Volume 7, Issue 4, Pages 747-750 (April 2014) Nitric Oxide Is Required for Homeostasis of Oxygen and Reactive Oxygen Species in Barley Roots under Aerobic Conditions  Gupta Kapuganti J. , Hebelstrup Kim H. , Kruger Nicholas J. , George Ratcliffe R.   Molecular Plant  Volume 7, Issue 4, Pages 747-750 (April 2014) DOI: 10.1093/mp/sst167 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Scavenging of NO by Non-Symbiotic Hemoglobin 1 Leads to Increased Respiration, Decreased Internal Oxygen, Increased ROS Production, and Increased Glucose Consumption in Barley Roots. Data are presented as the mean ± SD of the specified number of measurements (n). WT and Hb+ plants were compared using Student’s t-test assuming unequal variance; * indicates a comparison for which P < 0.05 and ** for one where P < 0.01. (A) NO levels in roots of WT and Hb+ plants. NO was visualized through the intracellular production of fluorescent triazolofluorescein. Images of WT roots are shown in the left column, and those for Hb+ plant are in the right column; for each, the upper and lower rows show images of roots before and after treatment with cPTIO, respectively. NO-dependent fluorescence measurements of WT and Hb+ roots were quantified by analysis of digital images using ImageJ software (lower graph, n = 6). (B) NO emission from WT and Hb+ roots measured by gas phase chemiluminescence using a CLD-700 AL analyzer (Eco Physics, Switzerland) (n = 6). Root segments (2.2 g FW) were incubated in 50 mM HEPES buffer containing 0.5 mM nitrite, pH 7.2. (C) Respiration rates of WT and Hb+ roots measured using a Clark-type oxygen electrode (n = 4). Root segments (60mg FW) were incubated in freshly prepared growth medium containing 0.5 mM nitrite, pH 7.2. (D) Internal oxygen distribution in WT and Hb+ barley roots. The upper panels show representative images of the oxygen content of WT roots (left) and Hb+ roots (right); the values on the images indicate the internal oxygen concentrations determined using VisiSens software. The oxygen concentration in WT and Hb+ roots is compared quantitatively in the lower graph (n = 9). (E) Release of 14CO2 by WT and Hb+ roots supplied with [14C]glucose labeled in specific carbon positions (n = 4). (F) ROS levels in WT and Hb+ roots, determined by quantification of the production of dichlorofluorescein (n = 6). Roots were incubated in 5 μM 2′,7′-dichlorodihydro-fluorescein diacetate, 10 mM Tris-Cl buffer, pH 7.2 for 10min in the dark, and then washed with buffer before recording images with a Leica M165 FC microscope. Fluorescence was excited at 488 nm and detected at 522 nm; the emission was quantified per unit area of root using ImageJ. Molecular Plant 2014 7, 747-750DOI: (10.1093/mp/sst167) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions