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.

Proteome analysis of peroxisomes from dark‐treated senescent Arabidopsis leaves

“…Peroxisomes are metabolically and morphologically dynamic. Environmental factors, including light, are known to cause changes in peroxisome abundance (Lopez-Huertas et al 2000;Koh et al 2005;Desai and Hu 2008;Rodr ıguez-Serrano et al 2016;Desai et al 2017;Fahy et al 2017). The proteome and primary functions of plant peroxisomes vary, quantitatively and qualitatively, across developmental stages, and distinct names were given to the three major subtypes of plant peroxisomes.…”

Resistin inhibits neuronal autophagy through Toll-like receptor 4

“…Hence, by eliminating injured and aged mitochondria that constitute an important source of reactive oxygen species. Thus, in contrast with resistin action, autophagy reduces cellular inflammatory responses (De Lavera et al 2017, Fahy et al 2017. However, the impact of resistin on neuronal autophagy is unknown.…”

A PEX5 missense allele preferentially disrupts PTS1 cargo import into Arabidopsis peroxisomes

“…Thus, in seedlings with functioning peroxisomes, IBA treatment confers high‐auxin phenotypes including reduced root and hypocotyl elongation (Strader et al., ; Zolman et al., ), and IBA‐resistance screens have uncovered genes needed for peroxisome biogenesis and function (reviewed in Bartel et al., ). As salt increases Arabidopsis peroxisome abundance (Fahy et al., ; Frick & Strader, ; Mitsuya et al., ), we reasoned that screening for IBA resistance in the presence of salt might uncover factors impacting salinity‐induced peroxisome proliferation. We therefore identified seedlings with elongated roots on normally inhibitory concentrations of IBA and NaCl.…”