Citrus sinensis CBF1 Functions in Cold Tolerance by Modulating Putrescine Biosynthesis Through Regulation of ARGININE DECARBOXYLASE.

C-repeat (CRT) binding factors (CBFs) are well known to act as crucial transcription factors that function in cold stress response. Arginine decarboxylase (ADC)-mediated putrescine biosynthesis has been reported to be activated in plants exposed to cold conditions, but it remains elusive whether CBFs can regulate ADC expression and putrescine accumulation. In this study, we show that cold up-regulated ADC gene (CsADC) and elevation of endogenous putrescine content in sweet orange (Citrus sinensis). Promoter of CsADC contains two CRT sequences that are canonical elements recognized by CBFs. Sweet orange genome contains four CBFs (CsCBF1-4), in which CsCBF1 was significantly induced by cold. CsCBF1, located in the nucleus, was demonstrated to bind directly and specifically to the promoter of CsADC and acted as a transcriptional activator. Overexpression of CsCBF1 led to notable elevation of CsADC and putrescine level in sweet orange transgenic plants, along with remarkably enhanced cold tolerance, relative to the wild type (WT). However, pretreatment with D-arginine, an ADC inhibitor, caused prominent reduction of endogenous putrescine level in the overexpressing lines, accompanied by greatly compromised cold tolerance. Taken together, these results demonstrate that CBF1 of sweet orange directly regulates ADC expression and modulates putrescine synthesis for orchestrating the cold tolerance. Our findings shed light into the transcriptional regulation of putrescine accumulation through targeting the ADC gene in the presence of cold stress. Meanwhile, this study illustrates a new mechanism underlying the CBF-mediated cold stress response.