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SUMMARY: ERFs (ethylene‐responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold‐induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold‐hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus‐induced gene silencing)‐mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress‐responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6‐like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP‐glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110‐overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110‐silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110‐ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress. Significance Statement: The transcription factor ERF110 of Poncirus trifoliata L. Raf. functions positively in cold tolerance due to modulation of sugar and sterol biosynthesis by regulating SPS4, ERD6L16, and UGT80B1. [ABSTRACT FROM AUTHOR]

Summary: Plant ethylene‐responsive factors (ERFs) play essential roles in cold stress response, but the molecular mechanisms underlying this process remain poorly understood. In this study, we characterized PtrERF9 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold‐hardy plant. PtrERF9 was up‐regulated by cold in an ethylene‐dependent manner. Overexpression of PtrERF9 conferred prominently enhanced freezing tolerance, which was drastically impaired when PtrERF9 was knocked down by virus‐induced gene silencing. Global transcriptome profiling indicated that silencing of PtrERF9 resulted in substantial transcriptional reprogramming of stress‐responsive genes involved in different biological processes. PtrERF9 was further verified to directly and specifically bind with the promoters of glutathione S‐transferase U17 (PtrGSTU17) and ACC synthase1 (PtrACS1). Consistently, PtrERF9‐overexpressing plants had higher levels of PtrGSTU17 transcript and GST activity, but accumulated less ROS, whereas the silenced plants showed the opposite changes. Meanwhile, knockdown of PtrERF9 decreased PtrACS1 expression, ACS activity and ACC content. However, overexpression of PtrERF9 in lemon, a cold‐sensitive species, caused negligible alterations of ethylene biosynthesis, which was attributed to perturbed interaction between PtrERF9, along with lemon homologue ClERF9, and the promoter of lemon ACS1 gene (ClACS1) due to mutation of the cis‐acting element. Taken together, these results indicate that PtrERF9 acts downstream of ethylene signalling and functions positively in cold tolerance via modulation of ROS homeostasis by regulating PtrGSTU17. In addition, PtrERF9 regulates ethylene biosynthesis by activating PtrACS1 gene, forming a feedback regulation loop to reinforce the transcriptional regulation of its target genes, which may contribute to the elite cold tolerance of Poncirus trifoliata. [ABSTRACT FROM AUTHOR]

Summary: Ethylene‐responsive factors (ERFs) have been revealed to play essential roles in a variety of physiological and biological processes in higher plants. However, functions and regulatory pathways of most ERFs in cold stress remain largely unclear. Here, we identified PtrERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) and deciphered its role in cold tolerance. PtrERF109 was drastically up‐regulated by cold, ethylene and dehydration, but repressed by salt. PtrERF109 was localized in the nucleus and displayed transcriptional activity, and the C terminus is required for the activation. Overexpression of PtrERF109 conferred enhanced cold tolerance in transgenic tobacco and lemon plants, whereas VIGS (virus‐induced gene silencing)‐mediated suppression of PtrERF109 in trifoliate orange led to increased cold susceptibility. PtrERF109 overexpression caused extensive transcriptional reprogramming of several suites of stress‐responsive genes. Prx1 encoding class III peroxidase (POD) was one of the antioxidant genes exhibiting the greatest induction. PtrERF109 was shown to directly bind to the promoter of PtrPrx1 (trifoliate orange Prx1 homologue) and positively activated its expression. In addition, the PtrERF109‐overexpressing plants exhibited significantly higher POD activity and accumulated dramatically less H2O2 and were more tolerant to oxidative stress, whereas the VIGS plants exhibited opposite trends, in comparison with wild type. Taken together, these results indicate that PtrERF109 as a positive regulator contributes to imparting cold tolerance by, at least partly, directly regulating the POD‐encoding gene to maintain a robust antioxidant capacity for effectively scavenging the reactive oxygen species. Our findings gain insight into better understanding of transcriptional regulation of antioxidant genes in response to cold stress. [ABSTRACT FROM AUTHOR]