title: Impact of salt stress, cell death, and autophagy on peroxisomes: quantitative and morphological analyses using small fluorescent probe N-BODIPY.
abstract: Plant peroxisomes maintain a plethora of key life processes including fatty acid β-oxidation, photorespiration, synthesis of hormones, and homeostasis of reactive oxygen species (ROS). Abundance of peroxisomes in cells is dynamic; however mechanisms controlling peroxisome proliferation remain poorly understood because measuring peroxisome abundance is technically challenging. Counting peroxisomes in individual cells of complex organs by electron or fluorescence microscopy is expensive and time consuming. Here we present a simple technique for quantifying peroxisome abundance using the small probe Nitro-BODIPY, which in vivo fluoresces selectively inside peroxisomes. The physiological relevance of our technique was demonstrated using salinity as a known inducer of peroxisome proliferation. While significant peroxisome proliferation was observed in wild-type Arabidopsis leaves following 5-hour exposure to NaCl, no proliferation was detected in the salt-susceptible mutants fry1-6, sos1-14, and sos1-15. We also found that N-BODIPY detects aggregation of peroxisomes during final stages of programmed cell death and can be used as a marker of this stage. Furthermore, accumulation of peroxisomes in an autophagy-deficient Arabidopsis mutant atg5 correlated with N-BODIPY labeling. In conclusion, the technique reported here enables quantification of peroxisomes in plant material at various physiological settings. Its potential applications encompass identification of genes controlling peroxisome homeostasis and capturing stress-tolerant genotypes.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, 99164, WA, USA.
authors.forename: Deirdre
authors.initials: D
authors.lastname: Fahy
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, 99164, WA, USA.|Department of Genetics and Cytology, National Research Center, Giza, Egypt.
authors.forename: Marwa N M E
authors.initials: MNME
authors.lastname: Sanad
authors.affiliation: Plant Physiology, University of Kaiserslautern, Erwin Schrödinger Straße, Kaiserslautern, D-67653, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Duscha
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, 99164, WA, USA.
authors.forename: Madison
authors.initials: M
authors.lastname: Lyons
authors.affiliation: Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK.
authors.forename: Fuquan
authors.initials: F
authors.lastname: Liu
authors.affiliation: Department of Chemistry and Biotechnology, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7015, Uppsala, SE-75007, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Bozhkov
authors.affiliation: School of Biological Sciences, Washington State University, Pullman, 99164, WA, USA.
authors.forename: Hans-Henning
authors.initials: HH
authors.lastname: Kunz
authors.affiliation: MSU-DOE Plant Research Laboratory, Michigan State University, 612 Wilson Road, East Lansing, 48824, MI, USA.
authors.forename: Jianping
authors.initials: J
authors.lastname: Hu
authors.affiliation: Plant Physiology, University of Kaiserslautern, Erwin Schrödinger Straße, Kaiserslautern, D-67653, Germany.
authors.forename: H Ekkehard
authors.initials: HE
authors.lastname: Neuhaus
authors.affiliation: Department of Chemistry, Durham University, Durham, DH1 3LE, UK.
authors.forename: Patrick G
authors.initials: PG
authors.lastname: Steel
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, 99164, WA, USA. andrei.smertenko@wsu.edu.|Institute for Global Food Security, School of Biological Sciences, Queen's University Belfast, 18-30 Malone Road, Belfast, BT9 5BN, UK. andrei.smertenko@wsu.edu.
authors.forename: Andrei
authors.initials: A
authors.lastname: Smertenko
mesh_terms: D017209:Apoptosis; D017360:Arabidopsis / Q000187:drug effects*; D001896:Boron Compounds / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D009574:Nitro Compounds / Q000032:analysis; D020675:Peroxisomes / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism*; D013194:Staining and Labeling / Q000379:methods*; D013312:Stress, Physiological
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D009574:Nitro Compounds; D012965:Sodium Chloride