observed qualitative
variations of 19 protein spots in four wheat cultivars.

Gel electrophoresis
Soluble proteins of whole seed were examined by twodimensional gel electrophoresis according to the protocolof O`Farrell (1975). Sample solutions (200 µg proteins)197

Fig 1. (A-D) 2D gel analyses of proteins extracted from mature wheat seeds (A; China-108, B; Yeonnon-78, C; Norin-61, D;
Kantou-107). First dimension was performed on IEF with pH 3-10. In the second dimension gels were used and proteins
were visualized using Coomassie Brilliant Blue (R-250). Circle shows that dissimilar protein spots among wheat cultivars
The adverse effect of temperature on starch synthesis is
responsible for the reduction in grain weight and hence
for the decreased in yield (Wallwork et al., 1998). We
also identified GTP-binding proteins, which are involved
in signal transduction mechanism in plant systems, andthese proteins regulate a flow of kinases that play a vitalrole in environmental stress signal transduction, and alsohigh temperature seem to up-regulate the synthesis of
GTP-binding proteins resulting in increased kinase
activity(Grover et al., 2003). Vacuolar ATP synthesis
proteins are induced by heat (Golldack and Dietz, 2001).
Some others identified proteins were the ribulose
biphosphate, elongation factors and eucaryotic translation
initiation factors. Ribulose biphosphate (Rubisco) is the
enzyme that converts CO2 to plant biomass. Rubisco
electrovated temaperature inhibits photosynthetic CO2
fixation in plant species. The temperature-induced
decreased in the activation of Rubisco due to thisinhibition resulting increased the temperature inchloroplast (Kobza and Edwards, 1987). Besides,
translational activity was involved in the stress response.
Some heat up regulated proteins showed the similarities
to elongation factors (EF) and eucaryotic translation
initiation factors (eIFs). Heat shock involves changes inthe expression patterns of the eIFs, the EF 1-beta, EF 1alpha, EF Tu and eIF (4A, 4B, 4E, 4E-1, 4E-2, 5A-1, 5A2, SUII), as were investigated in wheat leaves as a heatshock (Gallie et al., 1998). Nevertheless, we identified
chaperone, ferritin, annexin, calcium-binding proteins,thioredoxin and ascorbate peroxidase and tubulin in fourAmong them, the protein spots C1-2 were only found in
China-108 (Fig. 1A), D1-11 in Yeonnon-67 (Fig. 1B), E1-2
in Norin-61(Fig. 1C) and F1-4 in Kantou-107 (Fig. 1D).
These results strongly indicate that these identified
proteins are cultivar specific and show the difference
among these cultivars (Yahata et al., 2005; Kamal et al.,2009).
Abiotic stress responsive proteins identified by mass
spectrometry
Heat stress responsive proteins
Out of 39 heat stress responsive proteins, 8 proteins wereidentified in China- 108, 18 in Yeonnon-78, 31 in Norin61, 27 in Kantou-107 (Fig 2). We observed heat increases
or decreases in proteins by heat stress using proteomic
technique in wheat grain. These proteins include heatshock proteins , heat stress transcription factor, granulebound starch synthesis, GTP binding proteins, betaamylase, eucaryotic initaition factor, elongation factor,
ribulose biphosphate related proteins and so on(Supplementary table I). These results confirmed theresults previously by Majoul et al. (2004), demonstratingthat the synthesis of HSPs occurs in the full range of
wheat tissues including developing grains (Giornini andGalili, 1991) and mature grain (Blumenthal et al., 1990),
and heat stress transcription factor to be up regulated byheat( Qin et al., 2007). We also found some granulebound starch synthesis, as reported Mojoul et al. (2004).

198

wheat cultivars (Supplementary Table I). Heat shockproteins (HSPs) function as molecular chaperones inmaintaining homeostasis of protein folding and are
related to the acquisition of thermotolerance (Wang et al.,2004) and ferritin encoding heat regulated (Qin et al.,2008). Calcium is a universal molecule in both animals
and plants, and the transient increase in Ca2+ level during
heat stress is well documented in plant. Heat shock
triggers cytosolic Ca2+ bursts, which is transducted by
Ca2+ bindig proteins(CBP) such as calmodin(CaM),calcineurin (CBL), annexin and then up regulates
the expression of HSPs (Liu et al., 2003). Thioredoxin
and ascorbate peroxidase increase more during short-term
heat shock than during long-term heat treatments (Qin etal., 2008). Tubulin proteins are coupled to GTP binding
proteins, which play a role in heat resistance in plant(Segal and Feldman, 1996). We are also identified serinecarboxypeptidase,
glucose-1-phosphate,glucose-6phosphate and S-adenosyl-methionone synthetase
proteins (Supplementary table I).

Fig 2. Functional distribution of the total identified
abiotic stress responsive proteins among four wheat
cultivars
warrants further analysis, particularly since some of these
proteins increase in response to high temperature (Dupont
et al., 2006).

Drought stress responsive proteins
In response to water deficit, plants have developed
various strategies to cope with stress conditions through a
combination of metabolic, physiological and morphological changes. These drought adaptive changes rely
largely on alterations in gene expression. Out of 33
drought stress responsive, 9 proteins were identified in
China-108, 15 in Yeonnon-78, 20 in Norin-61, 26 in
Kantou-107 (Fig. 2). We identified different abscisic acidresponsive proteins, LEA protein such as chaperonin, cys
peroxiredoxin, ethylene response, and elongation factor
TU in four wheat cultivars, which is responsible fordrought stress (Supplementary Table I). We also
observed cyclin-dependent kinase like, zinc finger,
transcription factor like MYB, lipid transfer proteins and
WRKY in this study (Supplementary table I). Water
stress quickly reduced the mitotic activity of mesophyll
cells in the meristematic zone and reduced the zone ofcell division; the early decline in the Cdc2-like kinase
activity indicates that the activation of the enzyme was
directly affected by stress (Schuppler et al., 1998).
AtMYB2 and AtMYC2 (rd22BP1) are MYBand bHLHrelated transcription factors that bind to MYBRS andMYCRS, respectively (Abe et al., 2003). All thesetranscription factors function as transcriptional activatorsin the expression of stressinducible genes. Although
understanding of the down-regulation of gene expression
under stress conditions is also important for understanding molecular responses to abiotic stresses, little is
known about cis- and trans-acting factors involved in
repression of stress-down-regulated genes. Zinc finger orzinc finger motif was highly enhanced under drought,
cold and salinity stress in Arabidopsis (Sakamoto et al.,2004). Two genes (TaLTP1 and TaLTP2) encoding lipid
transfer proteins (LTPs) were isolated from wheat-rye
near-isogenic line (NIL). High levels of expression of
TaLTPs in the tissue layers between the vascular bundles
might play a role in the drought tolerance response of thewheat crown (Jang et al., 2002). WRKY transcriptional
proteins are implicated in responses to the abiotic stresses
of wounding (Cheong et al., 2002), the combination ofdrought and heat (Rizhsky et al., 2002), and cold (Huang
and Duman, 2002). We also detected triticin in four
wheat cultivars (Supplementary table I). The complement
triticin of globulin storage proteins is very complex andCold stress responsive proteins
Out of 27 cold stress responsive proteins, 8 proteins wereidentified in China-108, 12 in Yeonnon-78, 15 in Norin61, 21 in Kantou-107 (Fig. 2). We revealed some cold
stress related proteins such as cold shock proteins, ABA
inducible proteins, cyclophilin, low temperature regulated
proteins, mitogen activated, and translation initiation infour wheat cultivars (Supplementary table I). Several
cold-responsive genes of unknown function wereidentified from cold-acclimated wheat (Breton et al.,2003). WCSP1 (cold shock protein 1) induction was
cold-specific because neither drought and salinity, norheat stress induced WCSP1 expression (Karlson et al.,2002). Some major protein as cold shock protein, ABA
inducible proteins and so on, synthesized in vivo and invitro during cold acclimation (Houde et al., 1992).
Protein synthesis is necessary for the development offreezing tolerance, and several distinct proteins
accumulate during acclimation to cold, as a result ofchange in gene expression (Guy, 1999). ABA appears tohave an important role in inducing freezing tolerance inwheat, which is influenced by water shortage, increasing
their freezing tolerance (Xu et al., 2006). Cyclophilin,
aquaporin, chitinase plays important role in cold (upregulated) stress of wheat (Houde et al., 2006). The
effects of dehydration, cold-temperature treatment, andosmotic and salt stress on the expression of an abscisic
acid-responsive protein kinase mRNA (PKABA1) was
determined in wheat seedlings (Holappa and Simmons,
1995). Mitogen have three protein kinases, MAPKKK,MAPK and a ribosomal kinase homologue, increased
markedly and simultaneously when plants were treatedwith low temperature (Zhang et al., 2006). The
progressive increase in AP1 transcription was consistent
with the progressive effect of vernalization on flowering
time (Yan et al., 2003). Initiation Factor 1 levels
increased two to threefold upon cold shock , and somemutations in the infA gene coding for IF1 result in cold
sensitivity, further strengthening the possible role of S1
domain proteins in cold acclimation (Giuliodori et al.,2004).

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Salt stress responsive proteins
In conclusion, using two-dimensional electrophoresis,
this study identified proteins involved in heat, drought,
cold, salt and some others abiotic stress responses inwheat. Our findings reveal a proteomic profile of abiotic
stress in wheat, which may provide benefits in two major
areas, in the better understanding of abiotic stressproteins including their functions, and the understandingof stress related physiology in wheat grain.

Out of 29 salt stress responsive proteins, 7 proteins wereidentified in China-108, 10 in Yeonnon-78, 17 in Norin61, 22 in Kantou-107 (Fig. 2). We identified some salt
stress responsive proteins such as salt stress protein, ABA
inducible, aquaporin, bowman-brik type proteinase
inhibitor, calcineurin B like protein, cyclophilin,
potassium channel, and RNA binding proteins in four
wheat cultivars (Supplementary table I). Dooki et al.
(2006) identified several salt responsive proteins
including salt stress protein and ABA using 2-DE.
Aquaporins-like proteins transport water across cellular
membranes and play vital roles in all organisms undersalt stress (Forrest and Bhave, 2008). Peroxiredoxin (2Cys) used as antioxidant enzyme in roots and shoots ofsalt stressed seedling (Uniprot). This peptide encoded by
WRS15 (myb-transcription factor) contains a BowmanBirk domain sharing a high level of sequence, theexpressin level of WRS15 was increased in SR3 wheatroots exposed to salt stress (Shan et al., 2008).
Calcineurin is a Ca2+- and calmodulin-dependent
serine/threonine phosphatase, and notably, the expressionof the mouse calcineurin gene in rice resulted in its higher
salt stress tolerance than the non-transgenic rice (Ma etal., 2005). Cyclophilins is up-regulated under salt stress(Godoy et al., 2000). The physiological role of theirhomologues with putative zinc finger motif remains
unclear. Zinc finger proteins play important role ingrowth and development in plants, and werecharacterized as salt stressed proteins in Arabidopsis (Xuet al., 2006). NaCl is the most plentiful salt encounteredby plants under salinity stress, both Ca2+ and K2+ affectintracellular Na+ concentrations (Zhong and Lauchli,1994), finally calcium enhances K+/Na+ selectivity andincreases salt tolerance (Liu and Zhu, 1997). RNAbinding proteins have probably functioned in salt stress(Sha Valli Khan et al., 2007).

Acknowledgements
This work was supported by a grant from the BioGreen
21 Program (20070301034043), Rural Development
Administration, Korea (Republic of).
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Supplementary Table 1. List of identified abiotic stress-responsive proteins in four wheat cultivars including name of gene

FunctionsMW

pI

SpeciesAccession Number

Gene
Name

Cultivar Specific SC
(%)

Ca

Cb

Ja

Jb

-

17

22

-

HSP18.
2

-

-

11

24

-

20

13

-

PER1

-

35

21

-

Q9S8M5_WHEAT

-

-

-

-

36

HSP17.
6

17.6 kDa class I heat shock protein H

17590

5.36

Arabidopsis
thaliana

HSP12_ARATH

17.8 kDa class II heat shock protein H

17786

5.33

Zea mays

HSP22_MAIZE

18.2 kDa class I heat shock protein H

18123

6.77

HSP13_ARATH

1-Cys peroxiredoxin S,D

23875

6.30

2721

6.75

23443

4.89

Arabidopsis
thaliana
Triticum turgidum
subsp. durum
Triticum aestivum
Triticummonococcum

gi|147225062

-

-

-

-

21

23314

5.71

Triticum aestivum

BAS1_WHEAT

TSA

-

-

25

22

41700

5.83

Triticum aestivum

Q8LK78_WHEAT

ABFB

7

37

19

18267

8.63

Triticum aestivum

gi|4929080

Wrab19

-

25

64

36

Arabidopsis
thaliana
Triticum turgidum
subsp. durum

gi|149207533

ABI3

-

9

-

17

gi|82174009

atu3

-

21

-

49

-

-

-

-

13

200 kDa cold-induced protein C
23.5kDa heat-shock protein

H

2-Cys peroxiredoxin BAS1,
chloroplastic S
ABA response element binding factor
ABA-inducible protein WRAB1

C

D,S,C

gi|12247762

Abscisic acid insensitive protein 3 D,S,C

27534

4.10

Alpha-tubulin H

5581

5.55

49460

6.64

Secale cerealegi|77166842
ANXD4_ARATH

ANN4

-

-

18

-

gi|161897630
Q8LLM6_WHEAT

PIP1-8

-

17

-

-

44

11
-

gi|148529650

BAMY1

-

-

21

14

Aluminum-activated malate transporter
O

33992

8.93

Arabidopsis
thaliana
Triticum aestivum
Triticum aestivum
Triticummonococcum
Triticum aestivum

Q84VJ1_WHEAT

-

-

-

28

-

Blue copper-binding protein-like O
Bowman-Birk type proteinase inhibitor
II-4 D,S

19196

8.77

Oryza sativa

Q7F1H3_ORYSA

-

-

-

27

-

5941

8.45

Triticum aestivum

IBB2_WHEAT

-

26

42

56

23

Cadmium-induced protein AS8 O

18290

9.30

CDI8_ARATH

At4g190
70

-

-

29

31

Calcineurin B-like protein 10 H,S

29895

4.88

Arabidopsis
thaliana
Oryza sativa
subsp. japonica

CNBLA_ORYSJ

CBL10

-

-

28

10

Annexin D4 H

36198

6.88

Aquaporin D,S,C
Ascorbate peroxidase H

21140
15360

9.14
4.70

9615

6.10

Beta-amylase 1

H

Biostress-resistance-related proteinO203

Calcium-dependent protein kinase D,S,C

CPK1C

18

-

39

CALM_WHEAT

-

-

-

35

Arabidopsis
thaliana
Arabidopsis
thaliana
Triticum aestivum

CALX2_ARATH

At5g073
40

-

-

11

-

Q9FFH8_ARATH

-

-

-

12

19

CATA2_WHEAT

CATA

-

-

5

-

Triticum aestivum
Arabidopsis
thaliana

gi|117653943

-

-

-

12

-

DNJ13_ARATH

ATJ13

-

-

16

36

5.73

Triticum aestivum

Q75QN8_WHEAT

WCSP3

-

24

-

22

5.74

Triticum aestivum
Brassica rapa
subsp. chinensis
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Triticum aestivum
Oryza sativa
subsp. indica

gi|21322752

WCSP1

-

-

6

16

gi|82659775

ICE1

42

-

36

22

gi|18496854

-

-

-

-

28

CNGC1_ARATH

CNGC1

-

-

4

-

Q94JY0_ARATH

-

-

-

17

-

23

23

-

16

-

-

36

24

27

22

77

39

86

80

23

60

59722

6.20

Triticum aestivum

gi|164472660

16838

4.10

Triticum aestivum

Calnexin homolog 2 H

60451

4.74

Casein kinase I S

53569

9.86

Catalase I H

56440

6.58

CBFIVd-D9 C

28770

8.76

D,H

59191

8.89

Cold shock domain protein 3 C

21520
21334
16812

9.93

O

14395

9.39

Cyclic nucleotide-gated ion channel 1D

17126

4

Cyclin delta-3 D

36883

5.65

Calmodulin

H

Chaperone protein dnaJ 13

Cold shock protein-1

C

Cold-regulated protein C
Copper transporter-like protein COPT3

D

27
10
0

25361

8.84

D

47473

5.69

Cyclophilin D,C

13584

9.19

Triticum aestivum

gi|82547214

Cytokinin dehydrogenase O

1703

8.59

CKX_WHEAT

O

5694

8.51

DEF12_TRIMO

-

7

-

37

65

20897

9.38

Triticum aestivum
Triticummonococcum
Arabidopsis
thaliana

cdc2-1ACYCB2-2CYP23d-XERO2_ARATH

XERO2

-

-

24

44

77652

6.14

Triticum aestivum

Q58QF6_WHEAT

-

34

18

-

-

31664

8.29

Triticum aestivum

gi|208293840

-

-

-

13

3459

11.71

Triticum aestivum

gi|192898656

-

-

-

26

10
0

9932

9.44

Glycine latifolia

Q6XPS8_9FABA

-

-

-

-

23

28380

5.25

Oryza sativa

Q69JX7_ORYSA

P0569E

-

13

21

13

Cyclin dependent protein kinase
Cyclin-B2-2

Defensin Tm-AMP-D1.2
Dehydrin Xero 2 D

Delta 1-pyrroline-5-carboxylate
synthetase D,S
DEP1 O
Dof-type zinc finger protein D,SDrought-induced protein 1 D
Drought-induced S-like ribonucleaseD204

gi|86438780
CCB22_ORYSI

11.38Elongation factor 1-alpha H
C

49135

9.20

Triticum aestivum

EF1A_WHEAT

TEF1

12

4

-

32

9953

5.55

Triticum aestivum

EM1_WHEAT

EM

5

16

19

-

Ethylene receptor-like protein O
Eukaryotic initiation factor 4B H
Ferritin 2A H

28536
14845
9021

7.44
5.18
5.47

gi|29465662
gi|6739520
gi|210061145

Eif4B
-

21
43

16
-

24
42
23

28
67
11

Germin-like protein 1-3 O

23615

8.45

Triticum aestivum
Triticum aestivum
Triticum aestivum
Oryza sativa
subsp. japonica

GL13_ORYSJ

GER8

-

-

11

16

33236

5.13

Triticum aestivum

S 05078

AGA.1

16

31

8

23

66008

6.38

Triticum aestivum

Q9SQ58_WHEAT

-

-

22

14

26

GTP binding protein H

23041

6.96

Zea maysgi|163838698

41

57

17

8401

8.26

Glycine max

HSP12_SOYBN

-

-

39

44

Heat shock protein 16.9 H

2263

8.09

Triticum aestivum

Q42417_WHEAT

76

88

71

71

Heat stress transcription factor C-1b H

27209

8.93

Oryza sativa
subsp. japonica

HFC1B_ORYSJ

HSP6834-A
hsp16.917LC3
HSFC1
B

-

H

-

21

25

29

Initiation factor 2 alpha kinase-like H

59704

5.18

Q6Z658_ORYSA

-

-

-

8

-

L-ascorbate peroxidase 1, cytosolic S

27086

5.31

APX1_ORYSI

APX1

-

-

52

-

LEA D-11 dehydrin D,H

12803

7.21

gi|21624242

Wdhn13

-

-

45

21

9827

8.89

Oryza sativa
Oryza sativa
subsp. indica
Triticum aestivum
Triticum turgidum
subsp. durum

Q9FEK9_TRITU

ltp7.1

7

-

70

18

14456

6.37

Brassica anpus

JQ2281

-

-

-

-

31

19302

6.77

gi|125663927

-

-

-

-

14

42316

6.14

Triticum aestivum
Arabidopsis
thaliana

MTPB_ARATH

MTPB

-

-

-

22

7378

4.44

Triticum aestivum

MT1_WHEAT

ALI1

54

62

62

24

40091

9.46

M2K4_ARATH

MKK4

-

-

-

18

20944

5.68

Arabidopsis
thaliana
Oryza sativa
subsp. japonica

MOC2B_ORYSJ

MOCS2

-

-

10

-

58894

5.60

Triticum aestivum

Q8W2G8_WHEAT

waxy

-

19

-

-

Em protein

Glucose-1-phosphateadenylyltransferase H
Granule-bound starch synthase GBSSIIH

Heat shock protein 1

Lipid transfer protein precursor

D

Low-temperature regulated protein
BN26 C
Manganese superoxide dismutase O
Metal tolerance protein B

O

Metallothionein-like protein 1 O
Mitogen-activated protein kinase kinase
4 C,S
Molybdenum cofactor synthesis protein
2B O
Mutant granule bound starch synthase I
H

205

MYB transcription factor TaMYB1 D,S,C

31894

8.93

Triticum aestivum

Q27W75_WHEAT

NADH dehydrogenase subunit J O

14825

6.11

Triticummonococcum

gi|164453470

8780

8.85

Oryza sativa

O

74852

5.41

Potassium channel AKT2/3 S

91253

6.09

Probable calcium-binding protein
CML30 D,S,C

22682

4.20

Probable elongation factor 1-gamma 2 H

46374

5.55

24346

5.26

D

20791

8.39

Protein dehydration-induced 19
homolog 5 D

24536

4.73

Protein kinase D,S,C

60937

-

-

-

31

23

ndhJ

-

-

10
0

28

Q9LWT2_ORYSA

-

-

-

35

-

Triticum aestivum

gi|34485589

-

-

-

-

-

Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana
Arabidopsis
thaliana

AKT2_ARATH

AKT2

-

-

12

6

CML30_ARATH

CML30

-

-

-

20

EF1G2_ARATH

At1g577
20

-

-

13

-

-

-

-

24

-

WRK24_ARATH

WRKY
24

-

-

32

15

DI195_ARATH

DI19-5

-

-

21

14

9.14

Triticum aestivum

gi|110341792

wpk4

22

-

26

75

27779

4.97

Q9SR20_ARATH

-

-

-

-

11

26308

7.71

Arabidopsis
thaliana
Oryza sativa

Q69Y99_ORYSA

-

-

-

8

-

Putative cold shock protein-1
Putative cysteine proteinase inhibitor 9

18689

6.28

Q84UR8_ORYSA

-

55

-

12383

5.04

Os03g0210000

-

D,C

-

-

-

24

Putative ethylene-responsive protein O

18729

5.59

-

-

-

-

18

Putative germin-like protein 2-2 O

23645

6.49

-

4

-

Putative GTP-binding protein

44308

6.30

Os02g0491700
-

-

H

-

-

24

42

Putative heat shock protein 21

H

25337

8.48

Oryza sativa
Oryza sativa
subsp. japonica
Arabidopsis
thaliana
Oryza sativa
subsp. japonica
Oryza sativa
Arabidopsis
thaliana

gi|39104609

-

-

-

22

24

7821

5.58

Triticum aestivum

Q8H1Z1_WHEAT

-

28

-

-

-

80153

5.02

Q9CAV6_ARATH

-

-

-

-

21

22724

9.46

Arabidopsis
thaliana
Triticummonococcum

gi|207174006

-

28

37

-

36

Ozone-responsive stress-related proteinlike O
Plastidic alpha 1,4-glucan phosphorylase

Probable RAS type GTP-binding protein
H

Probable WRKY transcription factor 24

Putative calmodulin-domain protein
kinase, 5' partial H
Putative chaperonin 21D
C

Putative low temperature and salt
responsive protein C,SPutative mitogen activated protein
kinase kinase C,SPutative protein kinase D,S,C

206

G84723

CYT9_ORYSJ
Q8LGG8_ARATH
GL22_ORYSJ
Q6Z1J6_ORYSA

Putative RING protein; putative VWA
protein O

23035

8.16

Putative RNA-binding protein S

16287

8.20

Putative rubisco activase H

5599

4.65

94223

6.72

Putative stress responsive protein S

5627

4.33

Putative thioredoxin H

29754

8.60

Putative zinc-binding protein D,S

28239

9.30

R2R3MYB-domain protein D,S,C

4931

8.79

24063

9.53

52817

-

-

51

-

-

-

-

-

4

gi|62176924

rba1

-

88

-

-

Oryza sativa

gi|14488297

-

-

-

-

10

Arabidopsis
thaliana
Oryza sativa
subsp. japonica
Oryza sativa
subsp. japonica
Zea mays
Oryza sativa
subsp. japonica

Q8GWH0_ARATH

At4g306
60

-

52

-

-

Q6Z4N3_ORYSA

-

-

-

-

26

Q6Z8A4_ORYSA

-

-

-

-

15

gi|6165732

-

-

-

-

72

RAC4_ORYSJ

RAC4

-

-

57

23

6.22

Triticum aestivum

RBL_WHEAT

rbcL

-

19

-

8

13045

5.84

Triticum aestivum

RBS3_WHEAT

-

35

47

53

18

RING-H2 finger protein ATL1E O

33476

8.26

ATL1E_ARATH

ATL1E

-

-

16

16

S-adenosyl methionine synthetase 1 H

42793

5.61

METK1_TRIMO

SAMS1

-

24

35

29

Salt and low temperature response
protein S,C

8139

4.42

Q6GYJ6_BRARP

-

-

35

35

35

Salt stress root protein RS1 S

21782

4.92

-

33

22

26

S

17058

4.71

gi|63021412

OsI_001
009
SI

23

17

5

24

Senescence-associated protein-related O

29623

5.85

gi|22331260

-

-

-

14

Serine carboxypeptidase 3 H

CBP3_WHEAT

CBP3

-

16

16

21

Putative salt-inducible protein

S

Rac-like GTP-binding protein 4

O

Ribulose bisphosphate carboxylase large
chain H
Ribulose bisphosphate carboxylase
small chain clone 512 H

Triticum aestivum
Oryza sativa sub
sp. japonica
T. turgidum
subsp. durum

gi|40644800
Q6AU49_ORYSA

55296

5.88

Arabidopsis
thaliana
Triticummonococcum
Brassica rapa
subsp.pekinensis
Oryza sativa
subsp. indica
Triticum aestivum
Arabidopsis
thaliana
Triticum aestivum

S

45573

8.95

Triticum aestivum

gi|117646987

-

-

-

31

-

Small GTP-binding protein O
Small heat shock protein, chloroplastic H

22941
26579

6.90
9.64

gi|57547575
HS21C_WHEAT

HSP21

8

17

49
18

-

Stress-induced protein KIN2 C

6547

9.11

Triticum aestivum
Triticum aestivum
Arabidopsis
thaliana

KIN2_ARATH

KIN2

-

50

-

-

Stress-responsive protein, putative O

21573

8.86

Oryza sativa

Q8LN75_ORYSA

OSJNB
b01N21.

-

53

-

-

Salt tolerant protein

Shaggy-like kinase

207

SRS1_ORYSI

27
Thioredoxin H-type H
Thylakoid-bound ascorbate peroxidase O
Translation initiation factor IF-1,
chloroplastic C

13515
41305

5.12
5.48

Triticum aestivum
Triticum aestivum

TRXH_WHEATQ8GZC1_WHEAT

13104

9.45

Triticum aestivum

Triticin D

23536

6.48

Truncated cold acclimation protein
COR413-TM1C

18722

Tubulin beta-1 chain H
Ubiquitin D
Universal stress protein / early nodulin
ENOD18-like O

-

40
23

40
-

34
-

45
-

IF1C_WHEAT

infA

-

-

38

67

Triticum aestivum

gi|171027863

-

10
0

95

-

13

11.8

Zea mays

Q84LB3_MAIZE

-

-

-

16

-

50254

4.73

TBB1_ORYSJ

TUBB1

-

-

14

31

8520

6.56

Oryza sativa
subsp. japonica
Triticum aestivum

UBIQ_WHEAT

-

23

16

48

48

17867

8.74

Oryza sativa

Q6ZHE6_ORYSA

-

-

-

25

47

Wall-associated kinase 4 O

57611

6.61

Triticum aestivum

Q4U3Z7_WHEAT

WAK4

8

-

11

24

WRKY family transcription factor D

39741

6.03

gi|18417879

-

-

-

24

22

Zinc finger A20 and AN1 domaincontaining stress-associated protein D

18898

8.44

Arabidopsis
thaliana
Arabidopsis
thaliana

SAP9_ARATH

SAP9

-

42

44

27

Criteria: MW: Molecular weight; pI: Iso-electric Point; Ca: China-108; Cb: Yeonnon-78; Ja: Norin-61; Jb: Kantou-107; D: Drought stress; H: Heat stress; S: Salt stress; C: Cold stress; O: Other
abiotic stress responses, SC: Sequence Coverage

208