Skip to main content
SpringerLink
Log in

The interaction between AtMT2b and AtVDAC3 affects the mitochondrial membrane potential and reactive oxygen species generation under NaCl stress in Arabidopsis

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

Main conclusion

AtMT2b interacts with AtVDAC3 in mitochondria in Arabidopsis. The overexpression of the AtMT2b and AtVDAC3 T-DNA insertion mutant confers tolerance to NaCl stress in Arabidopsis. Both AtMT2b and AtVDAC3 are involved in the regulation of the mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) under NaCl stress.

Metallothioneins (MTs) are small, cysteine rich, metal-binding proteins that perform multiple functions, such as heavy metal detoxification and reactive oxygen species (ROS) scavenging. MTs have been reported to be involved in mitochondrial function in mammals. However, whether a direct relationship exists between MTs and mitochondrial proteins remains unclear. In the present study, we used yeast two-hybrid and bimolecular fluorescence complementation assays to demonstrate that AtMT2b, which is a type 2 MT in Arabidopsis, interacts with the outer mitochondrial membrane voltage-dependent anion channel AtVDAC3. AtMT2b bound AtVDAC3, leading to its co-localization in mitochondria. AtMT2b transgenic seedlings exhibited increased tolerance to salt stress, and the atvdac3 mutant showed a similar phenotype. The mitochondrial membrane potential (MMP) was maintained, and ROS generation was reduced following AtMT2b overexpression and AtVDAC3 knockout under NaCl stress. Both AtMT2b and AtVDAC3 were shown to be involved in MMP regulation and ROS production under NaCl stress but showed opposite effects. We conclude that AtMT2b might negatively interact with AtVDAC3 in mitochondria, and both proteins are involved in the regulation of MMP and ROS under NaCl stress.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

BiFC:

Bimolecular fluorescence complementation

MMP:

Mitochondrial membrane potential

MTs:

Metallothioneins

OMM:

Outer mitochondrial membrane

ROS:

Reactive oxygen species

VDACs:

Voltage-dependent anion channels

Y2H:

Yeast two-hybrid

References

  • Al Bitar F, Roosens N, Smeyers M, Vauterin M, Van Boxtel J, Jacobs M, Homble F (2003) Sequence analysis, transcriptional and posttranscriptional regulation of the rice vdac family. Biochim Biophys Acta 1625:43–51

    Article  CAS  PubMed  Google Scholar 

  • Batandier C, Leverve X, Fontaine E (2004) Opening of the mitochondrial permeability transition pore induces reactive oxygen species production at the level of the respiratory chain complex I. J Biol Chem 279:17197–17204

    Article  CAS  PubMed  Google Scholar 

  • Cai L, Wang Y, Zhou G, Chen T, Song Y, Li X, Kang YJ (2006) Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J Am Coll Cardiol 48:1688–1697

    Article  CAS  PubMed  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  • Costello LC, Guan Z, Franklin RB, Feng P (2004) Metallothionein can function as a chaperone for zinc uptake transport into prostate and liver mitochondria. J Inorg Biochem 98:664–666

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Desai MK, Mishra RN, Verma D, Nair S, Sopory SK, Reddy MK (2006) Structural and functional analysis of a salt stress inducible gene encoding voltage dependent anion channel (VDAC) from pearl millet (Pennisetum glaucum). Plant Physiol Biochem 44:483–493

    Article  CAS  PubMed  Google Scholar 

  • Dong F, Li Q, Sreejayan N, Nunn JM, Ren J (2007) Metallothionein prevents high-fat diet induced cardiac contractile dysfunction: role of peroxisome proliferator activated receptor coactivator 1 and mitochondrial biogenesis. Diabetes 56:2201–2212

    Article  CAS  PubMed  Google Scholar 

  • Feng W, Cai J, Pierce WM, Franklin RB, Maret W, Benz FW, Kang YJ (2005) Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts. Biochem Biophys Res Commun 332:853–858

    Article  CAS  PubMed  Google Scholar 

  • Fu Z, Guo J, Jing L, Li R, Zhang T, Peng S (2010) Enhanced toxicity and ROS generation by doxorubicin in primary cultures of cardiomyocytes from neonatal metallothionein-I/II null mice. Toxicol In Vitro 24:1584–1591

    Article  CAS  PubMed  Google Scholar 

  • Futakawa N, Kondoh M, Ueda S, Higashimoto M, Takiguchi M, Suzuki S, Sato M (2006) Involvement of oxidative stress in the synthesis of metallothionein induced by mitochondrial inhibitors. Biol Pharm Bull 29:2016–2020

    Article  CAS  PubMed  Google Scholar 

  • Godbole A, Mitra R, Dubey AK, Reddy PS, Mathew MK (2011) Bacterial expression, purification and characterization of a rice voltage-dependent, anion-selective channel isoform, OsVDAC4. J Membr Biol 244:67–80

    Article  CAS  PubMed  Google Scholar 

  • Godbole A, Dubey AK, Reddy PS, Udayakumar M, Mathew MK (2013) Mitochondrial VDAC and hexokinase together modulate plant programmed cell death. Protoplasma 250:875–884

    Article  CAS  PubMed  Google Scholar 

  • Han D, Antunes F, Canali R, Rettori D, Cadenas E (2002) Voltage-dependent anion channels control the release of the superoxide anion from mitochondria to cytosol. J Biol Chem 278:5557–5563

    Article  PubMed  Google Scholar 

  • Heazlewood JL, Pan X, Chen Z, Yang X, Liu G (2014) Arabidopsis voltage-dependent anion channel 1 (AtVDAC1) is required for female development and maintenance of mitochondrial functions related to energy-transaction. PLoS One 9(9):e106941

    Article  CAS  Google Scholar 

  • Kondoh M, Inoue Y, Atagi S, Futakawa N, Higashimoto M, Sato M (2001) Specific induction of metallothionein synthesis by mitochondrial oxidative stress. Life Sci 69:2137–2146

    Article  CAS  PubMed  Google Scholar 

  • Kusano T, Tateda C, Berberich T, Takahashi Y (2009) Voltage-dependent anion channels: their roles in plant defense and cell death. Plant Cell Rep 28:1301–1308

    Article  CAS  PubMed  Google Scholar 

  • Lacomme C, Roby D (1999) Identification of new early markers of the hypersensitive response in Arabidopsis thaliana 1. FEBS Lett 459:149–153

    Article  CAS  PubMed  Google Scholar 

  • Lee SM, Hoang MHT, Han HJ, Kim HS, Lee K, Kim KE, Kim DH, Lee SY, Chung WS (2009) Pathogen inducible voltage-dependent anion channel (AtVDAC) isoforms are localized to mitochondria membrane in Arabidopsis. Mol Cells 27:321–327

    Article  CAS  PubMed  Google Scholar 

  • Li Z-Y, Xu Z-S, He G-Y, Yang G-X, Chen M, Li L-C, Ma Y (2013) The voltage-dependent anion channel 1 (AtVDAC1) negatively regulates plant cold responses during germination and seedling development in Arabidopsis and interacts with calcium sensor CBL1. Int J Mol Sci 14:701–713

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lindeque JZ, Levanets O, Louw R, van der Westhuizen FH (2010) The involvement of metallothioneins in mitochondrial function and disease. Curr Protein Pept Sci 11:292–309

    Article  CAS  PubMed  Google Scholar 

  • Martel C, Allouche M, Esposti DD, Fanelli E, Boursier C, Henry C, Chopineau J, Calamita G, Kroemer G, Lemoine A, Brenner C (2013) Glycogen synthase kinase 3-mediated voltage-dependent anion channel phosphorylation controls outer mitochondrial membrane permeability during lipid accumulation. Hepatology 57:93–102

    Article  CAS  PubMed  Google Scholar 

  • Miyayama T, Arai Y, Suzuki N, Hirano S (2013) Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate. Toxicology 305:20–29

    Article  CAS  PubMed  Google Scholar 

  • Moltó E, Bonzón-Kulichenko E, Gallardo N, Andrés A (2007) MTPA: a crustacean metallothionein that affects hepatopancreatic mitochondrial functions. Arch Biochem Biophys 467:31–40

    Article  CAS  PubMed  Google Scholar 

  • Robert N, d’Erfurth I, Marmagne A, Erhardt M, Allot M, Boivin K, Gissot L, Monachello D, Michaud M, Duchêne A-M, Barbier-Brygoo H, Maréchal-Drouard L, Ephritikhine G, Filleur S (2012) Voltage-dependent-anion-channels (VDACs) in Arabidopsis have a dual localization in the cell but show a distinct role in mitochondria. Plant Mol Biol 78:431–446

    Article  CAS  PubMed  Google Scholar 

  • Rostovtseva TK, Bezrukov SM (1998) ATP transport through a single mitochondrial channel, VDAC, studied by current fluctuation analysis. Biophys J 74:2365–2373

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rostovtseva TK, Bezrukov SM (2008) VDAC regulation: role of cytosolic proteins and mitochondrial lipids. J Bioenerg Biomembr 40:163–170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rostovtseva T, Colombini M (1997) VDAC channels mediate and gate the flow of ATP: implications for the regulation of mitochondrial function. Biophys J 72:1954–1962

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sampson MJ, Lovell RS, Craigen WJ (1997) The murine voltage-dependent anion channel gene family: conserved structure and function. J Biol Chem 272:18966–18973

    Article  CAS  PubMed  Google Scholar 

  • Sanchez Ferrer A, Santema JS, Hilhorst R, Visser AJ (1990) Fluorescence detection of enzymatically formed hydrogen peroxide in aqueous solution and in reversed micelles. Anal Biochem 187:129–132

    Article  CAS  PubMed  Google Scholar 

  • Simpkins C, Balderman S, Mensah E (1998a) Mitochondrial oxygen consumption is synergistically inhibited by metallothionein and calcium. J Surg Res 80:16–21

    Article  CAS  PubMed  Google Scholar 

  • Simpkins C, Lloyd T, Li S, Balderman S (1998b) Metallothionein-induced increase in mitochondrial inner membrane permeability. J Surg Res 75:30–34

    Article  CAS  PubMed  Google Scholar 

  • Takahashi Y, Tateda C (2013) The functions of voltage-dependent anion channels in plants. Apoptosis 18:917–924

    Article  CAS  PubMed  Google Scholar 

  • Tan W, Colombini M (2007) VDAC closure increases calcium ion flux. BBA Biomembr 1768:2510–2515

    Article  CAS  Google Scholar 

  • Tang W, Shaikh ZA (2001) Renal cortical mitochondrial dysfunction upon cadmium metallothionein administration to sprague-dawley rats. J Toxicol Environ Health Part A 63:221–235

    Article  CAS  PubMed  Google Scholar 

  • Tateda C, Yamashita K, Takahashi F, Kusano T, Takahashi Y (2008) Plant voltage-dependent anion channels are involved in host defense against Pseudomonas cichorii and in Bax-induced cell death. Plant Cell Rep 28:41–51

    Article  CAS  PubMed  Google Scholar 

  • Tateda C, Watanabe K, Kusano T, Takahashi Y (2011) Molecular and genetic characterization of the gene family encoding the voltage-dependent anion channel in Arabidopsis. J Exp Bot 62:4773–4785

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsugama D, Liu S, Takano T (2012) A putative myristoylated 2C-type protein phosphatase, PP2C74, interacts with SnRK1 in Arabidopsis. FEBS Lett 586:693–698

    Article  CAS  PubMed  Google Scholar 

  • Wandrey M, Trevaskis B, Brewin N, Udvardi MK (2004) Molecular and cell biology of a family of voltage-dependent anion channel porins in Lotus japonicus. Plant Physiol 134:182–193

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang GW, Klein JB, Kang YJ (2001) Metallothionein inhibits doxorubicin-induced mitochondrial cytochrome c release and caspase-3 activation in cardiomyocytes. J Pharmacol Exp Ther 298:461–468

    CAS  PubMed  Google Scholar 

  • Xue T, Li X, Zhu W, Wu C, Yang G, Zheng C (2009) Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. J Exp Bot 60:339–349

    Article  CAS  PubMed  Google Scholar 

  • Yan J, He H, Tong S, Zhang W, Li X, Yang Y (2009) Voltage-dependent anion channel 2 of Arabidopsis thaliana (AtVDAC2) is involved in ABA-mediated early seedling development. Int J Mol Sci 10:2476–2486

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang XY, Chen ZW, Xu T, Qu Z, Pan XD, Qin XH, Ren DT, Liu GQ (2011) Arabidopsis kinesin KP1 specifically interacts with VDAC3, a mitochondrial protein, and regulates respiration during seed germination at low temperature. Plant Cell 23:1093–1106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ye B, Maret W, Vallee BL (2001) Zinc metallothionein imported into liver mitochondria modulates respiration. Proc Natl Acad Sci USA 98:2317–2322

    Article  CAS  PubMed  Google Scholar 

  • Yi J, Moon S, Lee YS, Zhu L, Liang W, Zhang D, Jung KH, An G (2016) Defective tapetum cell death 1 (DTC1) regulates ROS levels by binding to metallothionein during tapetum degeneration. Plant Physiol 170:1611–1623

    Article  CAS  Google Scholar 

  • Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

    Article  CAS  Google Scholar 

  • Zalewska M, Trefon J, Milnerowicz H (2014) The role of metallothionein interactions with other proteins. Proteomics 14:1343–1356

    Article  CAS  PubMed  Google Scholar 

  • Zhang M, Takano T, Liu S, Zhang X (2015) Arabidopsis mitochondrial voltage-dependent anion channel 3 (AtVDAC3) protein interacts with thioredoxin m2. FEBS Lett 589:1207–1213

    Article  CAS  PubMed  Google Scholar 

  • Zheng Y, Shi Y, Tian C, Jiang C, Jin H, Chen J, Almasan A, Tang H, Chen Q (2004) Essential role of the voltage-dependent anion channel (VDAC) in mitochondrial permeability transition pore opening and cytochrome c release induced by arsenic trioxide. Oncogene 23:1239–1247

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by funding from the Changjiang Scholars and Innovative Research Team in University (PCSIRT) (IRT_17R99) to Shenkui Liu and a grant from the Fundamental Research Funds for the Central Universities (2572014DA06) to Xinxin Zhang. We thank professor Lixin Li for providing the BY2 cells.

Author information

Authors and Affiliations

  1. Key Laboratory of Saline-alkali Vegetation Ecology Restoration (SAVER), Ministry of Education, Alkali Soil Natural Environmental Science Center (ASNESC), Northeast Forestry University, Harbin, 150040, China

    Min Zhang & Xinxin Zhang

  2. School of Medicine, He University, Shenyang, 110163, China

    Min Zhang

  3. State Key Laboratory of Subtropical Silviculture, Zhejiang A & F University, Hangzhou, 311300, China

    Shenkui Liu

  4. Asian Natural Environment Science Center (ANESC), The University of Tokyo, 1-1-1 Midori Cho, Nishitokyo-shi, Tokyo, 188-0002, Japan

    Tetsuo Takano

Authors
  1. Min Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shenkui Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tetsuo Takano
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xinxin Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xinxin Zhang.

Ethics declarations

Conflict of interest

The authors have no conflicts of interest to declare.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 3404 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, M., Liu, S., Takano, T. et al. The interaction between AtMT2b and AtVDAC3 affects the mitochondrial membrane potential and reactive oxygen species generation under NaCl stress in Arabidopsis. Planta 249, 417–429 (2019). https://doi.org/10.1007/s00425-018-3010-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-018-3010-y

Keywords

Search

Navigation