Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat

Abstract

Cold acclimation is a complex biological process leading to the development of freezing tolerance in plants. In this study, we demonstrated that cold-induced expression of protease inhibitor FmASP in a Citrus-relative species kumquat [ Fortunella margarita (Lour.) Swingle] contributes to its freezing tolerance by minimizing protein degradation. Firstly, we found that only cold-acclimated kumquat plants, despite extensive leaf cellular damage during freezing, were able to resume their normal growth upon stress relief. To dissect the impact of cold acclimation on this anti-freezing performance, we conducted protein abundance assays and quantitative proteomic analysis of kumquat leaves subjected to cold acclimation (4^◦C), freezing treatment (−10^◦C) and post-freezing recovery (25^◦C). FmASP (Against Serine Protease) and several non-specific proteases were identified as differentially expressed proteins induced by cold acclimation and associated with stable protein abundance throughout the course of low-temperature treatment. FmASP was further characterized as a robust inhibitor of multiple proteases. In addition, heterogeneous expression of FmASP in Arabidopsis confirmed its positive role in freezing tolerance. Finally, we proposed a working model of FmASP and illustrated how this extracellular-localized protease inhibitor protects proteins from degradation, thereby maintaining essential cellular function for post-freezing recovery. These findings revealed the important role of protease inhibition in freezing response and provide insights on how this role may help develop new strategies to enhance plant freezing tolerance.

Acknowledgements

The authors would like to thank Drs. Xiangyang Lu (Hunan Agricultural University, China), Zhanguo Xin (USDA-ARS, Lubbock, TX, United States) and Huazhong Shi (Texas Tech University, Lubbock, TX, United States) for giving technical guidance and Drs. Zhanguo Xin and Xinbo Chen (Hunan Agricultural University, China) for revision on earlier versions of this manuscript. We also thank Drs. Jeff Mower and Arvind Dubey (University of Nebraska-Lincoln, United States) for their critical feedback and editing. This research was sponsored by the National Natural Science Foundation of China (No.31200963) and the Key Project of Hunan Provincial Education Department (No.18A091).

Author contributions

H.Y., X.X. and H.L. conceived and designed the experiments. H.Y., K.Q., J. T., J.C., and Y.Z. set up and carried out the experiments. H.Y., K.Q., J.T., Y.Z., L.R. and H.L. analyzed the data. H.Y., Y.Z. and H.L. wrote the paper. All authors reviewed the manuscript.

Data availability

All data supporting the findings of this work are included in the article and supplementary files. The plant materials used in this study are available from the corresponding authors upon request.

Conflict of interest statement

The authors declare that they have no conflict of interest.

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