Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Journal of Plant Physiology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Journal of Plant Physiology journal homepage: www.elsevier.com/locate/jplph

Physiology

Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents Zhi-Hui He a , Hong-Wei Li b , Yunkang Shen a , Zhen-Sheng Li b , Hualing Mi a,∗ a National Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 300 Fenglin Road, Shanghai 200032, China b State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

a r t i c l e

i n f o

Article history: Received 30 November 2012 Received in revised form 15 March 2013 Accepted 15 March 2013 Available online xxx Keywords: Chloroplast Proteomics Photosynthesis Cyclic electron transfer Wheat (Triticum aestivum L.)

a b s t r a c t To understand the photosynthetic basis in a single seed descent line 10 (SSDL10) of wheat contained high ATP in leaves, the chloroplast proteome was compared to SSDL10 and its parents using a combination of 2DE and MALDI-TOF MS and MS/MS. More than 300 protein spots could be reproducibly detected in the 2D gel. 18 spots were differentially expressed between SSDL10 and the parents, 16 of which were identified by MS with the localization in chloroplasts. These proteins are grouped into diverse functional categories, including Calvin cycle and electron transport in photosynthesis, redox homeostasis, metabolism, and regulation. In addition to Rubisco large subunit, the content of photosynthetic electron transfers such as chlorophyll a-b binding protein, ATP synthase ␦ subunit, ferredoxin-NADP+ oxidoreductase (FNR) was higher in SSDL10 than in its parents. Furthermore, cyclic electron transfer around photosystem I (CET) was faster in SSDL10 than in the parents. Analysis of NADPH-NBT oxidoreductase activity combined with immuno-detection further revealed that, the activity of two high molecular mass protein complexes containing FNR probably involved, the CET appeared higher in SSDL10 than in the parents. The possible mechanism for the regulative role of CET in photosynthesis in SSDL10 is discussed. © 2013 Elsevier GmbH. All rights reserved.

Introduction Light reaction in the oxygen-evolving photosynthesis converts light energy into chemical energy in the form of ATP and drives the production of NADPH. Two types of electron flow are involved in the reaction. The linear electron transfer from the water to NADP+ generates both ATP and NADPH, while cyclic electron transfer around photo system I (CET) is exclusively involved in ATP synthesis by generating the proton gradient across the thylakoid membrane. It is generally accepted that CET supplies the extra ATP needed for the CO2 concentration in C4 plant or during the CO2 assimilation limiting conditions (Rumeau et al., 2007). At least two distinct CET pathways have been suggested, the first one is the uncharacterized ferredoxin-plastoquinone oxidoreductase

Abbreviations: 2-cys Prx, 2-cysteine peroxiredoxins; ATPase, ATP synthase; CET, cyclic electron transfer; Cytb6 f, cytochrome b6 f; FNR, ferredoxin-NADP+ oxidoreductase; FQR, ferredoxin-plastoquinone reductase; GS, glutamine synthase; TRX m, m-type thioredoxin; MALDI TOF, matrix-assisted laser desorption/ionization time of flight; MS/MS, tandem mass spectroscopy; NDH, NAD(P)H dehydrogenase; OEC, oxygen-evolving complex; OEE1, oxygen-evolving enhancer 1; P700, the reaction center chlorophylls in PSI; PSI, photosystem I; PSII, photosystem II; RCA, rubisco activase; Rubisco, ribulose-1,5-bisphosphate carboxylase/oxygenase. ∗ Corresponding author. Tel.: +86 21 54924135; fax: +86 21 54924015. E-mail address: [email protected] (H. Mi).

(FQR)-dependent pathway. To date, the only characterized components of this route are the regulatory proteins PGR5 and PGRL1 (Dalcorso et al., 2008; Munekage et al., 2002). The other pathway is NAD(P)H dehydrogenase (NDH)-dependent route, however, the NDH complex concentration in the thylakoid membrane is very low (Burrows et al., 1998; Mi et al., 1995; Shikanai et al., 1998). It is noteworthy that during the grain filling stage of wheat, the ATP demand is always high. Thus CET probably contributes to the ATP supply for the plant. A single seed descent line 10 (SSDL10) of wheat was selected from above F6 progeny by crossing XY54 (maternal parent) with J411 (paternal parent). Our previous comparative studies showed that the ATP content in the leaves of SSDL10 was more than twofold higher than its parents during the heading and grain filling stages (Hu et al., 2007). These results imply that the intrinsic CET, one of the photo-protective and photosynthesis regulative pathways, is higher in SSDL10 than in its parents. Since the photosynthetic capacity difference was observed between SSDL10 and parent lines J411 and XY54, it seems necessary to investigate the chloroplast proteins pattern among SSDL10 and parent lines. Here we present the comparative chloroplast proteomics analysis between SSDL10 and parent lines from flag leaves at the grain filling stage. 18 spots were differentially expressed between SSDL10 and the parents, 16 of which were identified by MALDI-TOF/TOF. Based on the significant difference in proteins involved in CET, we

0176-1617/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2013.03.016

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

2

further found that CET activity was higher in SSDL10. The possible mechanism for photo-protective and the regulative role of the high CET in SSDL10 is discussed.

visualized by silver staining (Zhang et al., 2010). Three biological replicates of each sample were performed. Data analyses

Materials and methods Plant materials The winter wheat (Triticum aestivum L.) hybridization line SSDL10 (single-seed descent line 10) was selected from the above F6 progeny by crossing XY54 (Xiaoyan54, maternal parent) with J411 (Jing411 paternal parent). SSDL10 and the parent varieties XY54 and J411 were grown in a filed at Shanghai Institute of Plant Physiology and Ecology. Seeds were sown in 12 November 2007 and 2008. Flag leaves of the grain filling stage were used for all experiments. Chloroplast isolation and protein extraction For chloroplast isolation, flag leaves from the SSDL10 and the parent lines at the grain filling stage were collected. Intact chloroplasts were isolated according to the previous study (Wang et al., 2006) with slight modifications. Leaves were homogenized in cold medium containing 0.4 M sucrose, 50 mM Tris–HCl (pH 7.6), and 10 mM NaCl. The suspension was filtered through 2 layers of nylon cloth and centrifuged at 1000 × g for 3 min at 4 ◦ C. The pellet was resuspended in the medium and layered onto a 40% and 80% Percoll gradient. Intact chloroplasts were recovered from the 40%/80% Percoll interface and washed twice after centrifugation at 3 000 × g for 10 min at 4 ◦ C. Integrity of the chloroplasts was validated by measuring the O2 evolution after the addition of potassium ferricyanide (Heber and Santarius, 1970). Chloroplast proteins for IEF were prepared using a DTTtrichloroacetic acid (TCA)-acetone precipitation method. Lysis buffer (8 M urea, 2%, w/v thiourea, 5%, v/v Triton X-100, 50 mM NaCl, and 20 mM Tris pH 8.0) was added to the isolated chloroplast fraction. After vigorous shaking for 1 h, samples were centrifuged at 5000 × g for 15 min. The supernatant was precipitated by the acetone with 10% trichloroacetic acid again at −20 ◦ C for 1 h and centrifuged at 17,000 × g for 20 min at 4 ◦ C. The pellet was washed twice in the ice-cold acetone containing 0.07% DTT. The final protein precipitant was re-suspended in the protein sample buffer (8 M urea, 4%, w/v CHAPS, 65 mM DTT, and 0.2%, v/v Bio-lyte pH4-7). The samples were centrifuged at 15,000 × g for 20 min. The resulting supernatant was subjected to IEF. Protein concentration was quantified by the Bradford assay with the bovine serum albumin (BSA) as the standard (Bradford, 1976). IEF and 2-DE For IEF, 100 ␮g and 1 mg protein were loaded on analytical and preparative gels, respectively. Immobilized IPG strips (pH 4–7, 17 cm, Bio-Rad) were used for IEF for the first dimension. IEF was carried out in a PROTEAN IEF CELL (Bio-Rad, USA) with the following conditions: 14 h rehydration, 1 h 250 V, 1 h 1000 V, 2 h 10,000 V, and 6 h 10,000 V. System temperature was 20 ◦ C and the current was set to 50 ␮A per strip. For the second dimension, strips were placed in an equilibration buffer I (50 mM Tris–HCl pH 8.8, 6 M urea, 20%, v/v glycerol, 2%, w/v SDS, 2%, w/v DTT and trace bromophenol blue) and gently agitated for 15 min. After this, the strips were placed in the equilibration buffer II containing 2.5% (w/v) iodoacetamide instead of DTT with gentle agitation for another 15 min and rinsed in the SDS-PAGE buffer (25 mM Tris, 192 mM glycine, 0.1%, w/v SDS). SDS-PAGE was performed with 12% w/v acrylamide gels using the PROTEAN II XL CELL (Bio-Rad, USA). The protein spots were

The silver stained gels were scanned using Fluorescent Image Analyzer FLA-9000 (Fujifilm, Japan). The pixel size was set at 100 ␮m with the photo-multipliers (PMT) as 500 V. Spot detection and quantification were performed using Progenesis SameSpots (Nonlinear, USA). The protein spots intensities were calculated by the normalized volume percentage of three replicates of each sample, and were statistically analyzed by ANOVA. Only the spots which were reproducible at least two-fold difference of normalized percentage volume and statistical significance (p < 0.05) were considered to be differentially expressed protein spots. Protein identification Protein spots were excised from preparative gels and washed twice with ultra pure water and destained with 50% (v/v) acetonitrile (ACN) in 25 mM NH4 HCO3 . The gels were dehydrated 100% v/v ACN and dried by vacuum centrifugation. The sample was in-gel digested with trypsin (Promega, USA) in 25 mM NH4 HCO3 overnight at 37 ◦ C and the peptides were lyophilized. The lyophilized peptide was dissolved in 0.1% trifluoroacetic acid (TFA). MS analysis was conducted with a Bruker-Daltonics AutoFlex MALDI TOF-TOF Mass Spectrometer (Bruker, USA). Three of the protein spots gels were identified by using LC-ESI MS/MS with the RP18-C18 column (150 ␮m × 150 mm, Column Technology Inc., USA) coupled to the LTQ Obitrap mass spectrometer (Thermo Finnigan, USA). A database search was performed by MASCOT (Matrix Science, UK) using the local NCBInr database containing 9,487,487 protein sequences and 3,243,437,036 amino acid sequences (updated in August, 2009). The search parameters were set as follows: trypsin was the digest enzyme, carbamidomethyl (Cys) was the fixed modification, oxidation (Met) was variable modification, peptide tolerance was 100 ppm, fragment mass tolerance is 0.8 Da, and up to one missed cleavage was allowed. The keratin contamination was removed, and the MASCOT score above the 95% significance threshold (p < 0.05) was considered a positive hit. Oxido-reduction of P700 The redox state of P700 of the wheat leaves was monitored by absorbance changes at 810–830 nm, using the PAM-Fluorometer PAM-101 (Walz, Germany) equipped with an ED-P700-DW-E emitter-detector unit (Walz, Germany). Leaves were kept in the dark for 5 min prior to the measurement. P700 was oxidized by farred (FR) light (>705 nm, 5.2 ␮mol photons m−2 s−1 ) for 40 s, and the subsequent re-reduction of P700+ in darkness was monitored. The initial rate of P700+ re-reduction was calculated as described by Klughammer and Schreiber (1998). Thylakoid membrane proteins electrophoresis and protein blotting Thylakoid membranes were extracted as described previously (Wei et al., 2010). The wheat leaves were homogenized in cold STN medium (0.4 M sucrose, 50 mM Tris–HCl pH 7.6, 10 mM NaCl). The homogenate was filtered through two layers of nylon cloth and centrifuged at 200 × g for 2 min at 4 ◦ C. The supernatant was centrifuged at 6000 × g for 10 min at 4 ◦ C and washed twice with the same medium. Chlorophyll was determined as described (Porra et al., 1989). Native PAGE and NADPH-NBT activity staining was performed as per the previous description (Ma et al., 2006) with slight modifications: The thylakoid membranes containing the 0.5 mg/ml chl

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

were solubilized with 1.2% (w/v) n-dodecyl-␤-maltoside (DDM) by gentle agitation on ice for 1 h. After centrifugation at 15,000 × g for 10 min at 4 ◦ C, the samples were immediately subjected to Native-PAGE. Native-PAGE was performed with 7% polyacrylamide gel at a low constant current of 3 mA at 0 ◦ C. Following NativePAGE, gels were incubated in 20 mM Tris–HCl (pH 7.6) and 0.1% nitroblue tetrazolium (NBT) for 20 min. and then supplemented with 1 mM NADPH in the dark at room temperature for the activity staining. For the second dimension SDS-PAGE, the native gel strips were excised and incubated in Laemmli-buffer (5%, v/v ␤mercaptoethanol) (Laemmli, 1970), and subsequently applied to 12% SDS-PAGE. After being electro-blotted to the Nitrocellulose membrane (GE Healthcare, USA), the membrane was sequentially incubated with anti-FNR antibody at 1:10,000 dilution or antiNdhH antibody at 1:10,000 dilution and the alkaline phosphatase (AP)-conjugated secondary antibody at 1: 10,000 dilution. Signals were detected by alkaline phosphatase substrates (Sigma, USA). Results 2-DE analysis and MS identification of the chloroplast proteins To investigate the molecular mechanisms of higher ATP content of SSDL10, especially the photosynthetic capacity differences during the grain filling stage, the chloroplast proteins from the flag leaves of hybrid SSDL10 and parent varieties J411 and XY54 during the grain filling stage were analyzed by 2-DE with the pH range 4–7. Representative 2DE gel of the chloroplast proteins extracted from SSDL10 was shown in Fig. 1A. In total, more than 300 protein spots were reproducible detected on each gel. Quantitative analysis revealed that 18 spots were significantly differentially expressed between SSDL10 and the parents (p < 0.05), 10 of the spots have more abundance in SSDL10, while 8 of them have a lower expression in SSDL10 compared to the parents (Fig. 1B and C). All the 18 spots were recovered from the preparative gels, in gel digested by trypsin and analyzed by MALDI-TOF/TOF. 16 proteins were identified successfully with the localization in the chloroplasts. Spot 16 was not successfully identified by MS, while another spot was identified as the cytoplasmic protein. The protein identification, abundance, and details are summarized in Table 1. The MS analysis results were shown in supplementary Table S1. These identified proteins were classified into 5 functional categories, including Calvin cycle of photosynthesis (6 spots, three ribulose bisphosphate carboxylase large subunit, ribulose bisphosphate carboxylase activase B, phosphoribulokinase, phosphoglycerate kinase), photosynthesis electron transfer (5 spots, ATP synthase ␦ subunit, oxygen-evolving enhancer protein 1, ferredoxin-NADP+ oxidoreductase, chlorophyll a-b binding protein), redox homeostasis (3 spots, m-type thioredoxin, two 2cysteine peroxiredoxin), regulation/defense (1 spot harpin binding protein), metabolism (1 spot, plastid glutamine synthase) (Fig. 2). Most of the differentially expressed proteins were found to be related to photosynthesis, including Calvin cycle and electron transfer, which accounted for 37.5% and 37.2% of the identified proteins, respectively. Proteins involved in redox homeostasis also accounted for 18.8%. One protein of metabolism and one of defense were found. Comparison of CET It is implied that the high ATP content in the leaves of SSDL10 may result from the high cyclic photophosphorylation activity (Hu et al., 2007). To confirm this possibility, CET was compared between the leaves SSDL10 and parent lines. The dark re-reduction of P700+ reflects the rate of CET (Mi et al., 1992). The kinetics of the dark

3

reduction of P700+ after far-red light of SSDL10 and parents leaves are shown in Fig. 3A. The initial reduction rate (0–0.2 s) of P700+ is calculated. The re-reduction rate of SSDL10 is significantly faster than the parents during the grain filling stage (Fig. 3B), but not so during the seedling stage (data not shown), indicating the CET functions to supply extra ATP in the high ATP demand physiological processes. NADPH-NBT oxidoreductase activity staining and protein blot Since the ferredoxin-NADP+ oxidoreductase (FNR) was identified in SSDL10 with higher expression (Fig. 1B), native PAGE of the thylakoid membrane proteins from SSDL10 and the parents was carried out. It has been reported that FNR could interact with PGRL1 (Dalcorso et al., 2008), cytochrome b6 f (Cytb6 f) complex (Zhang et al., 2001), NDH complex (Guedeney et al., 1996; Quiles et al., 1996) participating in CET. Thylakoid membrane protein complex with the attached FNR could be stained as the dark purple band in the presence of NADPH and nitroblue tetrazolium (NBT). DM-solubilized thylakoid membranes from the SSDL10 and parents during the grain filling stage were subjected to Native-PAGE, followed by the NADPH-NBT activity staining. Two activity bands with the molecular mass of approximately 500 kDa were revealed in the native gel (Fig. 4A), and the staining of the activity bands was more intense in SSD10 thylakoid membrane sample, indicating the higher diaphorase activity in SSDL10. Furthermore, to identify and investigate the amount of FNR in the thylakoid membrane proteins of SSDL10, protein blotting was performed with an antibody against FNR after the protein complexes bands separated with Native-PAGE were subjected to second a 2D SDS-PAGE. The position electroblotted from the gel corresponding to the activity band reacted with the FNR and NdhH antibody strongly (Fig. 4B). The result suggests the higher activity band in SSDL10 is attributed to the higher expression of the Ndh complexe associated with FNR. Discussion Chloroplast serves as the important organelle in higher plants, where photosynthesis as well as the metabolism of carbohydrates, amino acid, and lipids takes place. In the present study, by using the 2DE analysis of the intact chloroplast proteins, comparison of the chloroplast expression patterns at the grain filling stage of SSDL10 and parent lines allowed us to identify 18 differentiated protein spots to further understand the molecular basis of the higher ATP level and higher photo-oxidative resistance of SSDL10 (Fig. 1). Among them, 10 proteins were found up-regulated, while 8 proteins were down-regulated in SSDL10 (Table 1). All the identified 16 proteins were classified to 5 categories, including Calvin cycle and photosynthetic electron transfer, redox homeostasis, metabolism, and defense in photosynthesis (Fig. 2). Photosynthesis related proteins Many chloroplast proteins are involved in photosynthesis. In this study, 6 proteins (37.5%) and 5 proteins (31.2%) were identified as Calvin cycle proteins and electron transfer-related proteins, respectively. Spot 1, identified as an ATP synthase ␦ subunit, has remarkably enhanced abundance in SSDL10 (Fig. 1A and B and Table 1). Chloroplast ATP synthase (cpATPase), located in the thylakoid membrane, is the main enzyme for ATP synthesis in the presence of a proton gradient across the membrane, containing the intrinsic CF0 and catalytic extrinsic CF1 segments. cpATPase ␦ subunit is essential for the complex stability. In Arabidopsis, in the absence of ␦ subunit, the entire cpATPase complex is destabilized (Maiwald,

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9 4

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

Fig. 1. 2-DE gel image of the proteins extracted from the wheat (Triticum aestivum L.) SSDL10 and parents J411, X54 flag leaves chloroplast. (A) Representative 2DE gel of the chloroplast proteins extracted from SSDL10. Chloroplast proteins were separated with a linear pH gradient 4–7 for the first dimension and a 12% SDS-PAGE in the second dimension. Protein spots were visualized with silver staining. Molecular weight standards are indicated on the left side of the gel. The differently expressed proteins are numbered with arrows as in Table 1. The protein spots up-regulated (B) or down-regulated (C) in hybrid compared with the parents lines of J411, XY54 are shown with arrows.

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

Spota

1

3

8

11

12

13

14

17

21

23

25

Protein name

Functional categories

Experimental/ theoretical Pi

Experimental/ theoretical MW

Scorec

Peptide matches

Oryza sativa

gi|115448701

ATP synthase ␦ subunit

Photosynthesis electron transfer

4.2/5.0

24/26.2

59

1

Triticum aestivum

Triticum aestivum

Triticum aestivum

Triticum aestivum

Hordeum vulgare

Triticum aestivum

Triticum aestivum

Triticum aestivum

Triticum aestivum

Triticum aestivum

Triticum aestivum

Triticum aestivum

gi|12344

gi|131388

gi|12344

gi|4138592

gi| 1076722

gi |20302471

gi |71362455

Rubisco large subunit

Oxygen-evolving enhancer protein 1

Rubisco large subunit

m-type thioredoxin

2-cystein peroxiredoxin

Ferredoxin-NADP+ oxidoreductase

Plastid glutamine synthetase

gi |21839

Phosphoribulokinase

gi |38679331

Harpin binding protein 1

gi|1805351

2-cystein peroxiredoxin

gi|1805351

gi|7960277

Phosphoglycerate kinase

Ribulose bisphosphate carboxylase activase B

Photosynthesis Calvin cycle

Photosynthesis electron transfer

Photosynthesis Calvin cycle

Redox homeostasis

Redox homeostasis

Photosynthesis electron transfer

Metabolism

Photosynthesis Calvin cycle

Regulation/Defense

Redox homeostasis

Photosynthesis Calvin cycle

Photosynthesis Calvin cycle

6.2/6.2

4.9/8.7

6.0/6.2

4.7/8.7

4.9/5.5

6.3/8.3

5.0/5.4

5.0/5.7

5.2/9.5

4.6/5.7

5.6/6.6

5.7/6.9

31/52.8

31/34.7

31/52.8

12/19.1

21/23.2

35/38.9

42/46.7

39/45.1

28/29.4

21/23.3

43/49.8

44/47.8

64

-

43

26

114

29

51

49

97

80

109

21

Histogram d

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

SSDL10

J411 XY54

4

4

2

1

2

1

2

3

Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

6

Accession no.b

3

2

3

1

ARTICLE IN PRESS

4

Organism

G Model

JPLPH-51713; No. of Pages 9

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

Table 1 MS identification of the differentiated expressed proteins between SSDL10 and parents lines J411, XY54.

5

ARTICLE IN PRESS

G Model JPLPH-51713; No. of Pages 9

Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

1

J411 XY54 SSDL10

c

d

Spot numbers correspond to Fig. 1. Accession number of the identified protein. Mascot MOWSE score for MS-based identification. Mean values of the normalized percentage volume from 3 replicates of the protein spots from the SSDL10 and parents are shown in the diagram. Error bars indicate SE. a

b

26/28.2 5.0/5.7 Photosynthesis electron transfer Chlorophyll a–b binding protein Triticum aestivum 33

gi|115782

14/52.8 5.0/6.2 Photosynthesis Calvin cycle Rubisco large subunit Triticum aestivum 27

gi| 132065

Triticum aestivum 26

gi|225690804

Chlorophyll a-b binding protein

Photosynthesis electron transfer

5.7/5.1

26/28.5

22

1

SSDL10

J411 XY54

9

SSDL10

Histogram d Organism Spota

Table 1 (Continued)

Accession no.b

Protein name

Functional categories

Experimental/ theoretical Pi

Experimental/ theoretical MW

Scorec

Peptide matches

J411 XY54

6

2003). It is reported that the N-terminal and C-terminal of the subunit could interact with the cpATPase CF1 and CF0 , respectively (Ni et al., 2004). Transgenic tobacco expressing an antisense gene directed at the gene encoding the cpATPase ␦ subunit, showed reduction of the electron transport rate and photosynthetic capacity (Yamori et al., 2010). Our previous studies demonstrate that in the leaves of SSDL10, the ATP content was generally higher than the parents, and was more than two-fold higher during the heading and grain filling stages (Hu et al., 2007). Therefore, the up regulation of the cpATPase ␦ subunit might be responsible for the greater abundance of the ATP concentration in the leaves of hybrid SSDL10. Another important electron transport related protein, FNR, was identified in spot 12 (Fig. 1A and B and Table 1). FNR is a ubiquitous enzyme involved in various electron transfer pathways, harboring one molecule of non-covalently bound flavin adenine dinucleotide (FAD). Leaf-type FNRs carry out the last step of linear photosynthetic electron transport in which they catalyze a two-electron transfer from two independent Fd molecules to a single molecule of NADP+ (Arakaki et al., 1997). FNR has been found to associate with NDH to use NADPH as a substrate (Guedeney et al., 1996; Quiles et al., 1996). The association of FNR with cytochrome b6 f (Cytb6 f) or PGRL1 is believed to enable these proteins to direct electrons to the CET via a FQR-dependent route (Dalcorso et al., 2008; Zhang et al., 2001). During the grain filling stage, the CET activity reflected by the initial reduction rate of P700+ was significantly faster than the parents in SSDL10 leaves (Fig. 3B). Meanwhile, Native-PAGE followed by NADPH-NBT activity staining of the thylakoid membrane proteins from SSDL10 and parents revealed that two activity bands with the molecular mass of approximately 500 kDa were more intense in SSDL10 than the parents (Fig. 4A). Subsequent 2D SDS-PAGE of the gel strips demonstrated that the activity bands corresponded to the membrane-bound complex containing FNR, which might participate in the CET (Fig. 4B). In addition, it was reported that the formation of the FNR-cytbf complex would contribute to the CET process (Joliot and Johnson, 2011). Thus, the higher activity (Fig. 4A) and amount of FNR (Table 1) in SSDL10 would contribute to its higher activity of CET (Fig. 3). Two protein spots (spot 26, 33) up-regulated in SSDL10 were identified as chlorophyll a-b binding protein, suggesting the higher efficiency of light harvesting processed in SSDL10 leaves (Fig. 1B and Table 1). Spot 4 was identified as the oxygen-evolving enhancer 1 (OEE1), also named PsbO, which was down-regulated in SSDL10. The oxygen-evolving complex (OEC) of higher plant photo-system II (PSII) consists of an inorganic Mn4 Ca cluster and three nuclearencoded proteins, PsbO, PsbP, and PsbQ (Slowik et al., 2011). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn4 Ca cluster against photo-inhibition. The low amount of the OEE1 in SSDL10 might be a result of the inherence of parent line J411, which has less of an abundance of OEE1. Ribulose bisphosphate carboxylase (Rubisco) is the major photosynthesis enzyme in plants. Three protein spots (spots 3, 6, 27) were identified as partial degradation products of the Rubisco large unit (Table 1). These 3 spots appeared to have more abundance in SSDL10 (Fig. 1B), indicating higher expression of Rubisco in SSDL10 or more liability to breakdown in the SSDL10 sample preparations. Intriguingly, one protein (spot 25) was identified as the Rubisco activase (Fig. 1C and Table 1), which has a lower expression in SSDL10. Rubisco activase (RCA) is a nuclear-encoded, cytosol-synthesized chloroplast protein that regulates the activity of Rubisco by promoting the ATP-dependent removal of inhibitory sugar phosphate from Rubisco active sites (Portis, 1995). In most species studied, RCA is found in two forms, the longer a (43–47 kDa) form and the shorter b (41–42 kDa) form, both of which are

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

7

Fig. 2. Functional classification of the identified 17 differentially expressed chloroplast proteins between the SSDL10 and the parents lines of J411, XY54.

Fig. 3. Redox change of P700+ in SSDL10 and parent lines of J411, XY54. (A) Kinetics of P700+ in the dark. P700 was oxidized by far-red (FR) light for 40 s and after termination of FR illumination, P700+ re-reduction was monitored in the dark. Black line: SSDL10; gray line: J411; narrow gray line: XY54. (B) Initial P700+ re-reduction rate (0–0.2 s) after the FR illumination of SSDL10 and parents lines J411, XY54. Values are means of 5 replicates, error bar indicates SE. Statistically significant values relative to the parent samples are indicated by asterisk (*p < 0.05).

capable of Rubisco activation (Ristic et al., 2009). In this study the b form is identified (Table 1). Another two identified Calvin cycle proteins (spot 14, 23), phosphoribulokinase and phosphoglycerate kinase, were also found down-regulated in SSDL10 (Fig. 1C and Table 1). Phosphoribulokinase is believed to catalyze the phosphorylation of ribulose-5-phosphate with one molecule ATP, producing the ribulose 1, 5-bisphosphate, which is the substrate of Rubisco. The enzyme phosphoglycerate kinase catalyze the phosphorylation of phosphoglycerate (3PGA) by ATP, with the 1, 3bisphosphoglycerate as the product. Now, it is not clear why these two enzymes are down-regulated in SSDL10. Redox homeostasis proteins Three proteins involved in redox homeostasis were identified, 2 (spot 11, 21) of which were the same protein, 2-cysteine peroxiredoxin, might be caused by post-translational modification. They had the enhanced abundance in SSDL10 (Table 1). The chloroplast has a highly variable reduction potential and a very active metabolism, depending on environmental parameters such as light, temperature, and acceptor availability. Reactive oxygen species (ROS) production could damage macromolecules and membranes (Asada, 1999). 2-cysteine peroxiredoxins (2Cys Prx), one of the subgroup of the peroxiredoxins, belong to the enzymic antioxidants, which play a photo-protective role in

leaves by detoxification of peroxide in the photosynthetic electron flux (Konig, 2002). The 2-Cys Prx are ubiquitous enzymes that reduce a broad range of peroxides by intermolecular thiodisulfide transition (Chae et al., 1994). In addition to reduction of H2 O2 , Prx proteins also detoxify alkyl hydroperoxide and peroxinitrite, despite the fact that significant differences exist in substrate specificity and kinetic properties (Dietz, 2006). Partial suppression of 2-Cys Prx expression caused impairment of photosynthesis and increased oxidative damage of chloroplast proteins during early plant development (Dietz et al., 2002). Over-expression of chloroplast 2-Cys Prx protected yeast cells from reactive nitrogen species (Sakamoto et al., 2003). On the other hand, spot 8 was identified as m-type thioredoxin (TRX m), which showed expression of down-regulation (Fig. 1C and Table 1). Thioredoxins (TRXs) are small oxidoreductases of ca. 12 kDa found in all free living organisms. In plants, two chloroplastic TRXs, named TRX f and TRX m, were originally identified as light dependent regulators of several carbon metabolism enzymes including Calvin cycle enzymes (Lemaire et al., 2007). Earlier studies also suggested more tolerance to photo-inhibition in a wheat hybrid (Yang et al., 2006). The combination of these results, it could be concluded that the overall anti-oxidative defense metabolism in the hybrid exhibit higher capacity than the parent lines, especially in the later reproductive stages, when plants are often exposed to high light conditions.

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

8

Physiological significance of the differentially expressed protein spots

Fig. 4. NADPH-NBT oxidoreductase activity analysis of the thylakoid membrane proteins from SSDL10 and the parent lines J411, XY54. (A) Thylakoid membrane proteins from SSDL10 and parents were subjected to Native-PAGE, followed by the NADPH-NBT activity staining. An 6-␮g portion of chlorophyll was loaded in each well. Arrows indicate the activity band. Molecular mass standards were shown in the left side of the gel. (B) Thylakoid membrane proteins of SSDL10 were separated with Native-PAGE, run on a 2D SDS-PAGE, and thereafter protein blotting with the FNR and NdhH antibody was performed.

CET has been suggested to be required for the efficiency photosynthesis (Munekage et al., 2002). In the early time, it has been suggested that the CET plays a role in providing ATP for carbon assimilation (Schurman et al., 1972; Slovacek and Hind, 1981). Actually, the ATP supply could not always satisfy the demand of the plant during the later grain filling stage (Shen, 1994). Thus, CET would be enhanced to generate the sufficient proton gradient across the thylakoid membrane in this demand. Thus, the higher activity of CET (Fig. 3), combined with the thylakoid membrane complex containing FNR or NDH complex (Fig. 4) in SSDL10, would produce more proton gradient across the thylakoid membrane. Consequently, it could supply sufficient ATP for regulation of photosynthesis, so that it showed superior photosynthesis capacity, especially in the grain-filling stage (Cheng et al., 2009). In addition, the grain numbers per panicle of SSDL10 was more than its parents by about 30–50%, although its height was lower than its parents by about 15–20% and the panicles per plant was lower by about 9%. It is generally accepted that CET is indispensable for the higher plants, especially during a stressful environment, when CO2 assimilation is limited. In tobacco (Nicotiana tabacum), NDH complex has been suggested to supply extra pH for optimal photosynthesis under stressed conditions when CO2 assimilation is limited (Shikanai, 2007; Wang et al., 2006). The higher activity of CET in SSDL 10 (Fig. 3) implies it has stronger tolerance to stressed conditions. In conclusion, the comparative analysis of the chloroplast protein pattern from a wheat single seed descent line SSDL10 and its parents allowed us to identify a set of differentiated proteins among them. The elevated amount of proteins involved in CET, the higher activity of this alternative electron transport pathway, and higher anti-oxidative metabolism system capacity in SSDL10 would function in its efficient photosynthesis and photo-inhibition resistance, especially during the physiological processes of the later reproductive stages. Our results give new insight to the molecular basis of the higher photo-phosphorylation activity and photo-inhibition resistance of the SSDL10. Acknowledgements

Proteins related to metabolism and defense Another two protein spots (spot 13, 17) were identified as plastid glutamine synthase (GS) and harpin binding protein 1, both of which were down-regulated in SSDL10 compared with the parent lines (Table 1). Glutamine synthase (GS) functions as the major assimilatory enzyme for ammonium produced from N fixation, and nitrate or ammonia nutrition. GS is distributed in different sub-cellular locations (chloroplast and cytoplasm) and in different tissues and organs. The plastidic form of GS (GS2) is widely distributed in the chloroplast, the major role of which is thought to be re-assimilating the NH3 generated in photorespiration (Miflin and Habash, 2002). The down-regulation of the GS2 in SSDL10 might be associated with the less NH3 generation from the photorespiration, although this hypothesis needs further experimental evidence. Spot 17, identified as harpin binding protein 1, was downregulated as well in SSDL10. Two proteomics studies about the rice leaves stress-responsive proteins also identified the harpin binding protein 1 (Ma et al., 2011; Wan and Liu, 2008). The harpin protein group, first found in Erwinia amylovora, may elicit multiple plant responses (Wei et al., 1992). Harpin binding protein 1 is believed to play important roles in plant disease and drought resistance (Zhang et al., 2011). Nevertheless, the reason why the harpin binding protein 1 was down-regulated in SSDL10 is unknown.

This work was supported by funds from the State Key Basic Research and Development Plant 973 [2009CB118504] and the National Natural Science Foundation of China [30870183, 31070215]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jplph.2013.03.016. References Arakaki A, Ceccarelli E, Carrillo N. Plant-type ferredoxin-NADP+ reductases: a basal structural framework and a multiplicity of functions. FASEB J 1997;11:133–40. Asada K. The water–water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annu Rev Plant Biol 1999;50:601–39. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 1976;72:248–54. Burrows PA, Sazanov LA, Svab Z, Maliga P, Nixon PJ. Identification of a functional respiratory complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J 1998;17:868–76. Chae HZ, Chung SJ, Rhee SG. Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem 1994;269:27670–8.

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016

G Model JPLPH-51713; No. of Pages 9

ARTICLE IN PRESS Z.-H. He et al. / Journal of Plant Physiology xxx (2013) xxx–xxx

Cheng J, Ma W, Chen G, Hu M, Shen Y, Li Z, et al. Dynamic changes of photosynthetic characteristics in Xiaoyan 54, Jing 411, and the stable selected superior strains of their hybrid progenies. Acta Agric Sin 2009;35:1051–8. Dalcorso G, Pesaresi P, Masiero S, Aseeva E, Schunemann D, Finazzi G, et al. A complex containing PGRL1 and PGR5 is involved in the switch between linear and cyclic electron flow in Arabidopsis. Cell 2008;132:273–85. Dietz KJ. The function of peroxiredoxins in plant organelle redox metabolism. J Exp Bot 2006;57:1697–709. Dietz KJ, Horling F, König J, Baier M. The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation. J Exp Bot 2002;53:1321–9. Guedeney G, Corneille S, Cuiné S, Peltier G. Evidence for an association of ndh B, ndh J gene products and ferredoxin-NADP+ reductase as components of a chloroplastic NAD(P)H dehydrogenase complex. FEBS Lett 1996;378:277–80. Heber U, Santarius K. Direct and indirect transfer of ATP and ADP across the chloroplast envelope. Z Naturforsch B 1970;25:718–28. Hu M, Wang Y, Zhang L, Wang C, Shen Y, Li Z, et al. Photosynthetic characteristics of different wheat cultivars and their offspring of hybridization. Acta Agric Sin 2007;33:1879–83. Joliot P, Johnson GN. Regulation of cyclic and linear electron flow in higher plants. Proc Natl Acad Sci USA 2011;108:13317–22. Klughammer C, Schreiber U. Measuring P700 absorbance changes in the near infrared spectral region with a dual wavelength pulse modulation system. In: Grab G., editor. Photosynthesis: mechanism and effects, 5. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1998. p. 4357–60. Konig J. The plant-specific function of 2-Cys peroxiredoxin-mediated detoxification of peroxides in the redox-hierarchy of photosynthetic electron flux. Proc Natl Acad Sci USA 2002;99:5738–43. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–5. Lemaire S, Michelet L, Zaffagnini M, Massot V, Issakidis-Bourguet E. Thioredoxins in chloroplasts. Curr Genet 2007;51:343–65. Ma W, Deng Y, Ogawa T, Mi H. Active NDH-1 complexes from the cyanobacterium Synechocystis sp. strain PCC 680. Plant Cell Physiol 2006;47:1432–6. Ma W, Song C, Zeng F, Feibo W, Zhang G. Proteomic analysis of nitrogen stressresponsive proteins in two rice cultivars differing in N utilization efficiency. J Integr OMICS 2011;1:78–87. Maiwald D. Knock-out of the genes coding for the Rieske protein and the ATP-synthase (subunit of Arabidopsis. Effects on photosynthesis, thylakoid protein composition, and nuclear chloroplast gene expression. Plant Physiol 2003;133:191–202. Mi H, Endo T, Ogawa T, Asada K. Thylakoid membrane-bound, NADPH-specific pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol 1995;36:661–8. Mi H, Endo T, Schreiber U, Asada K. Donation of electrons from cytosolic components to the intersystem chain in the cyanobacterium synechococcus sp. PCC 7002 as determined by the reduction of P700+ . Plant Cell Physiol 1992;33:1099–105. Miflin BJ, Habash DZ. The role of glutamine synthetase and glutamate dehydrogenase in nitrogen assimilation and possibilities for improvement in the nitrogen utilization of crops. J Exp Bot 2002;53:979–87. Munekage Y, Hojo M, Meurer J, Endo T, Tasaka M, Shikanai T. PGR5 is involved in cyclic electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell 2002;110:361–71. Ni Z-L, Shi X-B, Wei J-M. Functional consequences of N- or C-terminal deletions of the (subunit of chloroplast ATP synthase. Biochemistry 2004;43:2272–8. Porra R, Thompson W, Kriedemann P. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectrometry. Biochim Biophys Acta 1989;975:384–94.

9

Portis AR. The regulation of Rubisco by Rubisco activase. J Exp Bot 1995;46:1285–91. Quiles MJ, Albacete ME, Sabater B, Cuello J. Isolation and partial characterization of the NADH dehydrogenase complex from barley chloroplast thylakoids. Plant Cell Physiol 1996;37:1134–42. Ristic Z, Momcilovic I, Bukovnik U, Prasad PVV, Fu J, DeRidder BP, et al. Rubisco activase and wheat productivity under heat-stress conditions. J Exp Bot 2009;60:4003–14. Rumeau D, Peltier G, Cournac L. Chlororespiration and cyclic electron flow around PSI during photosynthesis and plant stress response. Plant Cell Environ 2007;30:1041–51. Sakamoto A, Tsukamoto S, Yamamoto H, Ueda-Hashimoto M, Takahashi M, Suzuki H, et al. Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species. Plant J 2003;33:841–51. Schurman P, Buchanan BB, Arnon DI. Role of cyclic photophosphorylation in photosynthetic carbon-dioxide assimilation by isolated chloroplasts. Biochim Biophys Acta 1972;267:111–24. Shen Y. Dynamic approaches to the mechanism of photosynthesis. Photosynth Res 1994;39:1–13. Shikanai T. Cyclic electron transport around photosystem I: genetic approaches. Annu Rev Plant Biol 2007;58:199–217. Shikanai T, Endo T, Hashimoto T, Yamada Y, Asada K, Yokota A. Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I. Proc Natl Acad Sci USA 1998;95:9705–9. Slovacek RE, Hind G. Correlation between photosynthesis and the transthylakoid proton gradient. Biochim Biophys Acta 1981;635:393–404. Slowik D, Rossmann M, Konarev PV, Irrgang K-D, Saenger W. Structural investigation of PsbO from plant and cyanobacterial photosystem II. J Mol Biol 2011;407:125–37. Wan X-Y, Liu J-Y. Comparative proteomics analysis reveals an intimate protein network provoked by hydrogen peroxide stress in rice seedling leaves. Mol Cell Proteomic 2008;7:1469–88. Wang P, Duan W, Takabayashi A, Endo T, Shikanai T, Ye JY, et al. Chloroplastic NAD(P)H dehydrogenase in tobacco leaves functions in alleviation of oxidative damage caused by temperature stress. Plant Physiol 2006;141:465–74. Wei J, Xu M, Zhang D, Mi H. The role of carotenoid isomerase in maintenance of photosynthetic oxygen evolution in rice plant. Acta Biochim Biophys Sin 2010;42:457–63. Wei Z, Laby R, Zumoff C, Bauer D, He S, Collmer A, et al. Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 1992;257:85–8. Yamori W, Takahashi S, Makino A, Price GD, Badger MR, von Caemmerer S. The roles of ATP synthase and the cytochrome b6 /f complexes in limiting chloroplast electron transport and determining photosynthetic capacity. Plant Physiol 2010;155:956–62. Yang X, Chen X, Ge Q, Li B, Tong Y, Zhang A, et al. Tolerance of photosynthesis to photoinhibition, high temperature and drought stress in flag leaves of wheat: a comparison between a hybridization line and its parents grown under field conditions. Plant Sci 2006;171:389–97. Zhang H, Whitelegge JP, Cramer WA. Ferredoxin:NADP+ oxidoreductase is a subunit of the chloroplast cytochrome b6 f complex. J Biol Chem 2001;276: 38159–65. Zhang L, Xiao S, Li W, Feng W, Li J, Wu Z, et al. Overexpression of a harpinencoding gene hrf1 in rice enhances drought tolerance. J Exp Bot 2011;62: 4229–38. Zhang M, Li G, Huang W, Bi T, Chen G, Tang Z, et al. Proteomic study of Carissa spinarum in response to combined heat and drought stress. Proteomics 2010;10:3117–29.

Please cite this article in press as: He Z-H, et al. Comparative analysis of the chloroplast proteomes of a wheat (Triticum aestivum L.) single seed descent line and its parents. J Plant Physiol (2013), http://dx.doi.org/10.1016/j.jplph.2013.03.016