Abstract
Ascorbic acid is a ubiquitous water soluble antioxidant that plays a critical role in plant growth and environmental stress tolerance. It acts as a free radical scavenger as well as a source of reducing power for several cellular processes. Because of its pivotal role in regulating plant growth under optimal as well as sub-optimal conditions, it becomes obligatory for plants to maintain a pool of reduced ascorbic acid. Several cellular processes help in maintaining the reduced ascorbic acid pool, by regulating its synthesis and regeneration processes. Current study demonstrates that monodehydroascorbate reductase is an important enzyme responsible for maintaining the reduced ascorbate pool, by optimizing the recycling of oxidized ascorbate. Cloning and functional characterization of this important stress inducible gene is of great significance for its imperative use in plant stress management. Therefore, we have cloned and functionally validated the role of monodehydroascorbate reductase gene (mdar) from a drought tolerant variety of Eleusine coracana. The cloned Ecmdar gene comprises of 1437bp CDS, encoding a 478 amino acid long polypeptide. The active site analysis showed presence of conserved Tyr348 residue, facilitating the catalytic activity in electron transfer mechanism. qPCR expression profiling of Ecmdar under stress indicated that it is an early responsive gene. The analysis of Ecmdar overexpressing Arabidopsis transgenic lines suggests that monodehydroascorbate reductase acts as a key stress regulator by modulating the activity of antioxidant enzymes to strengthen the ROS scavenging ability and maintains ROS homeostasis. Thus, it is evident that Ecmdar is an important gene for cellular homeostasis and its over-expression could be successfully used to strengthen stress tolerance in crop plants.
Highlights
Ascorbic acid is a vital antioxidant molecule that plays a crucial role in removal of excessive reactive oxygen species (ROS), by enzymatic as well as non-enzymatic scavenging mechanisms [1]
There are various isoforms of Monodehydroascorbate reductase that exist in a cell; out of which cytosolic and peroxisomal isoforms are considered to be the regulatory points for maintaining ascorbate pool [27]
In our endeavor to understand the dynamics of this essential process, we have cloned and characterized the mdar from a stress tolerant variety of Eleusine coracana
Summary
Ascorbic acid is a vital antioxidant molecule that plays a crucial role in removal of excessive reactive oxygen species (ROS), by enzymatic as well as non-enzymatic scavenging mechanisms [1]. It participates in numerous developmental processes such as cell division, cell expansion and cell wall growth [2,3]. Ascorbate is oxidized to monodehydroascorbate (MDHA) and to dehydroascorbate (DHA), by enzymatic action of ascorbate peroxidase (APX) and ascorbate oxidase (AAO) respectively and non-enzymatic reaction like detoxification of toco-trienoxyl radical during the process of ROS scavenging [11]. Monodehydroascorbate reductase (MDAR EC 1.6.5.4) appears to be an important enzyme regulating the reduced AsA pool [13], and is a key component in the stress tolerance profile of a plant
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