Skip to main content
SpringerLink
Log in

An improved immunofluorescence staining method for chloroplast proteins

  • Original Article
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

An improved immunofluorescence staining method significantly facilitates the visualization of the subcellular localization of interested proteins in chloroplasts.

Abstract

As an important technical approach, immunofluorescence staining is widely used in the subcellular localization study of interested proteins. During the study of the functions of chloroplast division proteins, immunofluorescence staining was frequently adopted. Previously, a method has been developed to study the localization of a chloroplast division protein, FtsZ. However, it is laborious and time-consuming. In this study, we report a modified immunofluorescence staining method, in which protoplasts were isolated from leaf tissues, and then fixed for immunofluorescence staining. The time of the experiment was significantly reduced to several hours. Furthermore, we used correction pen in the fixation procedure and a new way to coat the slide, which greatly saved the cost of the experiment. With the chloroplast division protein ARC6 as an example, we can get a good fluorescence signal. Moreover, the localization of ARC6 in two chloroplast division mutants, arc3 and arc5, and three other plant species, such as cabbage, radish and pea, was also successfully analyzed with our new method. Overall, the immunofluorescence staining method we reported here is very practical, and it significantly facilitates the visualization of the subcellular localization of interested proteins in plant cells.

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

References

  • Agbulut O, Coirault C, Niederländer N, Huet A, Vicart P, Hagège A, Puceat M, Menasché P (2006) GFP expression in muscle cells impairs actin–myosin interactions: implications for cell therapy. Nat Methods 3:331

    Article  CAS  PubMed  Google Scholar 

  • Agbulut O, Huet A, Niederländer N, Puceat M, Menasché P, Coirault C (2007) Green fluorescent protein impairs actin–myosin interactions by binding to the actin-binding site of myosin. J Biol Chem 282:10465–10471

    Article  CAS  PubMed  Google Scholar 

  • Celler K, Fujita M, Kawamura E, Ambrose C, Herburger K, Holzinger A, Wasteneys GO (2016) Microtubules in plant cells: strategies and methods for immunofluorescence, transmission electron microscopy, and live cell imaging. Cytoskeleton methods and protocols: methods and protocols, pp 155–184

  • Chang N, Gao Y, Zhao L, Liu X, Gao H (2015) Arabidopsis FHY3/CPD45 regulates far-red light signaling and chloroplast division in parallel. Sci Rep 5:1–6

    Google Scholar 

  • Gao H, Kadirjan-Kalbach D, Froehlich JE, Osteryoung KW (2003) ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery. PNAS 100:4328–4333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gao Y, Liu H, An C, Shi Y, Liu X, Yuan W, Zhang B, Yang J, Yu C, Gao H (2013) Arabidopsis FRS4/CPD25 and FHY3/CPD45 work cooperatively to promote the expression of the chloroplast division gene ARC5 and chloroplast division. Plant J 75:795–807

    Article  CAS  PubMed  Google Scholar 

  • Glynn JM, Froehlich JE, Osteryoung KW (2008) Arabidopsis ARC6 coordinates the division machineries of the inner and outer chloroplast membranes through interaction with PDV2 in the intermembrane space. Plant Cell 20:2460–2470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gould SB, Waller RF, McFadden GI (2008) Plastid evolution. Plant Biol 59:491–517

    Article  CAS  Google Scholar 

  • Hanson DA, Ziegler SF (2004) Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule. J Negat Results Biomed 3:1–3

    Article  Google Scholar 

  • Kam WW-Y, Middleton R, Lake V, Banati RB (2013) Green fluorescent protein alters the transcriptional regulation of human mitochondrial genes after gamma irradiation. J Fluoresc 23:613–619

    Article  CAS  PubMed  Google Scholar 

  • Keeling PJ (2013) The number, speed, and impact of plastid endosymbioses in eukaryotic evolution. Annu Rev Plant Biol 64:583–607

    Article  CAS  PubMed  Google Scholar 

  • Maliga P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55:289–313

    Article  CAS  PubMed  Google Scholar 

  • Mcandrew RS, Olson BJ, Kadirjan-Kalbach DK, Chi-Ham CL, Vitha S, Froehlich JE, Osteryoung KW (2008) In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex. Biochem J 412:367–378

    Article  CAS  PubMed  Google Scholar 

  • Miyagishima S-y, Froehlich JE, Osteryoung KW (2006) PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site. Plant Cell 18:2517–2530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ovečka M, Šamajová O, Baluška F, Šamaj J (2014) Immunofluorescent localization of MAPKS in Steedman’s wax sections. Plant MAP kinases: methods and protocols, pp 117–130

  • Pan D, Shi Y, Liu X, Gao Y, Liu Z, Gao H (2013) Genetic mapping and isolation of two arc3 alleles in Arabidopsis. Plant Cell Rep 32:173–182

    Article  PubMed  Google Scholar 

  • Pyke KA, Leech RM (1994) A genetic analysis of chloroplast division and expansion in Arabidopsis thaliana. Plant Physiol 104:201–207

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pyke KA, Rutherford SM, Robertson EJ, Leech RM (1994) arc6, a fertile Arabidopsis mutant with only two mesophyll cell chloroplasts. Plant Physiol 106:1169–1177

    CAS  PubMed  PubMed Central  Google Scholar 

  • Robertson E, Pyke K, Leech R (1995) arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices. J Cell Sci 108:2937–2944

    CAS  PubMed  Google Scholar 

  • Shimada H, Koizumi M, Kuroki K, Mochizuki M, Fujimoto H, Ohta H, Masuda T, K-i Takamiya (2004) ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase. Plant Cell Physiol 45:960–967

    Article  CAS  PubMed  Google Scholar 

  • Vitha S, McAndrew RS, Osteryoung KW (2001) FtsZ ring formation at the chloroplast division site in plants. J Cell Biol 153:111–120

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vitha S, Froehlich JE, Koksharova O, Pyke KA, van Erp H, Osteryoung KW (2003) ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2. Plant Cell 15:1918–1933

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang Y, Glynn JM, Olson BJ, Schmitz AJ, Osteryoung KW (2008) Plastid division: across time and space. Curr Opin Plant Biol 11:577–584

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Yoshida Y, S-y Miyagishima, Kuroiwa H, Kuroiwa T (2012) The plastid-dividing machinery: formation, constriction and fission. Curr Opin Plant Biol 15:714–721

    Article  CAS  PubMed  Google Scholar 

  • Zhang F, Hackett NR, Lam G, Cheng J, Pergolizzi R, Luo L, Shmelkov SV, Edelberg J, Crystal RG, Rafii S (2003) Green fluorescent protein selectively induces HSP70-mediated up-regulation of COX-2 expression in endothelial cells. Blood 102:2115–2121

    Article  CAS  PubMed  Google Scholar 

  • Zhang M, Schmitz AJ, Kadirjan-Kalbach DK, TerBush AD, Osteryoung KW (2013) Chloroplast division protein ARC3 regulates chloroplast FtsZ-ring assembly and positioning in Arabidopsis through interaction with FtsZ2. Plant Cell 25:1787–1802

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang Q, Jia N, Li H, Gao H-B (2015) Identification and analysis of ARC6 gene in a chloroplast division mutant cpd44 in Arabidopsis thaliana. Plant Physiol J 51:501–508

    CAS  Google Scholar 

  • Zhang M, Chen C, Froehlich JE, TerBush AD, Osteryoung KW (2016) Roles of Arabidopsis PARC6 in coordination of the chloroplast division complex and negative regulation of FtsZ assembly. Plant Physiol 170:250–262

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Science and Technology Innovation Projects of Beijing Forestry University (BLX2014-24), the National Natural Science Foundation of China (31070162, 31501090), and the Beijing Natural Science Foundation (5154031).

Author information

Authors and Affiliations

  1. College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, 100083, China

    Yiqiong Li, Qingqing Sun, Xiaomin Liu & Hongbo Gao

  2. Beijing Key Lab of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China

    Yue Feng

Authors
  1. Yiqiong Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qingqing Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yue Feng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiaomin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongbo Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Xiaomin Liu or Hongbo Gao.

Ethics declarations

Conflict of interest

The authors claim no competing interests.

Additional information

Communicated by Q. Zhao.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Li, Y., Sun, Q., Feng, Y. et al. An improved immunofluorescence staining method for chloroplast proteins. Plant Cell Rep 35, 2285–2293 (2016). https://doi.org/10.1007/s00299-016-2034-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-016-2034-7

Keywords

Search

Navigation