Blueberry VcLon1 protease increases iron use efficiency by alleviating chloroplast oxidative stress



Aim to unveil the functions of VcLon1 in plant Fe use efficiency.


RT-PCR was used to analyze the expression profile of VcLon1. VcLon1 was expressed in Nicotiana benthamiana, and its homologous tobacco gene was silenced using RNAi. The differences in biomass growth, oxidative stress and chloroplast ultrastructure between wild type and transgenic Nicotiana benthaminana were analyzed after being subjected to Fe deficiency stress.


The RT-PCR showed that the expression of VcLon1 was significantly higher in young leaves than in old leaves or in leaves treated with Fe deficiency stress, indicating that VcLon1 is involved in reactive oxygen species (ROS) homeostasis induced by senescence or Fe deficiency. Compared with wild-type, overexpression of VcLon1 in Nicotiana benthamiana reduced damage to chloroplast structure induced by Fe deficiency. Furthermore, the contents of H2O2, MDA and carbonylated protein in leaves were kept at a low level, and antioxidant enzyme activities in chloroplasts such as SOD and APX are also generally higher in chloroplasts with VcLon1 overexpression. In contrast, the oxidative stress levels in NbLon1 RNAi silenced Nicotiana benthamiana leaves showed opposite trends.


Under Fe deficiency stress, VcLon1 reduces oxidative damage in plants by degrading carbonylated proteins in organelles such as chloroplasts and effectively maintains the structure and function of organelles and the activity of functional proteins, contributing to Fe use efficiency in plants.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9



Superoxide Dismutase


Ascorbate Peroxidase


Electron transport chain


Reactive oxygen species


Plasma membrane


Ferric chelate reductase


Ferrochelatases 2





NJ method:

Neighbor-joining method


Oxidative phosphorylation pathway


Tricarboxylic Acid


  1. Augustyniak E, Adam A, Wojdyla K, Rogowska-Wrzesinska A, Willetts R, Korkmaz A, Atalay M, Weber D, Grune T, Borsa C (2015) Validation of protein carbonyl measurement: a multi-centre study. Redox Biol 4:149–157

    CAS  PubMed  Google Scholar 

  2. Bertamini M, Muthuchelian K, Nedunchezhian N (2002) Iron deficiency induced changes on the donor side of PS II in field grown grapevine (Vitis vinifera L. cv. Pinot noir) leaves. Plant Sci 162:599–605

    CAS  Google Scholar 

  3. Besche H, Tamura N, Tamura T, Zwickl P (2004) Mutational analysis of conserved AAA + residues in the archaeal Lon protease from Thermoplasma acidophilum. FEBS Lett 574:161–166

    CAS  PubMed  Google Scholar 

  4. Bissonnette SA, Izarys RR, Sauer RT, Baker TA (2010) The IbpA and IbpB small heat-shock proteins are substrates of the AAA+ Lon protease. Mol Microbiol 75:1539–1549

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Bota DA, Davies KJA (2002) Lon protease preferentially degrades oxidized mitochondrial aconitase by an ATP-stimulated mechanism. Nat Cell Biol 4:674–680

    CAS  PubMed  Google Scholar 

  6. Bota DA, Van RH, Davies KJ (2002) Modulation of Lon protease activity and aconitase turnover during aging and oxidative stress. FEBS Lett 532:103–106

    CAS  PubMed  Google Scholar 

  7. Chen YB, Li YQ, Sun L, Shen YW, Chen WR, Liu X, Guo WD (2015) Effects of non-acid rhizosphere pH on the iron elements uptakes and expressions of iron metabolism related genes in blueberry. Acta Horticulturae Sinica 42:233–242

    Google Scholar 

  8. Chen WR, Shao JY, Ye MJ, Yu KD, Bednarek SY, Duan XW, Guo WD (2017) Blueberry VcLON 2, a peroxisomal LON protease, is involved in abiotic stress tolerance. Environ Exp Bot 134:1–11

    CAS  Google Scholar 

  9. Chenna R, Sugawar H, Koike T, Lopez R, Gibson TJ, Higgins DG, Thompson JD (2003) Multiple sequence alignment with the Clustal series of programs[J]. Nucleic Acids Res 2003(31):3497–3500

    Google Scholar 

  10. Cory S, Lei L, Polydefkis H, Millar AH (2012) Loss of Lon1 in Arabidopsis changes the mitochondrial proteome leading to altered metabolite profiles and growth retardation without an accumulation of oxidative damage. Plant Physiol 160:1187–1203

    Google Scholar 

  11. Covarrubias JI, Rombolà AD (2015) Organic acids metabolism in roots of grapevine rootstocks under severe iron deficiency. Plant Soil 394:165–175

    CAS  Google Scholar 

  12. Czégény G, Wu M, Dér A, Eriksson LA, Strid Å, Hideg É (2014) Hydrogen peroxide contributes to the ultraviolet-B (280-315 nm) induced oxidative stress of plant leaves through multiple pathways. FEBS Lett 588:2255–2261

    PubMed  Google Scholar 

  13. Jeeyon J, Christopher C, Loubna K, Marinus P, Connolly EL, Mary Lou G (2008) Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions. Proc Natl Acad Sci U S A 105:10619–10624

    Google Scholar 

  14. Jelali N, Donnini S, Dell'Orto M, Abdelly C, Gharsalli M, Zocchi G (2014) Root antioxidant responses of two Pisum sativum cultivars to direct and induced Fe deficiency. Plant Biol 16:607–614

    CAS  PubMed  Google Scholar 

  15. Jeong J, Connolly EL (2009) Iron uptake mechanisms in plants: functions of the FRO family of ferric reductases. Plant Sci 176:709–714

    CAS  Google Scholar 

  16. Karl R, Marinus P (2013) Copper and iron homeostasis in plants: the challenges of oxidative stress. Antioxid Redox Signal 19:919–932

    Google Scholar 

  17. Ling Q, Jarvis P (2016) Plant signaling: ubiquitin pulls the trigger on chloroplast degradation. Curr Biol 26:R38–R40

    CAS  PubMed  Google Scholar 

  18. Liu W, Karemera NU, Wu T, Yang YF, Zhang XZ, Xu XF, Wang Y, Han ZH (2017) The ethylene response factor AtERF4 negatively regulates the iron deficiency response in Arabidopsis thaliana. PLoS One 12:e0186580

    PubMed  PubMed Central  Google Scholar 

  19. Ma Q, Wang J, Lu S, Lv Y, Yuan Y (2013) Quantitative proteomic profiling reveals photosynthesis responsible for inoculum size dependent variation in Chlorella sorokiniana. Biotechnol Bioeng 110:773–784

    CAS  PubMed  Google Scholar 

  20. Magdalena B, Chris W, Alexey K, Łukasz O, CWT R, Van RDB, Marten V, Van Der Klei IJ (2012) Peroxisomal proteostasis involves a Lon family protein that functions as protease and chaperone. J Biol Chem 287:27380–27395

    Google Scholar 

  21. María Esther PP, Lemaire SD, Crespo JL (2012) Reactive oxygen species and autophagy in plants and algae. Plant Physiol 160:156–164

    Google Scholar 

  22. Miki K, Naoya H, Sadaki Y, Nobuyuki S, Tsuneo I, Hisaaki T (2004) Proteomic analysis of rat liver peroxisome: presence of peroxisome-specific isozyme of Lon protease. J Biol Chem 279:421–428

    Google Scholar 

  23. Molassiotis AN, Diamantidis GC, Therios IN, Tsirakoglou V, Dimassi KN (2005) Oxidative stress, antioxidant activity and Fe(III)-chelate reductase activity of five Prunusrootstocks explants in response to Fe deficiency. Plant Growth Regul 46:69–78

    CAS  Google Scholar 

  24. Nunez GH, Olmstead JW, Darnell RL (2015) Rhizosphere acidification is not part of the strategy I iron deficiency response of Vaccinium Arboreum and the southern highbush blueberry. HortScience 50:1064–1069

    CAS  Google Scholar 

  25. Payá-Milans M, Nunez GH, Olmstead JW, Rinehart TA, Staton M (2017) Regulation of gene expression in roots of the pH-sensitive Vaccinium corymbosum and the pH-tolerant Vaccinium arboreum in response to near neutral pH stress using RNA-Seq. BMC Genomics 18:580

    PubMed  PubMed Central  Google Scholar 

  26. Rahman M, Islam MS, Kabir AH, Haider SA, Paul NK (2014) Screening of Fe-deficiency tolerance in okra ( Abelmoschus esculentus L.) through hydroponic culture. Notulae Scientia Biologicae 6:363–367

    Google Scholar 

  27. Rigas S, Daras G, Tsitsekian D, Hatzopoulos P (2012) The multifaceted role of Lon proteolysis in seedling establishment and maintenance of plant organelle function: living from protein destruction. Physiol Plant 145:215–223

    CAS  PubMed  Google Scholar 

  28. Robinson NJ, Procter CM, Connolly EL, Guerinot ML (1999) A ferric-chelate reductase for iron uptake from soils. Nature 397:694–697

    CAS  PubMed  Google Scholar 

  29. Sellammal R, Robin S, Raveendran M (2014) Association and heritability studies for drought resistance under varied moisture stress regimes in backcross inbred population of rice. Rice Sci 21:150–161

    Google Scholar 

  30. Sen A, Alikamanoglu S (2013) Antioxidant enzyme activities, malondialdehyde, and total phenolic content of PEG-induced hyperhydric leaves in sugar beet tissue culture. In Vitro Cellular & Developmental Biology - Plant 49:396–404

    CAS  Google Scholar 

  31. Shinya W, Hiroyuki I, Masanori I, Kohki Y, Yoshinori O, Tadahiko M, Amane M (2009) Autophagy plays a role in chloroplast degradation during senescence in individually darkened leaves. Plant Physiol 149:885–893

    Google Scholar 

  32. Sieburth LE, Meyerowitz EM (1997) Molecular dissection of the AGAMOUS control region shows that cis elements for spatial regulation are located intragenically. Plant Cell 9:355–365

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Sigrun R (2004) Specification of the peroxisome targeting signals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiol 135:783–800

    Google Scholar 

  34. Souza IRPD, Oliveira ED, Peres MA, Oliveira ACD, Álvaro A, Purcino C (2003) Peroxidase activity in maize inbred lines resistant or susceptible to maize dwarf mosaic virus. Revista Brasileira De Milho E Sorgo 2:1–8

    Google Scholar 

  35. Stamatis R, Gerasimos D, Miriam L, Nikolas M, Constantinos F, Sweetlove LJ, Polydefkis H (2009a) Role of Lon1 protease in post-germinative growth and maintenance of mitochondrial function in Arabidopsis thaliana. New Phytol 181:588–600

    Google Scholar 

  36. Stamatis R, Gerasimos D, Sweetlove LJ, Polydefkis H (2009b) Mitochondria biogenesis via Lon1 selective proteolysis: who dares to live for ever? Plant Signal Behav 4:221–224

    Google Scholar 

  37. Sun CH, Wu T, Zhai LM, Li D, Zhang XZ, Xu XF, Ma HQ, Wang Y, Han ZH (2016) Reactive oxygen species function to mediate the Fe deficiency response in an Fe-efficient apple genotype: an early response mechanism for enhancing reactive oxygen production. Front Plant Sci 7:1726

    PubMed  PubMed Central  Google Scholar 

  38. Townsend AT (2000) The accurate analysis of the first row transition metals in water, urine, plant, tissue and rock samples by sector field ICP-MS. J Anal At Spectrom 15:307

    CAS  Google Scholar 

  39. Vashisth T, Johnson KL, Malladi A (2011) An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry. Plant Cell Rep 30:2167–2176

    CAS  PubMed  Google Scholar 

  40. Yang SS, Gao JF (2001) Influence of active oxygen and free radicals on plant senescence. Acta Botan Boreali-Occiden Sin 21:215–220

    CAS  Google Scholar 

  41. Yousfi S, Rabhi M, Abdelly C, Gharsalli M (2009) Iron deficiency tolerance traits in wild (Hordeum maritimum) and cultivated barley (Hordeum vulgare). Comptes Rendus Biologies 332:523–533

    CAS  PubMed  Google Scholar 

Download references


Our research is supported by the National Key Research and Development Program of China (2017YFD0801300); Zhejiang Science & Technology Project (2017C32046, 2015C32129) and the major program for science and technology of Zhejiang province (2018C02007, 2016C02052-9).

Author information



Corresponding authors

Correspondence to Wenrong Chen or Weidong Guo.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Responsible Editor: Miroslav Nikolic.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhong, J., Gu, J., Guo, Y. et al. Blueberry VcLon1 protease increases iron use efficiency by alleviating chloroplast oxidative stress. Plant Soil 445, 533–548 (2019).

Download citation


  • Blueberry
  • VcLon1 protease
  • Oxidative stress
  • Fe use efficiency