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

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,therebymaintaining 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.

PAPP2C Interacts with the Atypical Disease Resistance Protein RPW8.2 and Negatively Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis

Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell. The atypical resistance （R） protein RPW8.2 activates salicylic acid （SA）-dependent, haustorium-targeted defenses against Golovinomyces spp., the causal agents of powdery mildew diseases on multiple plant species. How RPW8.2 activates defense remains uncharacterized. Here, we report that RPWS.2 interacts with the phytochrome-associated protein phosphatase type 2C （PAPP2C） in yeast and in planta as evidenced by co- immunoprecipitation and bimolecular fluorescence complementation assays. Down-regulation of PAPP2C by RNA interfer- ence （RNAi） in Col-0 plants lacking RPWS.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway. Moreover, down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death, which correlates with elevated RPWS.2 expression. We further demonstrate that hemagglutinin （HA）-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity. In addition, silencing a rice gene （Os04g0452000） homologous to PAPP2C also results in spontaneous cell death in rice. Combined, our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.