A C2H2-type zinc finger protein ZAT12 of Poncirus trifoliata acts downstream of CBF1 to regulate cold tolerance.

The Cys2/His2 (C2H2)-type zinc finger family has been reported to regulate multiple aspects of plant development and abiotic stress response. However, the role of C2H2-type zinc finger proteins in cold tolerance remains largely unclear. Through RNA-sequence analysis, a cold-responsive zinc finger protein, named as PtrZAT12, was identified and isolated from trifoliate orange (Poncirus trifoliata L. Raf.), a cold-hardy plant closely related to citrus. Furthermore, we found that PtrZAT12 was markedly induced by various abiotic stresses, especially cold stress. PtrZAT12 is a nuclear protein, and physiological analysis suggests that overexpression of PtrZAT12 conferred enhanced cold tolerance in transgenic tobacco (Nicotiana tabacum) plants, while knockdown of PtrZAT12 by virus-induced gene silencing (VIGS) increased the cold sensitivity of trifoliate orange and repressed expression of genes involved in stress tolerance. The promoter of PtrZAT12 harbors a DRE/CRT cis-acting element, which was verified to be specifically bound by PtrCBF1 (Poncirus trifoliata C-repeat BINDING FACTOR1). VIGS-mediated silencing of PtrCBF1 reduced the relative expression levels of PtrZAT12 and decreased the cold resistance of trifoliate orange. Based on these results, we propose that PtrZAT12 is a direct target of CBF1 and plays a positive role in modulation of cold stress tolerance. The knowledge gains new insight into a regulatory module composed of CBF1-ZAT12 in response to cold stress and advances our understanding of cold stress response in plants.

The transcription factor ERF110 promotes cold tolerance by directly regulating sugar and sterol biosynthesis in citrus.

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.

Transcriptome and metabolome atlas reveals contributions of sphingosine and chlorogenic acid to cold tolerance in Citrus.

Citrus is one of the most important fruit crop genera in the world, but many Citrus species are vulnerable to cold stress. Ichang papeda (Citrus ichangensis), a cold-hardy citrus species, holds great potential for identifying valuable metabolites that are critical for cold tolerance in Citrus. However, the metabolic changes and underlying mechanisms that regulate Ichang papeda cold tolerance remain largely unknown. In this study, we compared the metabolomes and transcriptomes of Ichang papeda and HB pummelo (Citrus grandis 'Hirado Buntan', a cold-sensitive species) to explore the critical metabolites and genes responsible for cold tolerance. Metabolomic analyses led to the identification of common and genotype-specific metabolites, consistent with transcriptomic alterations. Compared to HB pummelo under cold stress, Ichang papeda accumulated more sugars, flavonoids, and unsaturated fatty acids, which are well-characterized metabolites involved in stress responses. Interestingly, sphingosine and chlorogenic acid substantially accumulated only in Ichang papeda. Knockdown of CiSPT (C. ichangensis serine palmitoyltransferase) and CiHCT2 (C. ichangensis hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase2), two genes involved in sphingosine and chlorogenic acid biosynthesis, dramatically decreased endogenous sphingosine and chlorogenic acid levels, respectively. This reduction in sphingosine and chlorogenic acid notably compromised the cold tolerance of Ichang papeda, whereas exogenous application of these metabolites increased plant cold tolerance. Taken together, our findings indicate that greater accumulation of a spectrum of metabolites, particularly sphingosine and chlorogenic acid, promotes cold tolerance in cold-tolerant citrus species. These findings broaden our understanding of plant metabolic alterations in response to cold stress and provide valuable targets that can be manipulated to improve Citrus cold tolerance.

Transcription factors ABF4 and ABR1 synergistically regulate amylase-mediated starch catabolism in drought tolerance.

ß-Amylase (BAM)-mediated starch degradation is a main source of soluble sugars that help plants adapt to environmental stresses. Here, we demonstrate that dehydration-induced expression of PtrBAM3 in trifoliate orange (Poncirus trifoliata (L.) Raf.) functions positively in drought tolerance via modulation of starch catabolism. Two transcription factors, PtrABF4 (P. trifoliata abscisic acid-responsive element-binding factor 4) and PtrABR1 (P. trifoliata ABA repressor 1), were identified as upstream transcriptional activators of PtrBAM3 through yeast one-hybrid library screening and protein-DNA interaction assays. Both PtrABF4 and PtrABR1 played a positive role in plant drought tolerance by modulating soluble sugar accumulation derived from BAM3-mediated starch decomposition. In addition, PtrABF4 could directly regulate PtrABR1 expression by binding to its promoter, leading to a regulatory cascade to reinforce the activation of PtrBAM3. Moreover, PtrABF4 physically interacted with PtrABR1 to form a protein complex that further promoted the transcriptional regulation of PtrBAM3. Taken together, our finding reveals that a transcriptional cascade composed of ABF4 and ABR1 works synergistically to upregulate BAM3 expression and starch catabolism in response to drought condition. The results shed light on the understanding of the regulatory molecular mechanisms underlying BAM-mediated soluble sugar accumulation for rendering drought tolerance in plants.

Front-line and second-line treatment for mantle cell lymphoma in clinical practice: A multicenter retrospective analysis.

BACKGROUND: There are few reports of clinical practice treatment patterns and efficacy in mantle cell lymphoma (MCL). MATERIALS AND METHODS: We retrospectively studied a large, multicenter, cohort of patients with MCL diagnosed between 2000 and 2020 in eight institutions. RESULTS: 536 patients were registered (73% male, median of 70 years). Front-line treatment was based on high-dose cytarabine, bendamustine, and anthracyclines in 42%, 12%, and 15%, respectively. The median PFS for all patients was 45 months; 68, 34, and 30 months for those who received high-dose cytarabine-based, bendamustine-based and anthracycline-based therapy. 204 patients received second-line. Bendamustine-based treatment was the most common second-line regimen (36% of patients). The median second-line PFS (sPFS) for the entire cohort was 14 months; 19, 24, and 31 for bendamustine-, platinum-, and high-dose cytarabine-based regimens, with broad confidence intervals for these latter estimates. Patients treated with cytarabine-based therapies in the front-line and those with front-line PFS longer than 24 months had a substantially superior sPFS. CONCLUSION: Front-line treatment in this cohort of MCL was as expected and with a median PFS of over 3.5 years. Second-line treatment strategies were heterogeneous and the median second-line PFS was little over 1 year.

The small molecule Zaractin activates ZAR1-mediated immunity in Arabidopsis.

Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.

Comparison of the positional accuracy of robotic guided dental implant placement with static guided and dynamic navigation systems: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: The development of robotic computer assisted implant surgery (r-CAIS) offers advantages, but how the positional accuracy of r-CAIS compares with other forms of guided implant surgery remains unclear. PURPOSE: The purpose of this systematic review and meta-analysis was to evaluate the positional accuracy of r-CAIS and to compare the positional accuracy of r-CAIS with s-CAIS and d-CAIS. MATERIAL AND METHODS: Five databases were systematically searched by 2 independent reviewers for articles published before May 2023. A manual search was also performed. Articles evaluating the positional accuracy of r-CAIS were included. The Cochrane risk of bias tool was used for the clinical studies, whereas the QUIN tool was used for the in vitro studies. A meta-analysis was performed to compare the positional accuracy of r-CAIS with d-CAIS. RESULTS: Thirteen studies were included, with 9 in vitro studies, 4 clinical studies, and a total of 920 dental implants. A high risk of bias was noted in 6 studies and low to moderate in 7 studies. R-CAIS showed greater accuracy for the coronal, apical, and angular deviations compared with d-CAIS. (-0.17 [-0.24, 0.09], (P<.001); -0.21 [-0.36, -0.06] (P=.006), and -1.41 [-1.56, -1.26] (P<.001)) CONCLUSIONS: R-CAIS can provide improved positional accuracy compared with d-CAIS when considering coronal, apical, and angular deviations. However, evidence to compare the positional accuracy of r-CAIS with s-CAIS was insufficient. These results should be interpreted with caution because of the limited data and the bias noted in several studies.

Tele-dentistry in COVID-19 era: A conduit for dental care in uncertain times.

Tele-dentistry encompasses all sorts of digital technologies that involve the exchange of patient's clinical data from a distant site for the provision of dental health care. Tele-dentistry has emerged from the concept of telemedicine, which has been in practice since the 19th century. In recent times, an upsurge in the digital technologies was noted, which has made the possibility of remote access to dental care. The outbreak of COVID- 19 pandemic has restricted the normal routine ways of clinical practice. In these challenging times, tele-dentistry serves as effective platform for providing dental health care. Tele-dentistry has vast applications across various disciplines of dentistry, including preventive dentistry, paediatric dentistry, oral medicine, and oral pathology etc. In these pandemic times, tele-dentistry can be efficiently used for identification of dental emergencies, allowing effective triage and subsequent management. There are different communication platforms available for tele-dentistry. The most common technologies used are web-based video conferencing and smart phone-based applications. As the clinicians are not aware of these digital technologies utilised in tele-dentistry, there are certain challenges associated with its use. In conclusion, tele-dentistry serves as an effective tool in providing health care in challenging times, but it has been underutilised by the dental fraternity. The legislative authorities should establish proper standard protocols to ensure the safety and confidentiality of patient information while using these digital platforms.

Nasopalatine canal cyst, a diagnostic twist.

We present a case of nasopalatine duct cyst in a 35-yearold female. The cyst was diagnosed based on the presence of only one clinical symptom and no obvious clinical signs, which is a relatively rare occurrence. However, the radiographic and histological presentation of this lesion was typical of a nasopalatine duct cyst. Therefore, this case report aims to highlight the variable presentations of the nasopalatine cyst, which is often misdiagnosed and treated as an endodontic infection.

Survival of patients with mantle cell lymphoma in the rituximab era: Retrospective binational analysis between 2000 and 2020.

Mantle cell lymphoma (MCL) is a rare peripheral B-cell lymphoma characterised by eventual relapse and progression towards a more aggressive disease biology. With the introduction of rituximab- and cytarabine-based immunochemotherapy regimens, the prognosis of the disease has changed dramatically over the last two decades. To assess the real-world survival of patients with MCL, we used a population-based cohort of 564 patients with MCL who were diagnosed and treated between 2000 and 2020. Patient data were collected from seven Finnish treatment centres and one Spanish treatment centre. For the entire patient population, we report a 2-year overall survival (OS) rate of 77%, a 5-year OS of 58%, and a 10-year OS of 32%. The estimated median OS was 80 months after diagnosis. MCL is associated with increased mortality across the entire patient population. Additionally, we assessed the survival of patients after MCL relapse with the aim of establishing a cut-off point of prognostic significance. Based on our statistical analysis of survival after the first relapse, disease progression within 24 months of the initial diagnosis should be considered as a strong indicator of poor prognosis.

Oral manifestations of Covid-19-A literature review.

Initially, it was reported that coronavirus 2019 disease (Covid-19) affects respiratory, gastrointestinal and neurological systems, but the oral, olfactory and integumentary systems are also involved. This review discusses various oral manifestations of Covid-19 reported in the literature along with possible underlying mechanisms. The reported manifestations include taste impairment, oral mucosal changes (petechiae, ulcers, plaque-like lesions, reactivation of herpes simplex virus 1(HSV1), geographical tongue and desquamative gingivitis) and dry mouth. The prominent location for mucosal lesions are tongue, palate and labial mucosa. The exact pathogenesis of these oral symptoms is not known. Angiotensin-converting enzyme 2 (ACE2) cell receptors are expressed in abundance on oral mucosa allowing severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) to infect them. Gustatory impairment along with olfactory changes is now listed as a symptom of Covid-19 by the World Health Organization, but further research is needed to confirm a link between reported additional oral symptoms and Covid-19. Dental professionals may encounter individuals with Covid-19 and be called upon to identify various oral manifestations of this disease.

Low educational attainment is associated with higher all-cause and cardiovascular mortality in the United States adult population.

INTRODUCTION: Educational attainment is an important social determinant of health (SDOH) for cardiovascular disease (CVD). However, the association between educational attainment and all-cause and CVD mortality has not been longitudinally evaluated on a population-level in the US, especially in individuals with atherosclerotic cardiovascular disease (ASCVD). In this nationally representative study, we assessed the association between educational attainment and the risk of all-cause and cardiovascular (CVD) mortality in the general adult population and in adults with ASCVD in the US. METHODS: We used data from the 2006-2014 National Death Index-linked National Health Interview Survey for adults ≥ 18 years. We generated age-adjusted mortality rates (AAMR) by levels of educational attainment (< high school (HS), HS/General Education Development (GED), some college, and ≥ College) in the overall population and in adults with ASCVD. Cox proportional hazards models were used to examine the multivariable-adjusted associations between educational attainment and all-cause and CVD mortality. RESULTS: The sample comprised 210,853 participants (mean age 46.3), representing ~ 189 million adults annually, of which 8% had ASCVD. Overall, 14.7%, 27%, 20.3%, and 38% of the population had educational attainment < HS, HS/GED, Some College, and ≥ College, respectively. During a median follow-up of 4.5 years, all-cause age-adjusted mortality rates were 400.6 vs. 208.6 and 1446.7 vs. 984.0 for the total and ASCVD populations for < HS vs ≥ College education, respectively. CVD age adjusted mortality rates were 82.1 vs. 38.7 and 456.4 vs 279.5 for the total and ASCVD populations for < HS vs ≥ College education, respectively. In models adjusting for demographics and SDOH, < HS (reference = ≥ College) was associated with 40-50% increased risk of mortality in the total population and 20-40% increased risk of mortality in the ASCVD population, for both all-cause and CVD mortality. Further adjustment for traditional risk factors attenuated the associations but remained statistically significant for < HS in the overall population. Similar trends were seen across sociodemographic subgroups including age, sex, race/ethnicity, income, and insurance status. CONCLUSIONS: Lower educational attainment is independently associated with increased risk of all-cause and CVD mortality in both the total and ASCVD populations, with the highest risk observed for individuals with < HS education. Future efforts to understand persistent disparities in CVD and all-cause mortality should pay close attention to the role of education, and include educational attainment as an independent predictor in mortality risk prediction algorithms.

Managing Monkeypox Virus: Characterizing Common Cutaneous Manifestations and Antiviral Efficacy.

BACKGROUND: The Monkeypox virus (MPX) has been detected in multiple non-endemic countries since May 2022. The cutaneous manifestations of MPX can have multiple distinct presentations, including pustular and vesicular. Although there are no approved treatments, three antivirals (brincidofovir, cidofovir, tecovirimat) have been utilized. The objective of our study was to conduct a systematic review to evaluate antiviral efficacy (first aim) and cutaneous manifestations of MPX (second aim). METHODS: Utilizing PRISMA guidelines, we searched PubMed and SCOPUS databases to identify studies utilizing antiviral treatment in human subjects for MPX and studies reporting cutaneous characteristics of MPX lesions. RESULTS: For our first aim, six articles met inclusion criteria. For our second aim, 27 met inclusion criteria. Eighty-eight percent had complete resolution with tecovirimat (n=28) which was well tolerated, and decreased hospitalization time (10 days) compared to brincidofovir (29 days). Forty-four percent of patients had <10 cutaneous lesions and 36% had 10-100 lesions. The most common lesion type was pustular (32%, n=380). CONCLUSION: This limited sample of studies suggests that tecovirimat is well tolerated and may be an effective antiviral for MPX treatment. Further studies are required to better understand the role of antivirals for MPX treatment among human patients. J Drugs Dermatol. 2023;22(3): doi:10.36849/JDD.7263.

Ethanol-Gas-Sensing Performances of Built-in ZrO2/Co3O4 Hybrid Nanostructures.

The development of novel nanomaterials as highly efficient gas-sensing materials is envisaged as one of the most important routes in the field of gas-sensing research. However, developing stable, selective, and efficient materials for these purposes is a highly challenging task requiring numerous design attempts. In this work, a ZrO2/Co3O4 composite is reported, for the first time, as a gas-sensing material for the detection of ethanol. The sensitive and selective detection of ethanol gas at 200 °C has been demonstrated for the ZrO2/Co3O4 (0.20 wt%/0.20 wt%)-based sensor. Furthermore, the sensor showed a very low response/recovery time of 56 s and 363 s, respectively, in response to a pulse of 20 ppm of ethanol and good stability. The interesting gas-sensing property of ZrO2/Co3O4 can be ascribed to both the porous structure, which facilitates the interaction between the target gas and the sensing site, and the p-p-junction-induced built-in electric field. These results indicate that the ZrO2/Co3O4 composite can serve as a heterostructured nanomaterial for the detection of ethanol gas.

The Implication of Stem Cells in Direct Pulp Capping: Where Do We Stand?

Direct pulp capping has been established as a more conservative alternative to root canal therapy, especially in case of an iatrogenic or traumatic exposure of the dental pulp. However, recent literature focuses on regeneration therapies to develop a physiological dentin barrier. The process of tissue regeneration through stem cell therapy involves a complex interaction between stem cells, growth factors and scaffolds known as the 'tissue engineering triad'. Recent advancements in stem cell therapy focus on the re programming of stem cells, development of scaffolds and enhancement of the regenerative potential of these stem cells, using appropriate growth factors and transfer media. This article provides a summary of the current evidence regarding the use of stem cell therapy in direct pulp capping. The sources of stem cells, types of scaffolds and growth factors have been described in detail along with the limitations and future prospects.

Evaluation of the Nasopalatine Canal (NPC) in a subset of Pakistani population, using Cone Beam Computed Tomography (CBCT).

OBJECTIVE: To evaluate variations in the shape, diameter, length and width of the nasopalatine canal along with the width of the buccal cortical bone anterior to it, using cone beam computed tomography imaging. METHODS: The retrospective, cross-sectional study was conducted at the Aga Khan University Hospital, Karachi, from September to October 2020, and comprised pre-existing cone beam computed tomography scans taken between 2015 and 2020 of patients of either gender aged 18-60 years who had maxillary central incisors present. The shapes and dimensions of the nasopalatine canal were observed along with the buccal bone anterior to the nasopalatine canal. Data was compared with respect to age and gender. Data was analysed using SPSS 23. RESULTS: Of the 90 scans evaluated, 46(51.1%) belonged to females with a mean age of 37.85±18.19 years, and 44(48.9%) belonged to males with a mean age of 38.07±13.58 years. The mean length and width of the nasopalatine canal was 11.28±1.90mm and 2.62±0.91mm, respectively. The nasopalatine canal was significantly longer (p<0.01) and wider (p=0.02) in males than females. The mean diameter of foramen of Stenson was 2.99±1.17mm and incisive foramen was 6.09±1.80mm. The mean width of the buccal cortical bone at the most coronal, middle and most incisal levels was 7.20±1.70mm, 6.12±1.31mm and 6.12±1.31mm, respectively. Buccal bone width was wider in males than females, but the difference was significant only at the midpoint (p<0.05). CONCLUSIONS: There was a significant difference in the dimensions of the width and length of the NPC with respect to gender. No significant differences were observed with respect to age.

The Dilemma Of Management Of Cystic Lesions; An Uncertain Way Forward: A Case Report.

Periapical diseases ranges from mild granulomatous lesions to large cystic ones, with the treatments corresponding to their respective pre-operative diagnoses. However, the determination of cause of periapical radiolucency is impossible on pre-operative clinical and radiographic examinations. We present a case highlighting the difficulties encountered in treating a periapical cyst using the current evidence in literature. It demonstrates the uncertainty involved in treating such lesions, owing to the impossible nature of determining the histopathological nature of the cyst, i.e., being either true cysts or pocket cysts. This case includes orthograde re-treatment; decompression of the cystic lesion, followed by peri-apical surgery of two teeth over a course of three years; and the uncertain outcomes encountered after each phase of the treatment.

Antimicrobial and antioxidant activity on Pakistan origin Launaea nudicaulis (L.) Hook f.: A promising halophyte.

Facultative type of Halophytes is those which can easily grow on both saline and non-saline soil conditions Launaea nudicaulis (L.) Hook f. belongs to family compositae [Asteraceae] is facultative halophyte with great medicinal values. The main objective of this study is to explore anti-oxidant and anti-bacterial potential on different morphological parts of Launaea nudicaulis (L.) Hook f. The results of the antibacterial activity of methanolic extracts of leaves stem and roots of Launaea nudicaulis (L.) Hook f. showed that the potent antibacterial activity observed in all three morphological parts against Staphylococcus aureus. DPPH scavenging activity of extracts of root, stem, leaves against standard i.e., ascorbic acid at different concentrations of 20, 40, 60, 80 and 100 showed that root extract have more significant activity when compared to the ascorbic acid. Documented potentials of halophytes are elevating its prominence which motivates scientist to further explore with extensive research.

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.

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.