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Effect of Non-Symbiotic Hemoglobin Expression on Oxygen Content in Roots
Oxygen Imaging and Respiration Measurement in Roots with VisiSens
Jagadis Gupta Kapuganti
Biochemistry & Systems Biology, Department of Plant Sciences, University of Oxford, GB
This project aimed to define the role of plant non-symbiotic hemoglobin and regulation of respiration by nitric oxide in plant roots. Wild-type and hemoglobin over-expressing barley roots were used for experiments. Oxygen imaging in cut roots as well as respiration measurements of roots placed in Eppendorf tubes was performed with the VisiSens™ system. Previous findings showed, that nitric oxide production is reduced in hemoglobin over-expressing plants compared to wild-type plants. Furthermore, nitric oxide inhibits mitochondrial respiration. In these experiments hemoglobin over-expressing roots showed internal oxygen concentrations of 67 % air sat., whereas in the wild-type roots 85.6 % air sat. could be measured. Root respiration measurements revealed that hemoglobin over-expressing roots had a respiration rate of 960 nmoles/gfw/h while the wild-type roots showed a respiration rate of 730 nmol/gfw/h. This is in accordance with previous findings and shows how nitric oxide inhibits respiration and increases oxygen homeostasis.
Plant hemoglobins constitute diverse groups of haem proteins and are divided into three groups: Class 1 hemoglobins possess a very high affinity to oxygen. Class 2 hemoglobins have a lower oxygen affinity and are involved in plant development. Class 3 hemoglobins are truncated and represent a class which is very distinct from class 1 and 2 hemoglobins.
Class 1 hemoglobins scavenge nitric oxide (NO) produced at very low oxygen levels in plants. Nitric oxide can be generated in various pathways, which are divided into oxidative and reductive enzymatic reactions. The reductive pathways operate under low oxygen conditions. Nitrate reductase (NR) reduces nitrate to nitrite and further to NO. Under low oxygen conditions (less than 1%) the mitochondrial electron transport chain also reduces nitrite to NO. The mitochondrial originated NO is very interesting as it is known to regulate plant respiration. NO binds to cytochrome c oxidase and inhibits respiration, by doing so it can increase the internal oxygen concentration. This is very important for short time survival of plants under conditions like flooding.
In this project it was investigated which effect non-symbiotic hemoglobin expression has on root respiration and internal oxygen concentrations to get new insights into the role of in vivo generated NO on plant respiration. The VisiSens™ oxygen imaging system was applied to visualize oxygen distributions in cut root and also for respiration measurement in roots in Eppendorf tubes. The planar oxygen sensor foils allowed integration in different set-ups.
Materials & Methods
Wild-type (WT) and hemoglobin over-expressing (Hb+) barley plants were grown on hydroponics according to Planchet et al. (2005). Roots were carefully excised from the plants and put into a beaker that contained hydroponic medium. 1 - 2 cm root slices were cut and placed on a slide that contained a few drops of nutrient solution. The oxygen-sensitive sensor foil (SF-RPSU4, PreSens) was cut into 1 x 2 cm pieces, placed on the roots and the internal oxygen distribution recoded with the VisiSens™ detector unit. For respiration measurement 200 mg of the roots were placed in 1.5 mL eppendorf tubes that contained 1 mL of HEPES pH 7.2.
Prior to adding roots, 0.5 x 0.5 cm sensor foil pieces were attached to the inner walls of the Eppendorf tube caps with silicone glue. The Eppendorf tubes equipped with the sensors along with roots were placed in a heat block and adjusted to a temperature of 25 °C. The VisiSens™ detector unit was fixed with a stand in 2 cm distance from the sensor foil to achieve best resolution (see Fig. 1).
Internal Oxygen & Respiration in Roots
In order to check the effect of in vivo generated NO on root internal oxygen content wild-type and non-symbiotic hemoglobin over-expressing barley plants were used. Roots from 3 week old plants were cut and the oxygen concentration measured as described above. Internal oxygen concentration of hemoglobin over-expressing barley roots was 67 % air saturation, whereas the wild-type roots showed oxygen concentrations of 85.6 % air saturation (see Fig. 2). 3D oxygen plots generated with the VisiSens AnalytiCal 1 software clearly indicate the oxygen concentrations in root tissues (Fig. 3). Results of a currently ongoing project (not shown here) indicate that in wild-type plants there is more nitric oxide (NO), while in hemoglobin over-expressing plants NO production is reduced to 30 % compared to the wild-type. NO inhibits mitochondrial respiration. By inhibiting respiration, NO can increase oxygen availability in the roots. This must explain increasing internal oxygen concentration of the wild-type roots in comparison to hemoglobin over-expressing roots. In order to check the effect of internal oxygen concentration on root respiration and vice versa respiration of wild-type and hemoglobin over-expressing roots was measured in a 20 minutes time series with VisiSens™. Interestingly, the respiratory rate of hemoglobin over-expressing roots was 960 mmoles/gfw/h (see also Fig. 4) whereas in the wild-type roots the respiratory rate was 730 mmoles/gfw/h. This clearly explains that nitric oxide inhibits respiration and increases oxygen homeostasis in roots.
The results in these experiments are in accordance with previous findings on NO production and its inhibiting effect on plant respiration. With the VisiSens™ system it was possible to visualize oxygen distributions in cut roots of wild-type and hemoglobin over-expressing barley. The oxygen imaging system also allowed respiration measurements. The planar sensor foils, which can be cut into any desired shape, make it possible to apply the system in different set-ups. The flexibility and easy control enable precise oxygen measurements. The results will contribute to the ongoing project and give further insight in regulatory processes in plants.