Improvement On The Thermal Stability Of Plant Cytosolic Ascorbate Peroxidase 1

Reactive oxygen species(ROS) are well recognized for playing a dual role in plant growth, development and responses to numerous environmental stresses: eliciting oxidative damages at high concentration, and mediating cellular signalling as second messenger at low concentration. Such a functional switch depends on the equilibrium between ROS production and scavenging, which is delicately monitored by an in vivo binary antioxidative system having both enzymic and nonenzymic components. Among the antioxidative enzymes, plant cytosolic ascorbate peroxidase 1(APX1) that is crucial in cellular redox homeostasis and ROS scavenging network, has been regarded as an important leverage of ROS roles and thus given with extensive research interest. However, the intrinsic thermolability of APX1 is proposed to cause plant heat-susceptibility and a thermostable APX1 variant would improve plant heat tolerance. At present, two major molecular methodologies are generally used to create thermostable protein variants: site-directed/random mutagenesis for a specific target protein, and fusing a thermostabilizing peptide partner to any client proteins of interest. Because of the simplicity, universal applicability and no requirements of protein high structural information, the latter fusion strategy has gained increasing concerns. In this work, several hyper-acidic fusion partners, as the C-terminal additive tails, were evaluated for their effects on the thermal stability and activity of APX1 from Jatropha curcas and Arabidopsis. The major results were shown as follows:(1) Gene cloning of APX1 from J. curcas and Arabidopsis and construction of their prokaryotic fusion expression vectorsTotal RNA were extracted from the seedlings of J. curcas and Arabidopsis by Trizol reagents, and reversely transcribed as the PCR templates. The gene coding region sequences(CDS) of J. curcas APX1(JcAPX1) and Arabidopsis APX1(AtAPX1) were successfully amplified with the corresponding specific primers, and subcloned into plasmid pET32a(+) to create their prokaryotic expression vectors pET(JcAPX1) and pET(AtAPX1) which were further verfied by sequencing. Then, the coding DNA fragments of several hyper-acidic parnters(ATS, ATTa, ATTb, Mb, ATYd) were individually in-frame fused behind APX1 CDS within the vector pET(JcAPX1) or pET(AtAPX1), to generate a series of E. coli fusion expression vectors of JcAPX1 or AtPAX1.(2) Hyper-acidic fusion partners can significantly improve the thermostability of both JcAPX1 and AtAPX1 and protect them from thermal inactivationAs judged by the SDS-PAGE-based thermostability analysis, all used hyper-acidic fusion partners(ATS, ATTa, ATTb, Mb, ATYd) could significantly enhance the solubility or thermostability of recombinant JcAPX1 protein fusions. Meanwhile, the results from In gel APX activity staining and spectrometry-based enzymatic assays indicated these hyper-acidic partners could efficiently protect JcAPX1 from thermal inactivation, with an elevated heat tolerance of at least 2℃higher. In addition, it was found from the similar analyses that the fusion partners ATS, ATTa could also remarkably improve the thermostability and thermo-activity of AtAPX1, with an increased heat tolerance of at least 3℃.As proposed, the thermostabilizing effects of these hyper-acidic fusion partners on the recombinant APX1 in E. coli should be reproduced in plants. Those thermostable APX1 variants created in this study should have potential uses to alleviate the heat-induced oxidative damages and improve plant heat tolerance as a new efficient route. Obviously, this study must be definitely regarded as the fundamental basis for the future work to improve plant heat tolerance through APX1-targeted plant genetic engineering.