Cold stress is a major factor restricting plant performance and fitness. Depending on the length and intensity of the cold stimulus, plants respond to the stress event by alleviating immediately occurring adverse effects of cold stress and by inducing cold acclimation. In the case of a timely limited cold stimulus, Arabidopsis thaliana responds with the formation of a cold memory that modifies its response to a future cold stimulus, a phenomenon which is called priming. Short-term cold priming was previously shown to attenuate chloroplast to nucleus signalling in the regulation of cold-induced ZAT10 expression. In the present study, this effect was mimicked by transient overexpression of tAPX, but not of sAPX at 20 °C, while counteracting priming-induced tAPX accumulation during the lag-phase abolished the priming effect. This demonstrated that cold priming is mediated at the thylakoid membrane and is regulated by post-priming tAPX expression. Electrolyte leakage assays demonstrated that the AP2/ERF-Ib transcription factors RAP2.4c and RAP2.4d, that have been proposed to regulate chloroplast APX gene expression, negatively regulate the direct cold response and cold acclimation. RNAseq analysis of rap2.4c and rap2.4d KO plants one hour after transfer to 4 °C revealed stronger induction of genes that are involved in JA/ET, JA and SA signalling pathways than in wild type Col-0 pants. Subsequent analyses did not show differences in hormone contents and in sensitivity to hormone signals in rap2.4c and rap2.4d plants, demonstrating that misregulation of gene expression in the knock-out lines is independent of hormone availability and sensing. Transient overexpression of RAP2.4c and RAP2.4d also did not influence hormone-related transcript levels. Additionally, the lack of RAP2.4c and RAP2.4d did not affect cold priming-dependent attenuation of ZAT10 regulation. The similarities of the expression patterns of rap2.4c and rap2.4d with that of an npr1 mutant in the cold indicate an upstream function of both transcription factors in NPR1-mediated gene expression regulation. A putative target is TRXH5, which is involved in quaternary structure regulation and, consequently, nuclear translocation of NPR1. This gene was strongly upregulated in the rap2.4d line. Besides a DRE-motif, which is a known RAP2.4d binding site, the TRXH5 promoter contains a RAP2.4d binding motif, which was identified in the present study based on a Yeast-One-Hybrid screen with RAP2.4d and random genomic DNA fragments of Arabidopsis thaliana. The present study highlights RAP2.4d, and to a lesser extent RAP2.4c, as cold-inducible inhibitors of TRXH5 expression attenuating the NPR1-mediated induction of JA/ET, JA and SA responses in the early cold response.
