Probing the role of Reactive Oxygen Species (ROS) in plant regeneration

Abstract

Plants have a remarkable capacity for wound-healing and regeneration, which can be investigated easily in Arabidopsis thaliana. Regenerative responses in Arabidopsis are broadly categorized as tissue culture-induced, or mechanical injury-induced regeneration. Regardless of the stimulus, regenerating tissue shows various stress responses before showing any visible changes in its morphology. Reactive oxygen species (ROS), accumulate as byproducts of aerobic metabolism, whose excess levels are harmful to plants, but they also serve as signalling molecules that are necessary for plants to initiate a repair and healing response. In order for organisms to survive and regenerate, ROS homeostasis must be tightly regulated. However, little is known about the in vivo spatiotemporal nature of ROS production and mitigation. In this study, we have used A. thaliana as a plant model system for studying the role of reactive oxygen species (ROS) in the plant regeneration. In this project, the mutant lines of stress marker family called Rboh (Respiratory burst oxidase homolog) are used to study ROS in leaf vein regeneration assay and de novo root regeneration (DNRR) assay. Also, the ROS responsive fluorescent reporter was cloned, utilizing the promoter of a well-known ROS-responsive transcription factor ZAT12 to drive the expression of free mCherry (Red fluorescence protein), in order to understand the dynamics of ROS and it’s biological role in plant regeneration. None of the ROS mutants (rbohD and rbohF) were found to be impaired in leaf vein regeneration. Strikingly, besides D-loop which is seen in wild type, a straight path was also observed in some of the regenerated samples for both the mutants. H2DCFDA fluorescent dye was used to monitor ROS dynamics post injury in these two mutants. ROS accumulation was observed in the cells lying in the immediate vicinity of the wound in wild type but not in rbohD and rbohF mutants. In case of DNRR, regeneration efficiency was severely compromised in these mutants. How ROS production signalling integrates with the auxin signalling and microtubules dynamics is still unclear. Generation of auxin and microtubule reporter lines in the rbohD and rbohF mutant background to study this integration is ongoing.