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Article|16 Feb 2023|OPEN
Protease inhibitor ASP enhances freezing tolerance by inhibiting protein degradation in kumquat
Hua Yang1,2 , , Ke-wei Qiao1 , Jin-jing Teng1 , Jia-bei Chen1 , Ying-li Zhong1 , Li-qun Rao1 and Xing-yao Xiong2,3 , , Huang Li,4 ,
1College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China
2Hunan Provincial Key Laboratory for Germplasm Innovation and Crop Utilization, Hunan Agricultural University, Changsha 410128, China
3Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China
4Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA
*Corresponding author. E-mail:,,

Horticulture Research 10,
Article number: uhad023 (2023)
Views: 156

Received: 24 Oct 2022
Accepted: 06 Feb 2023
Published online: 16 Feb 2023


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.