Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

Journal of Integrative Agriculture 2014, 13(12): 2616-2624 December 2014 RESEARCH ARTICLE Effect of Nitric Oxide on the Interaction Between Mitocho...

777KB Sizes 1 Downloads 48 Views

Journal of Integrative Agriculture 2014, 13(12): 2616-2624

December 2014

RESEARCH ARTICLE

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase LIU Yu-chen1, WANG Juan2, SU Pei-ying3, MA Chun-mei1 and ZHU Shu-hua1 1

College of Chemistry and Material Science, Shandong Agricultural University, Tai’an 271018, P.R.China Department of Horticultural and Forest Engineer, Heze University, Heze 274015, P.R.China 3 Tai’an Tumor Prevention and Treatment Hospital, Tai’an 271000, P.R.China 2

Abstract Mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) are sequential enzymes in Krebs cycle. mMDH, CS and the complex between mMDH and CS (mMDH+CS) were treated with nitric oxide solution. The roles of notric oxide (NO) on the secondary structures and the interactions between mMDH and CS were studied using circular diehroism (CD) and Fourier transform surface plasmon resonance (FT-SPR), respectivley. The effects of NO on the activities of mMDH, CS and mMDH+CS were also studied. And the regulations by NO on mMDH and CS were simulated by PyMOL software. The results of SPR confirmed that strong interaction between mMDH and CS existed and NO could significantly regulate the interaction between the two enzymes. NO reduced the mass percents of α-helix and increased that of random in mMDH, CS and mMDH+CS. NO increased the activities of CS and mMDH+CS, and inhibited the activity of mMDH. Graphic simulation indicated that covalent bond was formed between NO and Asn242 in active site of CS. However, there was no direct bond between NO and mMDH. The increase in activity of mMDH+CS complex depended mostly on the interaction between NO and CS. All the results suggested that the regulations by NO on the activity and interaction between mMDH and CS were accord with the changes in mMDH, CS and mMDH+CS caused by NO. Key words: Krebs cycle, nitric oxide, surface plasmon resonance, protein-protein interaction, citrate synthase, malate dehydrogenase

INTRODUCTION The tricarboxylic acid (TCA) cycle, also known as the Krebs cycle or the citric acid cycle, is at the center of cellular metabolism, playing a starring role in both the process of energy production and biosynthesis. At present, the TCA cycle is considered as not only a circle but also flux modes playing important roles in physiological processes in animals, plants and bacteria (Sweetlove et al. 2010; Meeks 2011; Zhang and Bryant 2011; McCarthy 2013; Nunes-Nesi et al. 2013). The intermediates

of the TCA cycle also involve in regulating physiological and biochemical processes, and supplement of the TCA cycle intermediates protects against cell death induced by high glucose/palmitate (Choi et al. 2011; Peti-Peterdi 2013). Those results suggest that the protection depends on the flux of the intermediates of the TCA cycle. Activities of the enzymes in the TCA cycle contribute to the flux of the intermediates. Malate dehydrogenase (MDH) and citrate synthase (CS) are two sequential enzymes in the TCA cycle. MDH catalyzes malic acid to oxaloacetic acid which is converted to citric acid by CS. In most ripe fruits, malic and citric acids are the

Received 4 November, 2013 Accepted 20 February, 2014 LIU Yu-chen, E-mail: [email protected]; Correspondence ZHU Shu-hua, Tel: +86-538-8247790, E-mail: [email protected] © 2014, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(13)60736-4

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

main organic acids contributed to fleshy fruit acidity which is an important component of fruit organoleptic quality, and affects postharvest softening (Centeno et al. 2011; Etienne et al. 2013). In recent years, mitochondrial malate dehydrogenase (mMDH) and CS are increasingly valued for their important roles in the plant TCA cycle (Sweetlove et al. 2010; Nunes-Nesi et al. 2013). Recently, complexes between several TCA cycle enzymes were identified in vivo interaction analyses in Bacillus subtilis (Meyer et al. 2011). New evidences confirm that mMDH lowers leaf respiration and alters photorespiration in Arabidopsis (Tomaz et al. 2010). The overexpression of mMDH gene can improve phosphorus acquisition by tobacco (Lü et al. 2012). Antisense inhibition of mMDH can not only enhance photosynthetic activity and the rate of carbon dioxide assimilation (Nunes-Nesi et al. 2005), but also alter root growth and architecture in tomato plant (van der Merwe et al. 2009). The enhanced photosynthetic performance and growth in transgenic tomato plants are also considered to be a consequence of decreasing mMDH activity (Nunes-Nesi et al. 2005). Overexpression of CS can improve plant growth under nutritional stress, such as aluminum and phosphorus tolerance (Koyama et al. 1999, 2000; Deng et al. 2009), and antisense repression of CS can inhibit the flower formation in transgenic potato plants (Landschutze et al. 1995). Structure and expression of CS from higher plants have been studied widely (La Cognata et al. 1996). Recently, the function of a citrate synthase gene (MaGCS), which is constitutively expressed in all organs with high levels in the fruit, during postharvest banana fruit ripening, is reported (Liu et al. 2013). The expression of MaGCS can be induced by ethylene and inhibited by the ethylene receptor inhibitor, improved by oxaloacetic acid and suppressed by citric acid, suggesting that MaGCS is associated with ethylene biosynthesis and plays an important role in postharvest banana fruit ripening (Liu et al. 2013). mMDH and CS of strawberry fruit are purified and the genes are cloned and identified (Iannetta et al. 2004). mMDH cDNA clones are also isolated from grape berries and the expression pattern are analysed throughout berry development (Or et al. 2000). Isolation and functional characterization of genes encoding citrate synthase are also studied in Malus (Han et al. 2012), pear fruit (Lu et al. 2011). As two sequential enzymes in the TCA cycle, the interaction between mMDH and CS

2617

has aroused the interests of the researchers. The interactions between mMDH and CS have been studied widely in animals, plants and microbes (Tompa et al. 1987; Morgunov and Srere 1998; Pettersson et al. 2000; Iannetta et al. 2004; Chow et al. 2005). Recently, it is found that bioconjugates formed by adding CS to the Au nanoparticles before MDH addition exhibits higher specific activities for both enzymes than those formed by adding the enzymes in the reverse order. These bioconjugates also have 3-fold higher per-particle sequential activity for conversion of malate to citrate (Keighron and Keating 2010). Those results suggest that exogenous treatments can affect the activities and interaction between mMDH and CS. As a small biomolecular, nitric oxide (NO) is considered to be a potent inhibitor of the mitochondrial electron transport chain (Brown and Borutaite 2002; Wang et al. 2010; Sarti et al. 2012), regulates the ripening processes of fruits (Manjunatha et al. 2010; Zhu et al. 2010), and affects fruit quality during storage (Duan et al. 2007; Sun et al. 2011). NO can regulate energy metabolism via the TCA cycle (Dai et al. 2013). Exogenous NO can decrease leaf citrate content and increase root citrate content of citrus, and inhibit CS activity in citrus leaves (Yang et al. 2012). Our previous research also found that NO makes a dramatic promotion of mMDH activity and slight increase in CS activity in peach fruit during storage (Ma et al. 2011). However, little works have been done to study the roles of NO in the interaction between mMDH and CS. In this paper, the effects of NO on the secondary structures and interaction between mMDH and CS were studied to explore the possible mechanism by which NO regulates the activities of mMDH and CS.

RESULTS Effects of NO on interactions between mMDH and CS Fig. 1 depicted the SPR response of the interactions between mMDH and CS in 10% PEG. The SPR wavenumber shift after step V means the change of the film’s thickness, which can reflect the protein-protein interactions. It could be found that there was a shift in the SPR resonance after NO treatment. The shift in © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

LIU Yu-chen et al.

2618

the control, suggesting that the inhibitory effect by NO on mMDH activity was slight. In contrast, the activity of CS treated with 80 mmol L-1 NO was 3.4 times higher than that of the control (Fig. 2-B). The activity of mMDH-CS complexes treated with NO was 1.25 times as high as that of the control (Fig. 2-C).

the SPR response upon the addition of the mMDH was attributed to the association of the amino group to the activated carboxylic acid in the gold chip (Fig. 1-A). Fig. 1-B depicted the response corresponding to the reflectance changes of the SPR sensor associated with mMDH upon addition of CS. The wavenumber shift of NO treatment was larger than the control in the step of combination of mMDH and CS (Fig. 1-B). The results demonstrated that the complex of CS and mMDH was obtained by the precipitation in PEG, and NO treatment could increase the interaction between CS and mMDH.

Effects of NO on secondary structures of mMDH and CS NO could significantly reduce the mass percent of α-helix and increase the mass percent of random of mMDH, CS and mMDH+CS (Table 1). However, no significant effects of NO on mass percent of β-sheet were found in mMDH, CS and mMDH+CS. There was no significant difference in β-turn of mMDH treated with/without NO. NO could decrease the mass percent of β-turn of CS and

Activities of mMDH, CS and mMDH-CS complex The result in Fig. 2-A showed that mMDH activity in the treatment with 80 mmol L-1 NO was 93.1% that of

SPR wavenumber shift (cm-1)

SPR wavenumber shift (cm-1)

8 900

9 000

A

8 950 8 900 8 850

1

8 800

2

8 750 8 700 8 650

0

B

8 880

1

8 860

2

8 840 8 820 8 800

160

10 20 30 40 50 60 70 80 90 100 110 Time (min)

180

200 Time (min)

220

9 000

SPR wavenumber shift (cm-1)

8 500

8 000

7 500

2 1

7 000

0

50

150

100

200

250

Time (min)

Fig. 1 Effect of nitric oxide (NO) on the interaction of mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS). A, the surface plasmon resonance (SPR) response of mMDH combined to the gold chip. B, the SPR response of CS coupled with mMDH. Line 1, the SPR response of the interactions between mMDH and CS; Line 2, the SPR response of the interactions between mMDH and CS treated with NO.

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

A

B

a a

100

C

b

350

b

120 a

300 100

80

60

40

mMDH+CS activity

CS activity

250 mMDH activity

2619

200 150 a 100

20

0 CK

CK

NO

60 40 20

50

0

80

0

NO

CK

NO

Fig. 2 Effects of NO on activities of mMDH, CS and mMDH-CS complex. Values (means±SD, number of replications=3) with different letters are significantly different at 0.05% (LSD).

Table 1 Effect of nitric oxide (NO) on the second structure of mitochondrial malate dehydrogenase (mMDH), citrate synthase (CS) and mMDH+CS Mass percent (%) α-Helix β-Sheet β-Turn Random

mMDH without NO 25.6±1.5 a 20.5±1.1 a 15.5±1.3 a 38.4±1.3 a

CS NO 21.0±1.1 b 19.9±0.8 a 15.3±1.6 a 43.8±0.6 b

without NO 25.7±0.9 a 22.3±1.3 a 25.8±0.8 a 26.2±1.2 a

NO 20.7±2.1 b 22.2±1.2 a 23.6±0.6 b 33.5±1.1 b

mMDH+CS without NO 23.6±1.2 a 13.5±0.9 a 25.7±1.0 a 37.2±1.5 a

NO 19.9±0.7 b 12.4±1.0 a 22.0±1.2 b 45.7±1.0 b

Values (means±SD, number of replications=3) with different letters are significantly different at 0.05% (LSD) for the same emzyme with different treatments in the same line.

mMDH+CS. After NO treatment, the negative-peak amplitudes of mMDH at 208 and 222 nm were decreased significantly, and the peak of mMDH became small and shifted to lower wave numbers (Fig. 3-A). The negative peak at 222 nm in circular diehroism (CD) spectra of CS was big and shifted to lower wave numbers with amplitude decreased (Fig. 3-B). The negative peak at 208 nm in CD spectra of mMDH+CS treated wtih NO shifted slightly to higher wave numbers and the amplitude increased (Fig. 3-C). On the contrary, the negative peak at 222 nm in CD spectra of mMDH+CS treated wtih NO shifted slightly to lower wave numbers and the amplitude decreased. No significant effect of NO on mMDH activity was found, while NO significantly changed the activities of CS and mMDH+CS (Fig. 3). The difference would lie in the different mass percents of β-turn in mMDH and CS (Table 1).

Graphic simulation NO was found to bind to Asn242 by covalent bond in

the active site of CS (Fig. 4-A), suggesting there was strong interaction force between NO and CS. However, the molecular modeling result showed that no strong and direct interaction force existed between NO and active site of mMDH.

DISCUSSION It has been demonstrated that the complexes of CS and mMDH obtained by the precipitation in PEG could remain a solid state for at least 2 min (Morgunov and Srere 1998). This allowed a study of the kinetics of these complexes. SPR results confirmed that the interaction between mMDH and CS was particularly strong, and NO also strongly affected the interaction between mMDH and CS. NO significantly changed the mass percents of the secondary structures of CS and mMDH+CS, but slightly changed that of mMDH, which coincided with the changes in the activities of mMDH, CS and mMDH+CS. The results of molecular modeling also confirmed that NO could affect CS more significantly than mMDH. © 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

LIU Yu-chen et al.

2620

A

2 0

CD (mdeg)

-2 -4

NO

-6 CK

-8 -10 190

200

210

220

230

240

250

260

240

250

260

240

250

260

Wavelength (nm)

B

2 0

CD (mdeg)

-2 -4 NO

-6 -8

CK

-10 -12 190

200

210

220

230

Wavelength (nm)

C 0

CD (mdeg)

-2 NO

-4

-6 CK -8 190

200

210

220

230

Wavelength (nm)

Fig. 3 Circular dichroism (CD) spectra of mMDH, CS, mMDH+CS before and after NO treatment. A, circular dichroism spectra of mMDH. B, circular dichroism spectra of CS. C, circular dichroism spectra of mMDH+CS.

NO could significantly increase the activities of CS and mMDH+CS, but slightly inhibit the activity of mMDH (Fig. 2). mMDH and CS are sequential enzymes in the Krebs cycle. The carboxyl terminal of subunits of CS and the amino terminal of subunits of mMDH could be brought together, which enables CS and mMDH be

docked together with a short linker region (Lindbladh et al. 1994). It has been shown that positive electrostatic potential exists between the active sites of the fusion proteins of mMDH and CS (Lindbladh et al. 1994; Elcock and McCammon 1996). These electrostatic forces could account for the channeling of OAA between the active sites of mMDH and CS. Thus, OAA, the product of mMDH might directly serve as the substrate for CS. The activity of the mMDH+CS complex was increased by NO, suggesting that NO could promote the interaction between mMDH and CS. Native conformation and special functional domain are important for enzyme activity, and realted to the contents of α-helix and random and the ratio of α/β (Karplus and Kuriyan 2005; Buxbaum 2011). The decrease in α-helix indicates the damages in hydrogen bond and other structures of enzyme and suggests that the peptide chains become loose. Random may cover the active site or the combination between binding sites and substrates, affect the binds between enzyme and substrates or inhibitors to change the activity of enzyme (Buxbaum 2011). The results of SPR indicated that NO could significantly affect the mass percents of the secondary structures in mMDH and CS, causing the changes in conformations of mMDH, CS and mMDH+CS, which was in accord with the changes in the activities of mMDH, CS and mMDH+CS. The catalytic site is highly conserved in CS, and contains 3 key residues: Asn242, His320 and Asp237 (Larson et al. 2009; Siriwardena et al. 2013). His238, Arg329 and Arg401 also involved in catalyzing oxaloacetic acid by CS (Daidone et al. 2004). The covalent bond between NO and Asn242 in active site of CS was strong interaction force, suggesting that NO could change significantly the native conformation of CS, which leading to the changes in CS acitivity. Asp and His are key residues in catalytic site of mMDH (Minarik et al. 2002; Wang et al. 2009). There was no significant interaction between NO and mMDH in molecular modeling (Fig. 4). Thus, the increase in mMDH+CS activity treated with NO depended on the regulations by NO on CS activity more than mMDH. However, mMDH activity was still inhibited by NO. Those results suggests there should be other pathways, such as S-nitrosylation (Gould et al. 2013), by which NO played its inhibitory effects on mMDH activity. However, further works should be done

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

A

2621

B

Fig. 4 Graphic simulations for the effect of NO on mMDH and CS. Only the active site of each enzyme is shown on this picture. A, the interaction between NO and mMDH. B, the interaction between NO and CS.

to deeply and thoroughly study the roles of NO in the interaction between mMDH and CS and the regulation by NO on Krebs cycle.

CONCLUSION NO could regulate the mass percents of the secondary structures in mMDH, CS and mMDH+CS, and increase the activities of CS and mMDH+CS by covalent bond between NO and Asn242 in active site of CS. The increase in activity of mMDH+CS complex depended mostly on the interaction between NO and CS.

MATERIALS AND METHODS Materials All chemicals were of analysis grade unless stated otherwise. Citrate synthase (CS, EC 2.3.3.1), mitochondrial malate dehydrogenase (mMDH, EC 1.1.1.37) (product no. M 2634), L-malic acid, oxaloacetic acid (OAA), acetyl coenzyme A trilithium salt, β-nicotinamide adenine dinucleotide hydrate (NAD+), β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), sodium bicarbonate, and (5,5´-dithiobis(2nitrobenzoic acid)) (DTNB) were purchased from SigmaAldrich Co. Ltd (Shanghai, China). 11-mercaptoundecanoic acid (COOH-thiol, 95%), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine were purchased from Aladdin Reagent Inc. (Shanghai, China). The gold-sputtered slide glass (18 mm×18 mm) used as a sensor chip was purchased from Thermo Electron Corp (USA). All solutions were filtered using Corning cellulose acetate membranes with 0.45 μm pores. Double-distilled water was used in all experiments.

Detection of the interaction between mMDH and CS The interaction between mMDH and CS and the effects of NO on the interaction were detected by Fourier transform surface plasmon resonance (FT-SPR). The FT-SPR measurements were performed with an SPR-100 module from Thermo equipped 107 with a flow sample cell mounted on a goniometer. It was inserted in a Thermo Nexus FT-IR 108 spectrometer using a near-IR tungsten-halogen light source. The incidence angle was adjusted to have minimal reflectivity located at 9 000 cm-1 at the beginning of each experiment so as to be in the best sensitivity region of the Indium Gallium Arsenide (InGaAs) detector. A peristaltic pump was used to pump the analyte or wash solution from a reservoir into the flow cell. PBS buffer solution (100 mmol L-1, pH 8.0) was used as a running buffer, and the flow rate was fixed at 0.8 mL min-1. The stable wavenumber shifts were recorded in real time using the SPR. In all experiments, all solutions were ultrasonicated. Before each binding measurement for the sample solutions, the SPR sensor chips were washed with PBS buffer in turn until a stable base line was obtained. Immobilization of proteins onto gold surface was carried out using a literature protocol (Madeira et al. 2011). The gold-sputtered slide glass, as a sensor chip, was dipped in 10 mL of freshly prepared piranha solution (70% H2SO4, 3% H2O2) for 2 min. After being rinsed with copious distilled water, the gold-sputtered slide glass was placed into 20 mL of 10 mmol L-1 11-mercaptoundecanoic acid in ethanol (Fig. 5 step I), and kept at 4ºC overnight. The carboxyl groups on the SPR sensor were activated by placing the chip into 10 mL PBS buffer containing 100 mmol L-1 EDC and 25 mmol L-1 NHS for 30 min (Fig. 5 step II). The chip was rinsed with ethanol and then double distilled water and finally dried by N2. Before being mounted into the SPR instrument, the chip was washed with PBS (100 mmol L-1, pH 8.0) at a flow rate of 0.8 mL min-1 until a stable base line was obtained. mMDH was diluted in immobilization buffer (100 μg mL-1). The intermediate product of the reaction between the carboxylic acid and the EDC was very labile and could be hydrolyzed

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

LIU Yu-chen et al.

2622

to oxaloacetate for mMDH in single and multienzyme bioconjugates, 100 mmol L-1 Tris-HCl (pH 8.1), 25 μL of 36 mmol L-1 malate and 25 μL of 60 mmol L-1 NAD+, for a final concentration of 0.3 mmol L-1 malate and 0.1 mmol L-1 NAD+, were mixed in a cuvette and equilibrated to room temperature (25ºC). The absorbance of NADH was monitored at 340 nm for 5 min, in accordance with previous reports (Keighron and Keating 2010). The activity of CS for conversion of oxaloacetate to citrate was assayed in similar fashion to mMDH with the exception that 25 μL of 12 mmol L-1 acetyl-CoA and 25 μL of 60 mmol L-1 OAA were used as substrates with 25 μL of 18 mmol L-1 DTNB, for a final concentration of 0.5 mmol L-1 OAA, 0.1 mmol L-1 acetyl-CoA, and 0.15 mmol L-1 DTNB. DTNB was added to monitor the production of coenzyme A by CS by adsorption at 412 nm. The overall reaction of malate to citrate catalyzed by bioconjugate as a PEG mMDH-CS precipitate, was monitored in 10 mmol L-1 malate, 4 mmol L-1 NAD+, and 0.1 mmol L-1 acetyl-CoA using 0.4 mmol L-1 DTNB at 412 nm in 100 mmol L-1 Tris-HCl (pH 8.1) buffer. Complexes of enzymes (2 g L-1 of solution) were incubated in 30% PEG at room temperature for 2 min in advance. Each assay was repeated at least three times.

O I

...

S

C OH Activation

EDC/NHS O S

II

C O

EDC H H

N

MDH

O III

S

...

C

N

MDH

H Ethanolamine (X)

O S

C O O

IV S

C

Detection for the secondary structures of mMDH and CS

X MDH

N H

O CS

C OH

O V

S

...

C

N

MDH

NH 4 ...... OOC

CS

H Protein-protin interaction

Fig. 5 The diagram for detection the intreactions between mMDH and CS by SPR.

quickly. The carboxylic acid could react with the ligand to yield a covalent amine bond. Thus, mMDH was immobilized (Fig. 5 step III). The immobilization consists in the formation of an amide bond between a primary amino group of the mMDH and the carboxylic acid groups of CS. And those in the middle indicate the interaction resulting from the fusion between the C-terminal end of CS and the N-terminal end of mMDH (Fig. 5 step V).

Enzyme activity assays To measure the rate of reaction for the conversion of malate

The changes in the secondary structures of mMDH and CS were detected on Jasco 810 circular dichroism (CD) spectropolarimeter (JASCO, Tokyo, Japan). mMDH and CS (0.1 g L-1) were dissolved in 10 mmol L-1 phosphate buffer (pH 8.0), respectively. The spectra were recorded using a 1-mm path length cell under constant nitrogen flush (∼30 L min-1) with a step size of 0.1 nm, bandwidth of 2 nm, and an average time of 3 s. The final spectrum reported was an average of five scans. For the NO denaturation experiments, mMDH and CS were incubated at final concentration of 80 µmol L-1 NO in phosphate-buffered saline (PBS) for 60 min, and the spectra were recorded from 180 to 260 nm at 25°C.

Molecular modeling The regulations by NO on mMDH and CS were modeled by PyMOL Molecular Graphics System (Schrodinger 2010). The structures of mMDH (PDB ID: 5MDH) and CS (PDB ID: 3ENJ) were download from RSCB Protein Data Bank (http://www.rcsb.org).

Statistical analysis The experiments were conducted in a completely randomized design. The data were expressed as means±SE, and processed

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

by analysis of variance (ANOVA), comparing treatments at a significance level of 0.05 according to the least significant difference (LSD) test.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (31270723, 31370686, 31470686) and the Science and Technology Development Planning of Shandong Province, China (2013CEX20109).

References

Brown G C, Borutaite V. 2002. Nitric oxide inhibition of mitochondrial respiration and its role in cell death. Free Radical Biology and Medicine, 33, 1440-1450 Buxbaum E. 2011. Protein secondary structure. In: Biophysical Chemistry of Proteins. Springer, US. pp. 291-302. Centeno D C, Osorio S, Nunes-Nesi A, Bertolo A L F, Carneiro R T, Araújo W L, Steinhauser M C, Michalska J, Rohrmann J, Geigenberger P, Olivera S N, Stitta M, Carrarid F, Roseb J, Ferniea A. 2011. Malate plays a crucial role in starch metabolism, ripening, and soluble solid content of tomato fruit and affects postharvest softening. The Plant Cell, 23, 162-184. Choi S E, Lee Y J, Hwang G S, Chung J H, Lee S J, Lee J H, Han S J, Kim H J, Lee K W, Kim Y. 2011. Supplement of TCA cycle intermediates protects against high glucose/ palmitate-induced INS-1 beta cell death. Archives of Biochemistry and Biophysics, 505, 231-241. Chow K M, Ma Z, Cai J, Pierce W M, Hersh L B. 2005. Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase. Biochimica et Biophysica Acta, 1723, 292-301. La Cognata U, Landschutze V, Willmitzer L, Muller-Rober B. 1996. Structure and expression of mitochondrial citrate synthases from higher plants. Plant Cell Physiology, 37, 1022-1029. Dai Z, Wu Z, Yang Y, Wang J, Satterfield M C, Meininger C J, Bazer F W, Wu G. 2013. Nitric oxide and energy metabolism in mammals. BioFactors, 39, 383-391. Daidone I, Roccatano D, Hayward S. 2004. Investigating the accessibility of the closed domain conformation of citrate synthase using essential dynamics sampling. Journal of Molecular Biology, 339, 515-525. Deng W, Luo K, Li Z, Yang Y, Hu N, Wu Y. 2009. Overexpression of Citrus junos mitochondrial citrate synthase gene in Nicotiana benthamiana confers aluminum tolerance. Planta, 230, 355-365. Duan X, Su X, You Y, Qu H, Li Y, Jiang Y. 2007. Effect of nitric oxide on pericarp browning of harvested longan fruit in relation to phenolic metabolism. Food Chemistry, 104, 571-576. Elcock A H, McCammon J A. 1996. Evidence for electrostatic channeling in a fusion protein of malate dehydrogenase and citrate synthase. Biochemistry, 35, 12652-12658. Etienne A, Genard M, Lobit P, Mbeguie A M D, Bugaud

2623

C. 2013. What controls fleshy fruit acidity? A review of malate and citrate accumulation in fruit cells. Journal of Experimental Botany, 64, 1451-1469. Gould N, Doulias P T, Tenopoulou M, Raju K, Ischiropoulos H. 2013. Regulation of protein function and signaling by reversible cysteine S-nitrosylation. Journal of Biological Chemistry, 288, 26473-26479. Han D G, Wang Y, Zhang L, Ma L, Zhang X Z, Xu X F, Han Z H. 2012. Isolation and functional characterization of MxCS1: a gene encoding a citrate synthase in Malus xiaojinensis. Biologia Plantarum, 56, 50-56. Iannetta P P M, Escobar N M, Ross H A, Souleyre E J F, Hancock R D, Witte C P, Davies H V. 2004. Identification, cloning and expression analysis of strawberry (Fragaria×ananassa) mitochondrial citrate synthase and mitochondrial malate dehydrogenase. Physiologia Plantarum, 121, 15-26. Karplus M, Kuriyan J. 2005. Molecular dynamics and protein function. Proceedings of the National Academy of Sciences of the United States of America, 102, 6679-6685. Keighron J D, Keating C D. 2010. Enzyme: Nanoparticle bioconjugates with two sequential enzymes: Stoichiometry and activity of malate dehydrogenase and citrate synthase on Au nanoparticles. Langmuir, 26, 18992-19000. Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D. 2000. Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphoruslimited soil. Plant and Cell Physiology, 41, 1030-1037. Koyama H, Takita E, Kawamura A, Hara T, Shibata D. 1999. Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant and Cell Physiology, 40, 482-488. Landschutze V, Muller-Rober B, Willmitzer L. 1995. Mitochondrial citrate synthase from potato: Predominant expression in mature leaves and young flower buds. Planta, 196, 756-764. Larson S B, Day J S, Nguyen C, Cudney R, McPherson A. 2009. Structure of pig heart citrate synthase at 1.78 A resolution. Acta Crystallographica Section F, 65, 430-434. Lindbladh C, Rault M, Hagglund C, Small W, Mosbach K, Bulow L, Evans C, Srere P. 1994. Preparation and kinetic characterization of a fusion protein of yeast mitochondrial citrate synthase and malate dehydrogenase. Biochemistry, 33, 11692-11698. Liu J H, Chi G H, Jia C H, Zhang J B, Xu B Y, Jin Z Q. 2013. Function of a citrate synthase gene (MaGCS) during postharvest banana fruit ripening. Postharvest Biology and Technology, 84, 43-50. Lu X P, Liu Y Z, Zhou G F, Wei Q J, Hu H J, Peng S A. 2011. Identification of organic acid-related genes and their expression profiles in two pear (Pyrus pyrifolia) cultivars with difference in predominant acid type at fruit ripening stage. Scientia Horticulturae, 129, 680-687. Lü J, Gao X, Dong Z, Yi J, An L. 2012. Improved phosphorus acquisition by tobacco through transgenic expression of mitochondrial malate dehydrogenase from Penicillium

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

2624

oxalicum. Plant Cell Reports, 31, 49-56. Ma C, Sun Z, Zhou J, Zhu S. 2011. Effects of exogenous nitric oxide on Krebs cycle-associated enzyme activities in Feicheng peach fruit. In: The 3rd Conference on Key Technology of Horticulture. Shenyang, China. London Science Publishing, UK. pp. 275-283. Madeira A, Vikeved E, Nilsson A, Sjögren B, Andrén P E, Svenningsson P. 2011. Identification of protein-protein interactions by surface plasmon resonance followed by mass spectrometry. Current Protocols in Protein Science, 65:19.21:19.21.1–19.21.9. http://onlinelibrary.wiley.com/ doi/10.1002/0471140864.ps1921s65/full Manjunatha G, Lokesh V, Neelwarne B. 2010. Nitric oxide in fruit ripening: Trends and opportunities. Biotechnology Advances, 28, 489-499. McCarthy N. 2013. Metabolism: Sensitivity to serine starvation. Nature Reviews Cancer, 13, 77-77. Meeks J C. 2011. Closing the cycle. Science, 334, 1508-1509. van der Merwe M J, Osorio S, Moritz T, Nunes-Nesi A, Fernie A R. 2009. Decreased mitochondrial activities of malate dehydrogenase and fumarase in tomato lead to altered root growth and architecture via diverse mechanisms. Plant Physiology, 149, 653-669. Meyer F M, Gerwig J, Hammer E, Herzberg C, Commichau F M, Völker U, Stülke J. 2011. Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon. Metabolic Engineering, 13, 18-27. Minarik P, Tomaskova N, Kollarova M, Antalik M. 2002. Malate dehydrogenases-structure and function. General Physiology and Biophysics, 21, 257-266. Morgunov I, Srere P A. 1998. Interaction between citrate synthase and malate dehydrogenase substrate channeling of oxaloacetate. Journal of Biological Chemistry, 273, 29540-29544. Nunes-Nesi A, Araújo W L, Obata T, Fernie A R. 2013. Regulation of the mitochondrial tricarboxylic acid cycle. Current Opinion in Plant Biology, 16, 335-343. Nunes-Nesi A, Carrari F, Lytovchenko A, Smith A M O, Ehlers Loureiro M, Ratcliffe R G, Sweetlove L J, Fernie A R. 2005. Enhanced photosynthetic performance and growth as a consequence of decreasing mitochondrial malate dehydrogenase activity in transgenic tomato plants. Plant Physiology, 137, 611-622. Or E, Baybik J, Sadka A, Saks Y. 2000. Isolation of mitochondrial malate dehydrogenase and phosphoenolpyruvate carboxylase cDNA clones from grape berries and analysis of their expression pattern throughout berry development. Journal of Plant Physiology, 157, 527-534. Peti-Peterdi J. 2013. Mitochondrial TCA cycle intermediates regulate body fluid and acid-base balance. The Journal of Clinical Investigation, 123, 2788-2790.

LIU Yu-chen et al.

Pettersson H, Olsson P, Bülow L, Pettersson G. 2000. Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase. European Journal of Biochemistry, 267, 50415046. Sarti P, Arese M, Forte E, Giuffrè A, Mastronicola D. 2012. Mitochondria and nitric oxide: chemistry and pathophysiology. In: Scatena R, Bottoni P, Giardina B, eds., Advances in Mitochondrial Medicine. Vol 942. Springer, Netherlands. pp. 75-92. Schrodinger, L L C. 2010. The PyMOL molecular graphics system. ver. 1.3rl. [2011-6-2]. http://sourceforge.net/ projects/pymol/files/. Siriwardena K, MacKay N, Levandovskiy V, Blaser S, Raiman J, Kantor P F, Ackerley C, Robinson BH, Schulze A, Cameron J M. 2013. Mitochondrial citrate synthase crystals: Novel finding in Sengers syndrome caused by acylglycerol kinase (AGK) mutations. Molecular Genetics and Metabolism, 108, 40-50. Sun Z, Li Y, Zhou J, Zhu S H. 2011. Effects of exogenous nitric oxide on contents of soluble sugars and related enzyme activities in ‘Feicheng’ peach fruit. Journal of the Science of Food and Agriculture, 91, 1795-1800. Sweetlove L J, Beard K F M, Nunes-Nesi A, Fernie A R, Ratcliffe R G. 2010. Not just a circle: Flux modes in the plant TCA cycle. Trends in Plant Science, 15, 462-470. Tomaz T, Bagard M, Pracharoenwattana I, Lindén P, Lee C P, Carroll A J, Ströher E, Smith S M, Gardeström P, Millar A H. 2010. Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis. Plant Physiology, 154, 1143-1157. Tompa P, Batke J, Ovadi J, Welch G R, Srere P A. 1987. Quantitation of the interaction between citrate synthase and malate dehydrogenase. Journal of Biological Chemistry, 262, 6089-6092. Wang X Y, Wang B, Hou S T, Zhu G P. 2009. Structure and function of malate dehydrogenases. Journal of Biology, 4, 69-72. Wang X, Li J, Liu J, He W, Bi Y. 2010. Nitric oxide increases mitochondrial respiration in a cGMP-dependent manner in the callus from Arabidopsis thaliana. Nitric Oxide, 23, 242-250. Yang L T, Chen L S, Peng H Y, Guo P, Wang P, Ma C L. 2012. Organic acid metabolism in Citrus grandis leaves and roots is differently affected by nitric oxide and aluminum interactions. Scientia Horticulturae, 133, 40-46. Zhang S, Bryant D A. 2011. The tricarboxylic acid cycle in cyanobacteria. Science, 334, 1551-1553. Zhu L Q, Zhou J, Zhu S H. 2010. Effect of a combination of nitric oxide treatment and intermittent warming on prevention of chilling injury of ‘Feicheng’ peach fruit during storage. Food Chemistry, 121, 165-170. (Managing editor WANG Ning)

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.