Detection and Imaging of Superoxide in Roots by an Electron Spin Resonance Spin-Probe Method

Abstract

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.

Singlet Oxygen Signatures Are Detected Independent of Light or Chloroplasts in Response to Multiple Stresses

“…Superoxide can react with H 2 O 2 via a Haber-Weiss mechanism to form singlet oxygen (H 2 O 2 +O 2 2 → OH 2 + OH$ + 1 O 2 ; Kellogg and Fridovich, 1975;Khan and Kasha, 1994), although the efficiency of singlet oxygen production by these ROS in vivo is not known (MacManus-Spencer and McNeill, 2005). Nonetheless, the accumulation of superoxide and H 2 O 2 is readily detected in local or systemic wounded tissue (Sagi et al, 2004;Warwar et al, 2011) and in the immune responses (Dubiella et al, 2013). A possible source could be NADPH oxidase-like and superoxide dismutase activity, as has been shown for elicitor and salt-induced accumulation of ROS (Leshem et al, 2006;Ashtamker et al, 2007;Suzuki et al, 2011).…”

“…For imaging and examination of stress-related transcripts in the background of ex1/ex2, in roots and application of flg22, 4-to 7-d-old seedlings were grown on 0.8% (w/v) solid agar (Duchefa) medium containing full-strength B5 Gamborg's nutrients (Duchefa) adjusted to pH 5.8. Seedlings were surface sterilized according to the method of Warwar et al (2011) and grown in vertically positioned plates under a 16-h-light/8-h-dark cycle at 21°C.…”

Reactive oxygen species: Reactions and detection from photosynthetic tissues

“…Warwar et al [284] were able to obtain onedimensional images of O 2 • production in a whole plant root using PTM-TC, and they also concluded that the probe stayed in the apoplastic space. More commonly used EPR probes, DMPO, BMPO and DEPMPO are thought to be membrane-permeable [298].…”

“…The spin traps 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) and perchlorotriphenylmethyl radical-tricarboxylic acid (PTM-TC) have been used to detect O 2 • from spinach thylakoids [282,283], respectively, and from plant roots [284]. O 2 • induces oxidation of OXANOH to OXANO, but it is also auto-oxidized in the presence of metal-ions [282].…”

Mitochondrial Activity as a Biomarker of Gamete and Embryo Health

“…The detection of superoxide fl ashes and their relationship to mitochondrial physiology, bioenergetic and calcium homeostasis, ROS signaling and redox state is a comparatively new area of mitochondrial research that has already contributed new insights into understanding normal cell function and the pathophysiology of disease [ 97,98 ] . Different analytical methods have been developed to detect superoxide fl ashes (i.e., the superoxide ion) including fl uorescence microscopy, chemiluminescence, and electron spin resonance with stable spin probes [ 99,100 ] . However, quantitation can be problematic and especially challenging with fl uorescent methods that use an endogenous superoxide biosensor, such as a circularly permuted yellow fl uorescent protein (cpYFP; 101), whose presence in the mitochondrial matrix currently requires genetically targeting transfection.…”