ERF109 of trifoliate orange (Poncirus trifoliata (L.) Raf.) contributes to cold tolerance by directly regulating expression of Prx1 involved in antioxidative process.

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 H2 O2 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.

Wang M ，Dai W ，Du J ，Ming R ，Dahro B ，Liu JH ... -  《-》

ERF108 from Poncirus trifoliata (L.) Raf. functions in cold tolerance by modulating raffinose synthesis through transcriptional regulation of PtrRafS.

Ethylene-responsive factors (ERFs) are plant-specific transcription factors involved in cold stress response, and raffinose is known to accumulate in plants exposed to cold. However, it remains elusive whether ERFs function in cold tolerance by modulating raffinose synthesis. Here, we identified a cold-responsive PtrERF108 from trifoliate orange (Poncirus trifoliata (L.) Raf.), a cold-tolerant plant closely related to citrus. PtrERF108 is localized in the nucleus and has transcriptional activation activity. Overexpression of PtrERF108 conferred enhanced cold tolerance of transgenic lemon, whereas virus-induced gene silencing (VIGS)-mediated knockdown of PtrERF108 in trifoliate orange greatly elevated cold sensitivity. Transcriptome profiling showed that PtrERF108 overexpression caused extensive reprogramming of genes associated with signaling transduction, physiological processes and metabolic pathways. Among them, a raffinose synthase (RafS)-encoding gene, PtrRafS, was confirmed as a direct target of PtrERF108. RafS activity and raffinose content were significantly increased in PtrERF108-overexpressing transgenic plants, but prominently decreased in the VIGS plants under cold conditions. Meanwhile, exogenous replenishment of raffinose could recover the cold tolerance of PtrERF108-silenced plants, whereas VIGS-mediated knockdown of PtrRafS resulted in cold-sensitive phenotype. Taken together, the current results demonstrate that PtrERF108 plays a positive role in cold tolerance by modulation of raffinose synthesis via regulating PtrRafS. Our findings reveal a new transcriptional module composed of ERF108-RafS underlying cold-induced raffinose accumulation in plants.

Khan M ，Hu J ，Dahro B ，Ming R ，Zhang Y ，Wang Y ，Alhag A ，Li C ，Liu JH ... -  《-》

ERF9 of Poncirus trifoliata (L.) Raf. undergoes feedback regulation by ethylene and modulates cold tolerance via regulating a glutathione S-transferase U17 gene.

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.

Zhang Y ，Ming R ，Khan M ，Wang Y ，Dahro B ，Xiao W ，Li C ，Liu JH ... -  《-》

A basic helix-loop-helix transcription factor, PtrbHLH, of Poncirus trifoliata confers cold tolerance and modulates peroxidase-mediated scavenging of hydrogen peroxide.

Huang XS ，Wang W ，Zhang Q ，Liu JH ... -  《-》

ICE1 of Poncirus trifoliata functions in cold tolerance by modulating polyamine levels through interacting with arginine decarboxylase.

ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix-loop-helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H2O2 and O2 (-), was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene.

Huang XS ，Zhang Q ，Zhu D ，Fu X ，Wang M ，Zhang Q ，Moriguchi T ，Liu JH ... -  《-》