Differential expression of proteins in maize roots in response to

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May 13, 2011 - X. Hu (&) 4 M. Lu 4 T. Liu 4 W. Wang 4 J. Wu 4 F. Tai 4. X. Li 4 J. .... TOF Pro mass spectrometer (GE Healthcare, USA). The ..... 53:195–200.

Acta Physiol Plant (2011) 33:2437–2446 DOI 10.1007/s11738-011-0784-y

ORIGINAL PAPER

Differential expression of proteins in maize roots in response to abscisic acid and drought Xiuli Hu • Minghui Lu • Chaohao Li • Tianxue Liu • Wei Wang • Jianyu Wu Fuju Tai • Xiao Li • Jie Zhang



Received: 21 November 2010 / Revised: 6 April 2011 / Accepted: 28 April 2011 / Published online: 13 May 2011 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2011

Abstract Roots are highly sensitive organ in plant response to drought, which commonly inhibits root growth. However, less is known about the effect of ABA on root protein expression induced by drought. To help clarify the role of ABA in protein expression of root response to drought, root protein patterns were monitored using a proteomic approach in maize ABA-deficient mutant vp5 and its wild-type Vp5 exposed to drought. Two-dimensional electrophoresis was used to identify droughtresponsive protein spots in maize roots. After coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on each gel, wherein 22 protein spots related to ABA or drought were identified using MALDI-TOF MS. Results showed that the 22 proteins are involved in such several cellular processes as energy and metabolism, redox homeostasis and regulatory. An anionic Communicated by Z.-L. Zhang. X. Hu (&)  M. Lu  T. Liu  W. Wang  J. Wu  F. Tai  X. Li  J. Zhang College of Life Science, Henan Agricultural University, Zhengzhou 450002, China e-mail: [email protected] M. Lu e-mail: [email protected] T. Liu e-mail: [email protected] W. Wang e-mail: [email protected] J. Wu e-mail: [email protected] F. Tai e-mail: [email protected]

peroxidase and two putative uncharacterized proteins were up-regulated by drought in ABA-dependent way; A glycine-rich RNA binding protein 2, pathogenesis-related protein 10, an enolase, a serine/threonine-protein kinase receptor and a cytosolic ascorbate peroxidase were up-regulated by drought in both ABA-dependent and ABAindependent way; a nuclear transport factor 2, a nucleoside diphosphate kinase, a putative uncharacterized protein and a peroxiredoxin-5 were up-regulated by drought in ABAindependent way; a superoxide dismutase 4A, a VAP27-2, a transcription factor BTF3, a glutathione S-transferase GSTF2 and a putative uncharacterized protein were up-regulated by drought in ABA-dependent way, but not exogenous ABA treatment in the absence of drought; a O-methyltransferase and a putative uncharacterized proteins were down-regulated by ABA and drought. The identification of some novel proteins in the drought response provides new insights that can lead to a better J. Zhang e-mail: [email protected] X. Hu Key Laboratory of Physiological Ecology and Genetic Improvement of Food Crops in Henan Province, Zhengzhou, China C. Li College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China e-mail: [email protected] C. Li Huanghuaihai Regional Innovation Center for Maize Technology, Ministry of Agriculture, Zhengzhou, China

X. Li e-mail: [email protected]

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understanding of the molecular basis of root drought tolerance. Keywords ABA  Drought stress  Roots  Zea mays L.  Proteomics Abbreviations ABA APX APRX CBB 2-DE DTT GRP2 GST IEF JA MALDI-TOF MS NTF2 NDPKs PMSF PVP PVPP pI SDS-PAGE TCA TFA OMT Prxs ROS SA SOD ZmPR10

Abscisic acid Ascorbate peroxidase Anionic peroxidase Coomassie brilliant blue Two-dimensional electrophoresis Dithiothreitol Glycine-rich RNA binding protein 2 Glutathione S-transferase Isoelectric focusing Jasmonate Matrix-assisted laser desorption/ionization time of flight Mass spectrometry Nuclear transport factor 2 Nucleoside diphosphate kinases Phenylmethanesulfonyl fluoride Polyvinylpyrrolidone Polyvinylpolypyrrolidone Isoelectric point Sodium dodecyl sulfate polyacrylamide gel electrophoresis Trichloroacetic acid Trifluoroacetic O-methyltransferase Peroxiredoxin Reactive oxygen species Salicylic acid Superoxide dismutase Maize pathogenesis-related protein 10

Introduction Drought is one of the most grievous environmental stress factors limiting plant growth and agricultural productivity in fields. Particularly, global climate change, in the form of rising temperature and altered soil moisture, has resulted in more severe drought in recent years and made water resources for agricultural uses become more limiting. As a result, increasing crop resistance to drought stress would be the most economical approach to improve its productivity and to reduce agricultural use of fresh water resources. To survive this adversity, land plants are equipped with various means of altering their metabolism, morphology and

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developmental processes (Jeong et al. 2010; Shao et al. 2008; Shinozaki and Yamaguchi-Shinozaki 2007; Taniguchi et al. 2010). To understand the mechanisms of plants adaptation to drought, it is a crucial step to identify the key genes that can be used for engineering transgenic crops with improved drought tolerance without reducing yield or biomass. It is generally believed that roots first perceive a dehydration stress signal when the water deficit reaches a certain level (Comstock 2002). However, the mechanisms of roots response to drought remain unclear, especially the effect of abscisic acid (ABA) on roots response to drought. The plant hormone ABA plays crucial roles in plant responses to stress, especially during drought stress. The involvement of ABA in plant response to drought has been revealed by genetic and physiological studies. Previous physiological studies indicated that primary root elongation in maize (Zea mays L.) seedlings exposed to drought requires ABA accumulation, which restricts ethylene production (for reviews, see Sharp and LeNoble 2002; Sharp et al. 2004; Spollen et al. 2000). Recently, molecular and biochemical studies have identified many of ABA- and stress-responsive genes in roots (Jeong et al. 2010; Xiong et al. 2006). By the study for drought inhibition of lateral root growth (dig) mutants with altered responses to drought or ABA in lateral root development, results showed that the DIG3 locus was required for ABA inhibition of lateral root growth as well as drought tolerance (Xiong et al. 2006). On the other hand, results demonstrated that root-specific overexpression of OsNAC10 enlarged rice roots, enhancing drought tolerance of transgenic plants, which increases grain yield significantly under field drought conditions. Moreover, OsNAC10 was induced by drought, high salinity and ABA (Jeong et al. 2010). These transgenic approaches are currently the mainstream method to bioengineer drought tolerance in crop plants, which requires better understanding of the molecular and genetic basis of drought tolerance. However, how the expression of protein is regulated by ABA when the roots perceive the physical signals of drought stress is still unclear. To identify these proteins associated with drought and ABA will be helpful to better understand the molecular and genetic basis of drought tolerance. High-resolution two-dimensional electrophoresis (2-DE) is very useful for separating complex protein mixtures. Proteome analysis has been employed to study alterations in protein expression in response to drought and ABA (Hu et al. 2010a) in leaves of maize seedlings. But to the best of our knowledge, there have not been any reports regarding the ABA roles of roots response to drought stress. The objectives of this study were to compare protein profiles of roots between ABA-deficient mutant vp5 and its wild-type Vp5 under drought stress using proteome analysis techniques, and to identify some novel proteins associated with both drought

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tolerance and ABA so as to provide a better understanding of the molecular basis of root drought tolerance.

Materials and methods

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performed in Ettan III system (GE Healthcare, USA) using pH 4-7 IPG strips (7 cm; GE Healthcare). The second dimension was carried out in 12.5% SDS polyacrylamide gels. 2-DE gels were stained with colloidal coomassie brilliant blue (CBB) G. Digital images of the gels were obtained using an ImageScanner.

Plant material and stress treatments Protein identification by mass spectrometry The ABA-deficient maize (Zea mays L.) vp5 mutant and its wild-type Vp5 were used in these studies. Seeds of the vp5 mutant and wild-type maize were obtained by selfing plants grown from heterozygous seed (Maize Genetics Stock Center, Urbana, IL, USA). Selfed ears with kernels segregating for the mutation were chosen; the maize vp5 mutants contain lowered amounts of ABA. The vp5 kernels can be distinguished from wild-type kernels early in development based on endosperm and embryo color. The vp5 mutant interrupts ABA biosynthesis early in the biosynthetic pathway (Robichaud et al. 1980). Homozygous recessive kernels (vp5/vp5) lack carotenoids, resulting in white endosperm and embryos, easily distinguished from the yellow wild-type kernels (Vp5/-). Since the recessive mutation is lethal in the homozygous state, it is maintained as heterozygotes. Mutant and wild-type seeds were washed in distilled water and germinated on moistened filter papers after seeds were surface sterilized for 10 min in 2% hypochlorite. Maize seedlings were grown in Hogland’s nutrient solution at 400 lmol m-2 s-1 photosynthetically active radiation, a 14/10 h (day/night) cycle, a temperature of 28/22°C (day/ night) and a relative humidity 75% in a light chamber. When the second leaves were fully expanded, seedlings were subjected to various treatments. Drought stress was imposed by placing the seedlings in a PEG6000 solution (-1.0 MPa) for 6 h at 28°C and 40% relative humidity. Control seedlings were kept at 28°C and 75% relative humidity. Afterwards, roots of treated and untreated seedlings were sampled, frozen immediately in liquid N2 and stored at -80°C until analysis. In addition, for inducer experiments, vp5 maize seedlings were pretreated with 100 lM ABA for 5 h, and then exposed to stress treatment for 6 h and sampled as described above. Each treatment of the experimental had three replicates. Maize root protein extraction Maize root samples were homogenized in liquid N2 in a mortar, soluble protein was extracted by SDS/phenol extraction protocol as described in Wang et al. (2006). Two-dimensional electrophoresis Proteins were analyzed by 2-DE gel electrophoresis as described (Wang et al. 2006). Isoelectrofocusing was

Proteins of interest in stained 2-DE gels were subjected to in-gel digestion with trypsin (Wang et al. 2009). The digested fragments were analyzed on an Ettan MALDITOF Pro mass spectrometer (GE Healthcare, USA). The ion acceleration voltage was 20 kV. Each spectrum was internally calibrated with the masses of two trypsin autolysis products. Mass spectra were used to search the UniProt Knowledgebase (Swiss-Prot and TrEMBL, http://www. expasy.org/) for homologous sequences with Mascot software (http://www.matrixscience.com). Theoretical Mr and pI of identified proteins were predicted at http://www. expasy.ch/tools/pI_tools.html.

Results 2-DE analysis of maize root proteins To investigate the unique expressed protein of the root of maize ABA-deficient mutant vp5 and its wild-type Vp5 plants response to the drought stress, proteins extracted from root were separated by 2-DE. In the case of the 2-DE analysis of protein samples, more than 450 protein spots were reproducibly detected in each CBB-stained gel (Fig. 1). Among these proteins, 22 specific proteins, which were regulated by drought, ABA or combined drought and ABA, were detected in maize Vp5 (Fig. 1a, b) or vp5 (Fig. 1c–f) roots subjected to drought treatment. Identification of drought and ABA-responsive protein spots In order to identify the differentially expressed proteins, spots were excised from the preparative gels, in-gel digested by trypsin and analyzed using a MALDI-TOF MS. In total, The 22 proteins were successfully identified by MALDI-TOF MS analysis (Table 1). All of the 22 spots contained only one protein. Some of the identified proteins were annotated as putative uncharacterized proteins without specific function in the maize database. To gain the functional information about these proteins, their homologs were searched with BLASTP (http://www.expasy.org/ tools/blast/) using their protein sequences as queries. The corresponding homologs with the highest homology are

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Fig. 1 A 2-DE analysis of maize root proteins. a and b were, respectively, a 2-DE gels of maize wild-type Vp5 roots subjected to distilled water (control) and drought treatment for 6 h; c, d, e and f were, respectively, a 2-DE gels of maize mutant vp5 roots subjected to distilled water (control), 100 lM ABA, drought, 100 lM ABA ? drought treatment for 6 h. After pretreated with 100 lM ABA for 5 h, the vp5 maize plants were exposed to drought treatment for 6 h. Protein loads were 800 lg. Gels were CBB G stained. Twentytwo root proteins were subjected to MALDI-TOF analysis. This is a representative figure from three biological replicas

shown in Table 2. Only spot no. 21 shared positives with homologs at the amino acid level, indicating that it might have similar function. Besides the unknown proteins, the rest of the identified proteins were classified into several functional categories including regulatory proteins, redox homeostasis-related proteins, energy and metabolism-related proteins and other proteins (Fig. 2). The largest functional category was proteins involved in regulation (36.3%), which was greatly affected by drought stress and ABA (Fig. 1). The redox homeostasis-related proteins (31.9%) belonged to these proteins of the second largest functional category, which suggest that reactive oxygen species (ROS) is readily produced under stress conditions

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and antioxidant defense systems that scavenge or reduce excessive ROS or ROS-induced toxic substances may play an important role in protecting the cells from damage under drought stress. The effect of drought and ABA on the expression of 22 proteins In the identified 22 proteins, compared with control (Fig. 1a), the rest spots except spot nos. 2, 18, 19 and 22 were all significantly strengthened in wide-type Vp5 roots exposed to drought stress (Fig. 1b). In order to determine the effects of ABA on these proteins induced by drought

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Table 1 The identification of differentially responsive proteins in maize roots subjected to drought Spot no.

Protein name

Accession no.

Exp. pI/mass

Theor. pI/mass

1

Nuclear transport factor 2

B4FC92a

5.5/13,800

5.70/13,565

a

Scoreb

%cov (matching peptides)c

73

39.5 (12) 11.8 (2)

2

Putative uncharacterized protein

B6SJL0

6.1/14,400

5.88/10,486

38

3

Glycine-rich RNA binding protein 2

B6STA5a

6.1/14,500

6.11/15,475

102

60 (9)

4

Nucleoside diphosphate kinase

B4FK49a

6.6/15,200

6.30/16,531

65

25 (5)

5

Pathogenesis-related protein 10

Q29SB6a

5.1/16,400

5.39/17,046

48

30 (4)

6

Putative uncharacterized protein

B4FAF8a

5.2/15,500

5.17/15,716

56

17.8 (3)

7 8

Superoxide dismutase [Cu–Zn] 4A VAP27-2

P23345a B6TRX1a

5.7/1,600 5.7/18,000

5.65/15,115 5.73/43,364

94 62

8.6 (2) 24 (7)

9

Superoxide dismutase [Cu–Zn] 4A

P23345a

6.1/1,500

5.65/15,115

61

22 (2)

10

Peroxiredoxin-5

B4FSM5a

6.4/1,800

8.45/20,735

81

42.5 (9)

11

Transcription factor BTF3

B4FIE5a

6.6/1,800

6.62/17,739

70

12.7 (1)

a

12

Enolase (EC 4.2.1.11)

B8A0W7

6/20,000

5.14/48,135

67

20 (5)

13

Serine/threonine-protein kinase receptor

B6T9K5a

6.5/24,000

6.80/46,683

49

12 (4)

14

Serine/threonine-protein kinase receptor

B6SGP4a

6.2/26,000

8.83/42,567

64

24 (7)

15

APx1–cytosolic ascorbate peroxidase

B6UB73a

5.8/27,000

5.65/27,385

189

49 (10)

16

APx1–cytosolic ascorbate peroxidase

B6TM55a

5.7/27,000

5.56/27,307

250

59 (12)

17

Glutathione S-transferase GSTF2

B6U1A7a

5.7/26,000

5.44/23,792

68

25 (2)

18

Putative uncharacterized protein

B4FV82a

5.4/32,000

5.57/32,016

79

14.7 (4)

19

Anionic peroxidase

O04710a

4.6/37,000

5.41/37,774

67

59.9 (26)

20

Putative uncharacterized protein

B4G1B0a

5.7/27,000

5.74/21,832

63

13.1 (2)

21

Putative uncharacterized protein

B4G019a

6.1/37,000

5.96/33,358

132

32 (7)

22

O-methyltransferase

Q6VWFa

5.5/39,000

5.48/38,865

202

37 (10)

a

Accession number in UniProt Knowledgebase (http://www.expasy.org/)

b

Score is a measure of the statistical significance of a match

c

Percentage of predicted protein sequence covered by matched peptides

Table 2 The homologs of the unknown proteins BLASTP (http://www.expasy.org/tools/blast/) was used to search the homologs of the unknown proteins in Table 1 Expasy accession no.a

Homolog Expasy accession no.b

Name

Organism

2

B6SJL0

Q9SM40

Putative glycine-rich protein

Sporobolus stapfianus (ressurection grass)

40

6

B4FAF8

D7LGX9

Nucleic acid-binding protein

Arabidopsis lyrata subsp. Lyrata

49 55

Spot no.

18

B4FV82

B6T315

Jasmonate-induced protein

Zea mays (maize)

20

B4G1B0







21

B4G019

B6TFB6

Stress-responsive protein

Zea mays (maize)

Score

– 107

The homologs with the highest homology are shown The accession number of the unknown proteins in Table 1

a

b

The accession number of the homologs

stress, the mutant vp5 plants were used. In roots of vp5 plants subjected to single 100 lM ABA treatment (Fig. 1d), compared with control (Fig. 1c), spot nos. 3, 5, 12, 13, 14, 15, 16, 18, 19 and 21 were up-regulated, spot nos. 2 and 22 were down-regulated, spot nos. 1, 4, 6, 7, 8, 9, 10, 11, 17 and 20 were not affected. In roots of vp5 plants subjected to single drought treatment (Fig. 1e),

compared with control (Fig. 1c), spot nos. 1, 3, 4, 5, 6, 10, 12,13, 14, 15 and 16 were up-regulated, spot nos. 2 and 22 were down-regulated, spot nos. 7, 8, 9, 11, 17, 18, 19, 20 and 21 were not significantly affected. In roots of maize mutant vp5 plants subjected to drought treatment after pretreatment with 100 lM ABA (Fig. 1f): (a) compared with control (Fig. 1c), the rest spots were strengthened

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unknown 22.7%

regulated by drought in ABA-dependent way. So, spot nos. 18, 19 and 21 were up-regulated by drought in ABAdependent way; (2) if protein expression in vp5 mutant is induced by drought, ABA and ABA ? drought, protein is up-regulated by drought in both ABA-dependent and ABAindependent way. So, spot nos. 3, 5, 12, 13, 14, 15 and 16 were up-regulated by drought both in ABA-dependent and ABA-independent way; (3) if protein expression in vp5 mutant is induced only by drought and ABA ? drought, but not induced by ABA, protein is up-regulated by drought in ABA-independent way. So, spot nos. 1, 4, 6 and 10 were only up-regulated by drought in ABA-independent way; (4) if protein expression in vp5 mutant is induced only by ABA ? drought, but not induced by drought and ABA, protein is up-regulated by drought in ABA-dependent way, but not exogenous ABA in the absence of drought. So, spot nos. 7, 8, 9, 11, 17 and 20 were only up-regulated by drought in ABA-dependent way, but not exogenous ABA treatment in the absence of drought; (5) spot nos. 2 and 22 were down-regulated by both drought and ABA.

regulatory proteins 36.3%

energy and metabolism 4.6%

other proteins 4.6% redox homeostasis 31.9% Fig. 2 The functional category distribution of the 22 identified proteins in maize roots subjected to drought

except that spot nos. 2 and 22 were down-regulated; (b) compared with 100 lM ABA treatment (Fig. 1d), the rest spots were strengthened except that spot nos. 18 and 19 were down-regulated; (c) compared with drought treatment (Fig. 1e), the rest spots were strengthened except that spot nos. 2, 4, 6 and 22 were not obviously affected. These results were more obviously shown in Fig. 3 according to the relative expression of proteins. Summarily, (1) if protein expression in vp5 mutant is induced only by ABA and ABA ? drought, but not induced by drought in the absence of ABA, protein is up-

Regulatory proteins Drought stress adversely affects plants growth and development, which also regulates a wide range of genes

A 18

CK

D

16

Relative expression of protein

Fig. 3 Histograms show the abundance ratio of the identified proteins in Fig. 1. a, b were, respectively, the abundance ratio of the identified proteins of maize wild-type Vp5 and mutant vp5 roots. CK distill water (control), D drought, ABA 100 lM ABA, ABA ? D 100 lM ABA ? drought. Each value represents the average of duplicate 2D gels

Discussion

14 12 10 8 6 4 2 0 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

Number of spot

Relative expression of protein

B

20 CK

ABA

D

ABA+D

15

10

5

0 1

2

3

4

5

6

7

8

9

10

11

12

13

Number of spot

123

14

15

16

17

18

19

20

21

22

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involved in many cellular responses. In this study, several proteins were identified including those involved in signal transduction, protein biosynthesis and processing. These proteins are categorized as regulatory proteins. Spot no. 1 was identified as a nuclear transport factor 2 (NTF2), which stimulates efficient nuclear import of a cargo protein. To the best of our knowledge, there was less report on NTF2 response to ABA or drought. The present results showed that NTF2 expression was up-regulated by drought in ABA-independent way, but not by exogenous ABA. Spot no. 3 was identified as a glycine-rich RNA binding protein 2 (GRP2). GRP2 belongs to the superfamily of glycine-rich proteins (GRPs) which is characterized by the presence of semi-repetitive glycine-rich motifs. In general, GRPs genes present developmentally regulated and tissue-specific expression pattern. The RNAbinding activity of RNA-binding GRPs has been biochemically demonstrated, suggesting that they may be involved in RNA stabilization, processing and transport. For some of those proteins, a RNA-chaperone activity has been demonstrated (for review, see Mangeon et al. 2010). GRP2 is localized into mitochondria. Interestingly, GRP2 presents transcription anti-termination activity, suggesting that it can act as a RNA chaperone (Kim et al. 2007). Our research results showed that GRP2 was up-regulated by drought in both ABA-dependent and ABA-independent way in maize roots. Several other RNA-binding GRPs are also modulated by drought and ABA treatment (for review, see Mangeon et al. 2010). However, some research showed that the transcript level of GRP2 extracted from whole Arabidopsis plants was not affected by drought (Kwak et al. 2005). The divergence could be explained as follows: (1) the difference between the response of maize and Arabidopsis to stress and (2) the difference of root and the whole plants response to drought, because GRPs genes have been proved to present developmentally regulated and tissue-specific expression pattern. Therefore, it might be more significant to analyze GRP2 expression of plant different organs responses to stress. The present results provide new evidence indicating that GRP2 plays important roles in maize root tolerance to drought stress. Spot no. 5 was identified as a maize pathogenesis-related protein 10 (ZmPR10), which is mainly expressed in root tissue with low expression in other tissues. The expression of ZmPR10 was induced by most abiotic stresses including salicylic acid (SA), CuCl2, H2O2, coldness, darkness and wounding (Xie et al. 2010). Earlier studies reported that PR10 was regulated by salt stress in rice (Moons et al. 1997), by drought stress in pine (Dubos and Plomion 2001), and by hormone treatment such as jasmonate (JA), SA, GA3 and ABA (Liu et al. 2006). In this study, ZmPR10 expression was significantly enhanced by drought in both ABA-dependent and ABA-independent

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way. Therefore, PR10 proteins may play important roles in plant defense against stress. Spot no. 11 was identified as a transcription factor BTF3, which forms a stable complex with RNA polymerase IIB and is required for transcriptional initiation. Previous results showed that BTF3 could be up-regulated by drought in rice (Gorantla et al. 2005). Presented results also proved that BTF3 could be up-regulated by drought in ABA-dependent way, but not by exogenous ABA treatment in the absence of drought. Spot no. 19 was identified as an anionic peroxidase (APRX, EC 1.11.1.7), which is a key enzyme involved in polymerization of phenolic monomers to generate the aromatic matrix of suberin. In maize seedlings the ZmAPl gene is expressed predominantly in roots, mesocotyl and coleoptile, but to a lower extent in node, whereas no expression was found in primary leaf (Teichmann et al. 1997). Previous analysis indicated that ABA induced the accumulation of APRX transcripts in potato and tomato callus tissues (Roberts and Kolattukudy 1989). In this study, APRX was up-regulated by drought in ABAdependent way in maize roots. The results suggest that ABA involves in the suberization by inducing APRX. Spot nos. 13 and 14 were identified as serine/threonineprotein kinase receptor, which belongs to the family of transferases, specifically those transferring phosphoruscontaining groups to protein kinases containing serine/ threonine. This enzyme participates in several metabolic pathways such as MAPK signaling pathway, cytokinecytokine receptor interaction and so on. In Arabidopsis, receptor serine/threonine protein kinase is up-regulated by SA treatment and Botrytis infection (Custers et al. 2002). In this study, serine/threonine-protein kinase receptor was up-regulated by drought in both ABA-dependent and ABA-independent way. Spot nos. 22 was identified as an O-methyltransferase (OMT), which is involved in lignin biosynthesis (GuilletClaude et al. 2004). Previous results showed that OMT was induced by ABA in rice callus (Yazaki et al. 2004), by drought stress in maize leaves and by JA in leaf segments of barley (Lee et al. 1996). However, other results indicated that OMT was not induced by ABA in leaf segments of barley (Lee et al. 1996), by drought stress in maize leaves (Vincent et al. 2005) and in rice roots (Yang et al. 2004). In this study, OMT was down-regulated by drought and ABA treatment in maize roots. Proteins involved in energy production and metabolism Spot no. 12 was identified as an enolase (EC 4.2.1.11), which is one of the key enzyme that catalyzes the conversion of 2-phosphoglycerate (2-PGA) to PEP in glycolysis. The present study showed that enolase was up-regulated by drought in both ABA-dependent and

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ABA-independent way. The up-regulation of the enolase has also been reported in roots response to salt tress, ABA treatment and drought (Forsthoefel et al. 1995). Taken together, these results suggest that the glycolytic pathway is highly disrupted by drought stress. ROS scavenging and detoxifying enzymes Plants exposed to drought generate significantly reactive oxygen species (ROS), which, on one hand can cause damage to cellular components, and on the other hand, can act as signaling molecules for stress responses (Apel and Hirt 2004; Hu et al. 2010b). Plants can regulate the ROS level through antioxidant defense enzyme scavenging them such as ascorbate peroxidase (APX), glutathione S-transferase (GST) and superoxide dismutase (SOD), of which three proteins were identified in this study (Table 1). Spot nos. 7 and 9 were identified as SOD (EC 1.15.1.1), which is considered to play an essential role in biological defense against oxygen toxicity. In maize young leaves, the gene transcript of cytosolic isozyme SOD-4A is up-regulated by ABA, but is not changed in response to osmotic stress (Guan and Scandalios 1998). In Sorghum bicolor, the gene transcript of SOD-4A is up-regulated by ABA and drought treatment in shoots, but up-regulated by ABA and down-regulated by drought treatment in root (Buchanan et al. 2005). In present study, SOD-4A was up-regulated by drought in ABA-dependent way, but not by exogenous ABA treatment in the absence of drought. Spot no. 10 was identified as peroxiredoxins (Prxs, EC 1.11.1.15), which are a ubiquitous family of antioxidant enzymes. Prxs constitute the most recently identified group of H2O2-decomposing antioxidant enzymes. In addition to the reduction of H2O2, Prxs also detoxify alkyl hydroperoxides and peroxinitrite, despite the fact that significant differences exist in substrate specificity and kinetic properties. Through this activity, Prx is likely to modulate oxolipid-dependent and NO-related signaling (Dietz et al. 2006). Early research showed that Prxs were induced by drought, ABA and salt stress (Mowla et al. 2002). In present study, Prxs were only up-regulated by drought in ABA-independent way. Taken together, these results indicate that Prxs are up-regulated by drought, but it is still to further study for the effect of ABA on Prxs. Spot no. 17 was identified as GSTF2 (EC 2.5.1.18). GSTs are abundant proteins which are encoded by a highly divergent, ancient gene family and have protective functions such as detoxification of herbicides, and the reduction of organic hydroperoxides formed during oxidative stress. Recent studies indicated that ATGSTU17 could be induced by ABA in Arabidopsis roots (Jiang et al. 2010); the GST activity could be strongly induced by ABA in maize roots (Kellos et al. 2008) and weakly induced by drought in

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barley roots (Haluskova´ et al. 2009). But to the best of our knowledge, there are few reports about GSTF2 in plant response to drought and ABA. Our present study showed that GSTF2 was slightly induced by drought in ABAdependent way, but not by exogenous ABA treatment in the absence of drought. Spot nos. 15 and 16 were identified as APX1. APX (EC 1.11.1.11) plays a key role in regulating H2O2 levels and H2O2 signaling in plant cells. Up-regulation of APX1 has also been reported in plants exposed to abiotic stresses including H2O2 (Davletova et al. 2005) and drought, heat, drought and heat combination (Koussevitzky et al. 2008). In the present investigation, APX1 was up-regulated by drought in both ABA-dependent and ABA-independent way. As for our knowledge, it was first reported that APX1 involved in plant roots response to ABA and drought stress. Based on these observations, it can be hypothesized that APX1 may use a gene of interest to generate drought-tolerant transgenic plants. However, to verify this hypothesis further research is needed. Others proteins Spot no. 4 was identified as a nucleoside diphosphate kinases (NDPKs), which is key metabolic enzymes that maintain the balance between cellular ATP and other nucleoside triphosphates (NTPs). The up-regulation of NDPKs has been reported in response to ABA (Cho et al. 2004), drought (Salekdeh et al. 2002; Hajheidari et al. 2005), heat and salt stress (Dooki et al. 2006; Lee et al. 2007). However, in this study it was up-regulated by drought in ABA-independent way. Spot no. 8 was identified as a VAP27-2, which is a SNARE-like protein that may be involved in vesicular transport to or from the ER. In this study, VAP27-2 was upregulated by drought in ABA-dependent way. But in bean roots, VAP27-2 was not regulated after drought treatment for 1 and 2 h (Torres et al. 2006). In conclusion, as one of the widely cultivated crops, maize is prone to high yield losses due to recurring droughts. In China, drought is a major constraint of maize production and accounts for as much as 13% of yield losses during recent years. Conventional crop breeding techniques though cumbersome and time-consuming, have been very helpful in releasing drought-tolerant varieties. However, this is not adequate to cope up with the future demand for maize, as drought seems to spread to more regions and seasons across the country. Understanding the genes that govern maize response to drought stress is urgently needed to enhance breeding maize with improved drought tolerance. In the present study, we identified some candidate proteins associated with drought stress response and ABA regulation by comparative proteomics. This will be useful to maize researchers as ready reference source for breeding

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through developing candidate gene markers associated with drought stress. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 30800667 to XL Hu), the Fok Ying-Tong Education Foundation, China (Grant No. 122032), the China Postdoctoral Science Foundation (Grant no. 20080440824 and No. 200902357 to XL Hu), the Foundation for University Key Teacher by the Ministry of Education (grant No.2009GGJS-028 to XL Hu) and the Foundation of Henan Major Public Projects (Grant No.091100910100).

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